J. MoL BioL (1995) 247, 618-635
Structural Transitions During Bacteriophage HK97
Head Assembly
Robert L. Duda ~, John HempeP, Hanspeter MicheP
Jeffrey Shabanowitz 3, Donald HunP ,4 and Roger W. Hendrix ~*
'Department of Biological
Sciences, University of
Pittsburgh, Pittsburgh
PA 15260, U.S.A.
2Department of Molecular
Genetics and Biochemistry
University of Pittsburgh
School of Medicine
Pittsburgh, PA 15261, U.S.A.
~Department of Chemistry
University of Virginia
Charlottesville, VA 22901
U.S.A.
4Department of Pathology
University of Virginia Health
Sciences Center
Charlottesville, VA 22908
U.S.A.
*Corresponding author
Bacteriophage HK97 builds its head shell from a 42 kDa major head protein,
but neither this 42 kDa protein nor its processed, 31 kDa form is found in
the mature head. Instead, each of the major head-protein subunits is
covalently cross-linked into oligomers of five, six or more by a protein
cross-linking reaction that occurs both in vivo and in vitro. Mutants that block
prohead maturation lead to the accumulation of one of two types of
proheads, termed Prohead I and Prohead II. Prohead I is assembled from
about 415 copies of the 42 kDa (384 amino acids) protein subunit and
accumulates in infections by mutant ainU4. Following assembl)4 the
N-terminal 102 amino acids of each subunit are removed, leaving a prohead
shell constructed of 31 kDa subunits, called Prohead II, which accumulates
in infections by mutant amC2. During DNA packaging, when the prohead
shell expands, all of the head protein subunits become covalently
cross-linked to other subunits. Purified Prohead II (or, less completely;
Prohead I) becomes cross-linked in vitro in response to any of a number of
conditions that induce shell expansion, including conditions commonly used
for protein analysis. 117vitro cross-linking occurs efficiently in the absence of
added cofactors of enzymes, and we propose that cross-linking is catalyzed
by shell subunits themselves. Shell expansion is easily monitored by
observing a decrease in electrophoretic mobility of Prohead II in agarose gels.
Using the mobility shift in agarose gel to monitor expansion and SDS/gel
electrophoresis to monitor cross-linking in vitro, we find that expansion
precedes and is required for cross-linking, and we propose that expansion
triggers the cross-linking reaction. Comparison of peptides isolated from
Prohead II and in vitro cross-linked Prohead II shows a single altered major
cross-link peptide in which a lysine, originating from lysine169 of the
protein sequence, is linked to asparagine356, presumably derived from
the neighboring subunit. Examination of the cross-link-containing peptide
by mass spectrometry shows that the cross-link bond is an amide between
the side-chains of. the lysine and the asparagine residues.
Keywords: bacteriophage assembly; virus capsids; proteolysis; protein
cross-linking; protein structure
Introduction
Assembly of the head of a dsDNA bacteriophage
entails arranging several hundred protein subunits
Present address: H. Michel, Department of Biotechnolog3,; Hoffmann-LaRoche Inc., 340 Kingsland Street,
Nutle~ NJ 07110, U.S.A.
Abbreviations used: ds, double stranded; TCA,
trichloroacetic acid; CAD, collision-activateddissociation; TPCK, L-l-tosylamido-2-phenylethyl
chloromethyl ketone; PEG, polyethylene glycol; IPTG,
isopropyl-~-D-thiogalactopyranoside;Mes, 2(N-morpholino)ethanesulfonic acid; UAc, uranyl acetate; KPTA,
potassium phosphotungstic acid.
0022-2836/95/140618-18 $08.00/0
into a precisely ordered array in the form of an
icosahedrally symmetric shell, which then must
recognize and condense a genomic DNA molecule
into its interior. The resulting head, after it joins a tail
to produce a mature virion, must provide a stable
structure that sequesters and protects the DNA from
environmental insults, but it must also be able to
release the DNA when the virus encounters an
appropriate host. Studies on a variety of these phages
indicate that the protein subunits of heads do not
initially assemble in the form that is found in mature
heads. Rather the proteins go through various
structural transitions between initial assembly and
appearance of mature heads. These structural
~') 1995 AcademicPress Limited
Phage HK97 Head Assembly
619
HK97 Head Assembly
head subunit
.~
47kD portal
25kD prolease
~
Prohead I
T=7
42 kD subunil
not oro~-linked
expansi.
Prohead I1
31 kD subunt!
not c r o ~ l i n k e d
. . .
Head 1
3! kD subunll
not cross-linked
lin~g
0
Head 11
31 kDsubunit
cross-linked
Figure 1. Proposed assembly pathway for HK97
heads.The properties of the head-related structures, the
descriptions of the cleavage, expansion and cross-linking
reactions, and the rationale for the arrangement of these
elements into this pathway are given in the text and in
Duda et al. (1995a).
transitions can include proteolytic cleavages, conformational changes, and covalent joining of subunits. It
is assumed that the successive properties the proteins
acquire as they progress through these structural
transitions enable them to carry out their successive
functions in head production, including shell assembl~ DNA packaging, DNA protection, and DNA
deliver34 as well as providing a pathway for achieving
the final structure. A more detailed understanding of
the nature and temporal order of the transitions that
occur in phage head assembly should lead to a more
complete appreciation of their individual roles in the
assembly process and a better understanding of the
overall logic of the assembly pathway
HK97 is a lambdoid bacteriophage that is proving
to be a tractable experimental system for investigating these aspects of viral assembly HK97 virions
have a morphology very similar to that of the well
studied phage ~, but there appear to be numerous
differences in the details of their head assembly
pathways. Most strikingl~ all of the major head
subunits of HK97 become covalently linked to their
neighbors during head maturation (Popa et al., 1991),
a phenomenon that has not been seen in any
previously studied virus. In this paper we further
characterize the cross-linking reaction, including the
chemical identity of the cross-link bond. In addition,
we show that the major head subunit, a 42 kDa
protein, is processed to a 31 kDa form prior to
cross-linking, and we give evidence for a major
conformational change in the subunits immediately
prior to cross-link formation.
Results
Head assembly pathway
We propose a pathway for the assembly of HK97
heads. This pathway is inferred from the properties
of heads and of head-related structures that we
believe may be intermediates in the pathway and
from structural transitions that these structures
undergo in vitro. We present the pathway here
(Figure 1) in order to provide a framework for
consideration of the data. Data that support the
various features of the pathway are described below
and in the accompanying paper (Duda et al., 1995a).
The first shell structure that we have identified in
the pathwa34 Prohead I, is composed of ~ 400 copies
of the 42 kDa major head subunit and 12 copies of the
portal subunit. (The portal is an annular structure at
one corner of the prohead which serves as the
attachment site for the tail and is thought to mediate
entry and exit of DNA (Bazinet & King, 1985).) In the
transition of Prohead I to the next structure, Prohead
II, each copy of the major subunit loses 102 amino
acids, about one-quarter of its mass, from its amino
terminus. Prohead II then undergoes an expansion
reaction in which the shell becomes thinner, more
angular, and larger in diameter. By analogy with
similar phages, we believe that expansion of Prohead
II in vivo is probably triggered by DNA packaging.
Expansion of the shell triggers a covalent cross-linking reaction in which each of the major subunits is
covalently linked to its neighbors. Note that HK97
head assembly differs from that of previously
characterized dsDNA phages in that there is no
separately encoded scaffolding protein that participates in shell assembly (Duda et al., 1995a; Z. Xie
and R. Hendrix, unpublished results).
Prohead composition and morphology
We previously described two amber mutants of
HK97 which cause accumulation of prohead like
structures in infected cells (Popa et al., 1991).
Figure 2A and B shows a comparison of SDS gels
of these proheads purified from infections by
HK97amU4 and HK97amC2. These structures
correspond to Prohead I and Prohead II of Figure 1,
respectively Both proheads have a minor component
of 47 kDa, which we have argued elsewhere (Popa
et al., 1991; Duda et al., 1995a) is the subunit of the
portal. The Prohead I lane shows a major component
of 42 kDa. Prohead II, on the other hand, has none of
the 42 kDa subunit but does have a comparable
amount of a 31 kDa subunit. We showed previously
(Popa et al., 1991) that the proteins of Proheads I and
II share many tryptic peptides and we show below
that the 31 kDa protein is a processed form of the
42 kDa subunit.
The compositions of these proheads can be
influenced by the conditions they experience during
purification and storage. In a previous publication
(Popa et al., 1991) we reported that all of the major
protein in Prohead II is found in higher molecular
mass, covalently cross-linked forms rather than the
31 kDa form shown in Figure 2B. We show below that
a number of treatments induce formation of these
cross-links in vitro. In our earlier purification scheme,
prohead-producing cells were exposed to chloroform to ensure complete lysis; chloroform is one of
the agents that we now know efficiently induces
cross-link formation in vitro. Thus we believe that the
proheads in our earlier experiments had become
cross-linked in vitro and that the uncross-linked form
of Prohead II shown in Figure 2B more closely
represents the form of Prohead II that is present in
infected cells.
The electron micrographs in Figure 3A and B
Phage HK97 Head Assembly
620
~,~,I,~-~
~'~ ~ b ~ ' ~~
+.,o~ b~
o~ ~
"¢'#~
"%¢¢~x~
b
_o*
~,~"
0
13 14 15 16 17 18 1 9
"2. • I
' , ,
2. . . .
~
Prohead II
gradient fractions
;
=1-5.6-m~
-=}-5,6-m~r
C"
, " .':(;~ " ' . : . , . ~ - - P o r t d
A
B
..'~ ~.~
,~ ~.,~.£a.
:,'~.h".9+r'~J--Pona
(3
Figure 2. Protein composition of HK97 Prohead I and
Prohead II. Lanes A and B show the protein compositions
of Prohead I and Prohead II, respectivel N analyzed on
stained SDS/polyacrylamide gels after TCA precipitation
and denaturation. Both structures contain the 47 kDa
portal protein. Prohead ! has a 42 kDa major head protein.
The major protein of Prohead II is the 31 kDa processed
form of the protein present in Prohead I. The difference in
mobility of the portal between the 2 gels is due to different
cross-linker concentrations in the gels: lane A was from a
normal 10% (Std-C) gel, while the gel in lane B was a 10%
(Low-C) gel that has less cross-linker (see Materials and
Methods). C shows 7 fractions from the region containing
the peak of proheads in a glycerol gradient used for
purifying Prohead II analyzed on a polyacrylamide
gel following TCA precipitation. These are fractions 13
through 19 out of a total of 35, numbered from the bottom.
The Prohead II peak is fraction 15. Several faint bands
above the 31 kDa position and also above the 47 kDa
position clearly are spillover from some contaminating
structure sedimenting higher in the gradient. One minor
band below the 47 kDa position is the 42 kDa uncleaved
form of the major head protein from a small fraction of the
faster sedimenting Prohead I present in this preparation of
Prohead II. The 24 kDa minor band below the 31 kDa
position was found (by N-terminal sequencing) to be a
proteolytic fragment of the major head protein starting at
residue 167. We conclude from these data that the only
minor species of prohead protein present in purified
Prohead II that co-sediments with the major head protein
is the portal protein and that all the properties we attribute
to the structure are due to the major head protein and the
portal. The gel contained 10% (Low-C) acrylamide.
reveal that Prohead I and Prohead II have similar
m o r p h o l o g y w h e n negatively stained with UAc; they
are both approximately spherical, thick-walled
structures with a d i a m e t e r of about 45 nm. P r o h e a d
I images have small ring-shaped capsomeres present
in the b a c k g r o u n d (Figure 3A); the relative n u m b e r
Figure 3. Morphology of HK97 Prohead I, Prohead II,
and expanded Prohead II (Head II). The Figure compares
electron micrographs of the different HK97 head
structures. A shows the approximately 45 nm diameter,
rounded, thick-walled Prohead I stained with UAc; note
the dissociated capsomeres in the background. B shows the
similar-appearing Prohead II, stained with UAc. C shows
Prohead II (made by plasmid expression and lacking the
portal) stained with KPTA. The thick stain preserves the
shape and shows the knob-studded surface. Both Prohead
II and Prohead II (plasmid) have nearly identical
morphologies and properties. D shows in vih'o expanded
Prohead II (Head II lacking DNA) stained with KPTA.
Following expansion the diameter increases to about
540 nm, the shell becomes thinner and smoother, and
changes to the same distinct angular shape observed in the
mature virion (Popa et al., 1991). Expansion was induced
with 7 M urea at pH 5 (see Table 2). E shows expanded
Prohead II stained with UAc, which causes distortion of the
structure, although the shell thickness change is apparent.
Proheads were expanded using chloroform (see Figure 7).
Note: UAc stain can also induce changes ill Prohead II,
while Prohead I is severely distorted by KPTA (not shown).
Image magnification is about 80,000.
of such capsomeres seen in Prohead I preparations
increases with time as the p r o h e a d s slowly dissociate
(data not shown). A variant of Prohead II, m a d e by
expression f r o m a plasmid in vivo (see u n d e r
Materials and Methods), is nearly identical, except
for the absence of the portal (Duda et al., 1995a).
W h e n Prohead II (plasmid) was p r e p a r e d using an
alternative stain (KPTA), the thick, nearly spherical
shells a p p e a r to have regularly textured inner and
outer surfaces (Figure 3C). E x p a n d e d proheads,
s h o w n in Figure 3D and E, are discussed below.
The portal is a universally conserved structure in
the d s D N A phages that have b e e n studied to date.
Portals f r o m several different phages have b e e n
s h o w n to have 12 protein subunits, though s o m e
Phage HK97Head Assembly
621
Table 1
Amino acid sequences
Source of
protein/peptide
Prohead I
Prohead II
CNBr/trypsin peptide from
uncross-linked protein, 34.3 rain
CNBr/trypsin peptide from
cross-linked protein, 40.2 rain
CNBr/V8 peptide from
uncross-linked protein, 24.5 min
CNBr/V8 peptide from
cross-linked protein, 32.8 rain
Sequence determined
SELALIQKAIEESQQKM
TQLFDAQKAelest
SLGSDADSAGSLIQPM
QIPGUMPGLRRLTIRDL
LAQGRTSSNALEYVRE
EVFTNNADVV
ALKPESDITFSK
AL( )PESDITFSK
Interpretation
Amino terminus of 42 kDa protein,
starting at position 2 of predicted
protein sequence
Amino terminus of 31 kDa protein,
starting at position 104 of predicted
protein sequence
Peptide from position 167 to 178
ALKPESDITFSK
I
NM
Peptide from position 345 to 357
VSREDxDNFVKN
KALKPE
t
VSREDRDNFVKNM
The Table shows the amino acid sequences that were determined in this study using the Edman degradation. The
experimental results are represented in the center column, using the single letter amino acid code. For sample 1, lower
case letters indicate weak signals for the corresponding amino acids in those cycles. For sample 4, the ( ) indicates
that none of the standard amino acids gave a significant signal in cycle 3. For sample 5, there was no readable signal in
cycle 6. For sample 6, there were strong signals for each of two amino acids in cycles 1, 2, 3, and 5; the aspartate signal in
cycle 12 was weak. The third column gives our interpretation of the structures of the samples.
V+ K,S+A, R+ L, E, D+ P,
E, D, N, F, V, K, (D)
cases have b e e n d o c u m e n t e d recently of portals with
13 subunits (Dube et al., 1993). If we a s s u m e that the
HK97 portal has 12 subunits, then the ratio of masses
in the portal and head subunit b a n d s on a gel such
as that in Figure 2B allows us to calculate h o w m a n y
subunits of the h e a d protein there are in a prohead.
We m e a s u r e d the ratio of radioactivity in these two
b a n d s in triplicate lanes of 3%-labeled Prohead II,
corrected for the n u m b e r s of sulfur a t o m s in the two
proteins, and d e t e r m i n e d that there w e r e 417 + 30
head protein subunits for each 12 portal protein
subunits. This corresponds very well to the
expectation for an icosadeltahedral shell built
according to the scheme of C a s p a r & Klug (1962)
with a triangulation n u m b e r (T) of 7. A T = 7 shell
should have 415 subunits (assuming that one corner
p e n t a m e r is displaced b y the portal), while the two
allowed triangulation n u m b e r s nearest to T = 7,
T = 4 and T = 9, would have 235 and 535 subunits,
respectively
Cleavage of the major head protein
We d e t e r m i n e d the a m i n o - t e r m i n a l a m i n o acid
sequences of the 42 kDa protein of Prohead I and the
31 kDa protein of P r o h e a d II, using purified
p r o h e a d s as substrates for the E d m a n degradation.
We obtained 31 residues of sequence f r o m the 42 kDa
protein, starting with the sequence SELALIQ
(Table 1). Figure 4 shows the a m i n o acid sequence of
the major head subunit protein of HK97 predicted
f r o m the D N A sequence of its gene (Duda et al.,
1995a). The a m i n o acid sequence of the 42 kDa
protein of P r o h e a d I matches the predicted sequence,
starting with the serine at position 2, and the
apparent molecular m a s s of the protein matches that
predicted f r o m the sequence. (As expected for a
protein with serine in position 2 (Dalboge et al.,
1990) the initiating methionine has b e e n lost.)
F r o m the 31 kDa protein of P r o h e a d II w e obtained
60 residues of a m i n o acid sequence beginning
SLGSDAD (Table 1). This matches the predicted
sequence beginning at residue 104. This result is
consistent with the hypothesis that the 31 kDa
protein is p r o d u c e d f r o m the 42 kDa protein b y the
proteolytic r e m o v a l of 102 a m i n o acids f r o m its
a m i n o terminus. Experiments described b y D u d a
et al. (1995a) which examine the kinetics of labeling
of the 31 kDa and 42 kDa proteins, a r g u e that the
HK97 Head Protein
M S E L A L IQKAI E E S Q Q K M T Q L F D A Q K A E I E S T G Q V S K Q L Q S D L M K V Q E E L
TKSGTRLFDLEQKLASGAENPGEKKSFSERAAEELIKSWDGKQGTFGAKT
5o
too
FNKS L G S D A D S A G S L 7 Q P M Q I P GI I I ~ G L R R L T I R D L L A Q G R T S S N A L E Y 15o
lcleavage
V R E E V F T N N A D V V A E K A L K P E S D I T F S K Q T A N V K T I A H W V Q A S R Q V M D D A 2OO
i crossqink
PMLQSY IN N R L M Y G L A L K E E G Q L L N G D G T G D N L E G L N K V
T A Y D T S L N A T 250
GDTRAD I IAHAIYQVTE SEFSASGIVLNP RDWHNIALLKDNEGRYIFGGP
3oo
QAF T S N I M W G L P V V P T K A Q A A G T F T V G G F D M A S Q V W D R M D A T V E V S R E D R
350
DNFVENMLTILCEE~YRPTAI
Across-link
IKGTFSSGS
385
Figure 4. Amino acid sequence of the HK97 head
protein. The sequence, which is given in the one letter
amino acid code, is derived from the DNA sequence of the
head protein gene reported in Duda et al. (1995a). Amino
acid sequence determination of peptides and of the cleaved
and uncleaved forms of the protein has directly confirmed
39% of the amino acid sequence. Arrows indicate the
positions of the cleavage (following Lysl03) and the two
amino acids that participate in cross-link formation
(Lys169 and Asn356).
622
Phage HK97 Head Assembly
Prohead I1"
cleaved
subunits
Prohead I
uncleaved
subunits
1
2
-
5 , 6 - m = ( =
4-mcr
--
2.met
~
3
-
~
~
4
-
5
6
Acetone TCA SDS
1
2
3
7
L---/
~
~
m
~,
:.;~_
""=-L
Portal
q L I
A
IJ
i
Figure 5. Spontaneous in vitro cross-linking of HK97 Prohead I and Prohead II in SDS/gel sample buffer. A, The gel
contained 10% (Low-C) acrylamide. Lane 1, highly purified amU4-derived Prohead I (uncleaved 42 kDa subunit) prepared
by mixing with 100°C SDS/gel sample buffer; lanes 2 and 3, same material allowed to incubate in SDS/gel sample buffer
at 23°C for 2 and 5 minutes prior to heating to 100°C. Higher-molecular-mass bands, which we interpret as multimers
of the 42 kDa subunit, form with time. Lane 6, highly purified amC2-derived Prohead II (cleaved 31 kDa subunit); lanes
7 and 8, the same experiment as above but carried out with Prohead II (cleaved 31 kDa subunits); samples were allowed
to incubate in SDS/gel sample buffer at 23°C for 2 and 5 minutes prior to heating to 100°C. Higher-molecular-mass bands,
which we interpret as multimers of the 31 kDa subunit, form with time. Lane 5, molecular-mass markers; lane 4, a mixture
of lanes 2 and 6. B, The gel contained 10% (Low-C) acrylamide. The 3 lanes show identical aliquots of uncross-linked
Prohead II that were prepared for gel electrophoresis by 3 different methods. Lane 1, proheads were precipitated with
90% acetone and the dried pellet was resuspended in SDS/gel buffer and immediately placed at 100°C; lane 2, the proheads
were TCA precipitated as described in Materials and Methods and resuspended in SDS/gel buffer as above; lane 3, the
sample was processed using normal procedures without special precautions: proheads were mixed with SDS/gel buffer
and then heated to 100°C.
31 kDa protein is in fact a post-translationally
processed form of the 42 kDa protein rather than the
result of an internal translational initiation.
In vitro cross-linking
Purified Prohead II undergoes spontaneous
cross-linking in vitro w h e n it is subjected to certain
solvent conditions. Figure 5A illustrates this for one
such condition, namely exposure to SDS/gel sample
buffer at room temperature. The sample in lane 6 was
prepared for electrophoresis by placing proheads in
SDS/gel sample buffer (2% SDS, w / v ) that was
preheated to 100°C. In lanes 7 and 8, the proheads
were placed in sample buffer at 25°C and incubated
at that temperature for two or five minutes,
respectivel~ before heating to 100°C. With time at
25°C, an increasing amount of the 31 kDa protein is
found in a ladder of bands extending u p the gel. We
interpret these bands as dimer, trimer, tetramer,
pentamer, and hexamer of the 31 kDa subunit. The
regularly spaced ladder stops with the hexamer
band, but there are some minor bands at a wider
spacing above the hexamer, and material accumulates with time in the sample well. The pentamer and
hexamer bands and the first minor band above the
hexamer are apparently identical to components of
mature heads and virions; none of the lower
oligomers of the head subunit appear in mature
structures.
A n u m b e r of other conditions induce the
formation of cross-links in Prohead II. Table 2 lists
the conditions we have investigated and their
effects on cross-linking. Figure 5B, lane 1 shows
the result of one of these conditions, precipitation with cold 90% acetone. With m a n y of the
reagents it is possible to achieve a level of
cross-linking comparable to the level found in
mature virions.
As Figure 5 illustrates, mixing proheads with
100°C SDS/gel buffer effectively blocks most
Phage HK97 Head Assembly
623
Table 2
Solvent, pH, a n d
d e n a t u r a n t effects u p o n H K 9 7 Prohead [I
Test
Prohead II
Expand
x-Link
Other conditions
P r o h e a d IIp1,~mi,J
Expand
x-Link
Organic solvents at 5%
Chloroform
Isopentyl alcohol, n-butanol
Isobutanol
Isopropanol, methylene chloride
Ethyl or butyl acetate, t-butanol
Methanol, ethanol, acetone,
acetonitrile, dimethyl sulfoxide
50
50
50
50
50
50
50
50
50
50
50
50
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
Tris-HCl
NaCI
Tris-HC1
NaCI
Tris-HCl
NaCI
Tris-HCI
NaCI
Tris-HCl
NaCI
Tris-HCl
NaC1
(pH 7.5),
+++
+++
+++
+++
(pH 7.5),
++
++
++
++
(pH 7.5),
++
++
(pH 7.5),
w
w
-
-
(pH 7.5),
-
-
-
-
(pH 7.5),
-
-
W
W
+
++
W
++
W
W
+++
++
W
++
W
++
+++
++,S
+++
W
Precipitating conditions
Acetone 90%, ethanol 95%
Trichloroacetic acid (TCA) 10%
++
Glycerol
10 to 60%
70%
80%
P h o s p h a t e buffered NaC1
Phosphate buffered NaC1
P h o s p h a t e buffered NaCI
+++
+++
+++
++
50
50
50
50
50
+++
+++
- ,D
++
++,a
w,a
- ,a
- ,a
++
++
+
+
GuanMine hydrochlorMe
1.0
1.5
2.0
2.5
5.0
M
M
M
to 3.0 M
to 7.0 M
mM
mM
mM
mM
mM
Tris-HC1
Tris-HC1
Tris-HCi
Tris-HC1
Tris-HCl
(pH
(pH
(pH
(pH
(pH
8.0)
8.0)
8.0)
8.0)
8.0)
Urea o1" salt, pH
1.7 to 3.4 M urea
5.0 M urea
6.7 M urea
2.0 M urea, 0.1 to 0.5 M KC1
5.0 M urea
6.0 M urea
7.0 M urea
2.0 M urea, 0.1 to 0.5 M KCI
5.0 M urea
6.0 M urea
7.0 M urea
2.0 M urea, 0.1 to 0.5 M KCI
5.0 M urea
6.0 M urea
7.0 M urea
0.1 M urea to 0.5 M KC1
2.0 M urea
5.0 M urea
6.0 M, 7.0 M urea
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
25 mM
25 mM
25 mM
25 m M
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 mM
50 m M
Tris-HCl (pH 7.5),
NaC1
Tris-HCl (pH 7.5),
NaC1
Tris-HCl (pH 7.5),
NaC1
Tris-HCl (pH 7.5)
Tris-HCI (pH 7.5)
Tris-HCl (pH 7.5)
Tris-HC] (pH 7.5)
Mes (pH 6.0)
Mes (pH 6.0)
Mes (pH 6.0)
Mes (pH 6.0)
sodium acetate (pH
sodium acetate (pH
sodium acetate (pH
sodium acetate (pH
sodium acetate (pH
sodium acetate (pH
sodium acetate (pH
s o d i u m acetate (pH
5.0)
5.0)
5.0)
5.0)
4.0)
4.0)
4.0)
4.0)
++
W
+++,d
+++,d
++
+
General conditions for these assays were as follows: for solvents, p r o h e a d s were diluted into the specified buffer a n d solvents were
a d d e d and mixed; for precipitating conditions p r o h e a d s were mixed with the specified solvent, set on ice for at least 20 m i n u t e s ,
centrifuged, and the pellet taken u p in SDS sample buffer a n d denatured; for glycerol, the glycerol was a d d e d by m a s s (1.26 g / m l ) , m i x e d
with buffer a n d p r o h e a d s in the s a m e buffer were a d d e d a n d mixed; for all other conditions p r o h e a d s were diluted into a m i x t u r e to
give the final concentrations. All incubations were at 22°C for 15 to 18 hours.
expand: Expansion was evaluated by r u n n i n g a sample on a native agarose gel; +++ complete expansion, ++ strong expansion,
+ m o d e r a t e expansion, w weak, b u t noticeable expansion, - no effect.
x-link: C r o s s q i n k i n g was evaluated by TCA precipitating a s a m p l e and analyzing it on a n S D S / p o l y a c r y l a m i d e gel as described u n d e r
Materials a n d Methods: +++ complete cross-linking (no m o n o m e r remaining), ++ strong cross-linking (a ladder of b a n d s w a s observed:
see Figure 5B, lane 1), + moderate cross-linking, w weak b u t noticeable cross-linking, - no effect.
Special notes: a, an abnormal d i m e r b a n d was observed ( r u n s slower in the SDS gel); d, moderate destructive effect: severe b a n d
smearing in agarose gel; D, severe destructive effect: no b a n d in agarose gel; s, special condition: expansion is complete in 60 m i n u t e s
u n d e r these conditions a n d there were no obvious destructive effects, b u t cross-linking is severely inhibited; if the p r o h e a d s are diluted
10 to 20-fold away from the urea into p H 7.5 buffer, cross-linking proceeds to completion in about 60 m i n u t e s (data not shown).
Phage HK97 Head Assembly
624
cross-link formation, although some dimer and a
trace of trimer are visible. An even more effective
method of preventing in vitro cross-link formation is
to precipitate proheads with cold 10% (w/v)
trichloroacetic acid (TCA: Figure 5B, lane 2) prior to
preparing them for SDS/gel electrophoresis. We
have used TCA precipitation to prepare samples for
all the SDS gels shown here except for those in
Figure 5.
In the purest preparations of Prohead II, no minor
bands are seen on an SDS gel except those
corresponding to the major head subunit and the
portal subunit (see Figure 2C). The sensitivity of
staining is such that we would expect to detect four
to six copies per prohead of any other protein
subunit if it were present. In vitro cross-linking of
Prohead II requires only the addition of one of the
reagents indicated in Table 2. Thus there is no
evidence for a requirement for a separate enzyme to
catalyze the cross-linking reaction. Furthermore,
experiments described by Duda et al. (1995a) show
that prohead-like structures made from a plasmid
carrying only the phage gene encoding the 42 kDa
head subunit are competent to cross-link; this argues
rigorously against catalysis of the cross-link by a
second phage-encoded protein. We propose that the
catalytic functions that might otherwise be provided
by an enzyme are provided by the head protein
subunits themselves and thus that the cross-linking
reaction is autocatalytic.
As Figure 5A also shows (lanes 1 to 3), Prohead I
can be induced to form cross-links in the same way
as Prohead II. A ladder of bands appears on the
SDS/gel in response to any of the same set of
treatments that induce cross-linking in Prohead II.
The ladder of bands is similar to that seen with
Prohead II but does not migrate as far into the gel,
and we interpret these as being monomer through
hexamer of the 42 kDa (uncleaved) form of the head
subunit. Unlike the case of Prohead II, we have never
seen the Prohead I cross-linking reaction go farther
than the partial cross-linking that gives a ladder
such as that shown in Figure 5. Thus it appears that
some feature of Prohead I, most likely the presence
of the amino-terminal segment that is cleaved off in
the transition to Prohead II, prevents the cross-linking from going to completion, and we suggest that
cross-linking of the uncleaved subunits of Prohead I
is not a normal reaction of the in vivo assembly
pathway
In vitro
expansion
It has been observed for a number of other tailed
phages that proheads increase in diameter and
become more angular in shape as they mature into
heads. In at least some cases, and very likely all, this
transition, known as shell expansion, occurs as DNA
is being packaged into the head shell. We find that
HK97 Prohead II also undergoes an expansion
reaction (Figure 3D). As with other phages, expansion
can be monitored as a characteristic change in size
and appearance by electron microscopy (see Fig-
UI~A
*
I
I
~;
t
I
I
I
,.
--
-
--
-
m
-"
~
m
.
concentration
I
, ~ (
.
-.'-- ~o,.
~
a
ded
Unexpanded
Figure 6. Urea treatment induces the expansion of HK97
Prohead II--using native agarose gel electrophoresis to
monitor conformational change. Purified Prohead II was
diluted into varying concentrations (noted in the Figure) of
urea buffered with 50mM Tris-HCl (pH 7.5) and
containing 50 mM NaC1. Samples were incubated at 23°C
for 18.5 hours and then diluted 2-fold with dyes and
glycerol, and electrophoresed in a 1.2% native agarose gel
as described under Materials and Methods. The starting
material contained mostly unexpanded proheads (the fast
migrating band) and a small fraction of expanded
proheads (the slower migrating band). At urea concentrations below 3.4 M, no change was seen in the proportion
of the 2 species, but at 5 M and 6.7 M urea the proheads
were induced to expand by the treatment.
ure 3D and E) or as a decrease in sedimentation
velocity (data not shown). Expansion can also be
monitored easily and quantitatively by an agarosegel assay. Figure 6 shows a 1.2% (w/v) agarose gel
which has been used to display Prohead II after
treatment with different concentrations of urea.
Untreated proheads form a discrete band which is
transformed to a second discrete band at higher urea
concentrations. When these and similar samples are
monitored by electron microscopy or sedimentation
velocit34 the fraction of expanded shells correlates
with the fraction of material in the slower-migrating
band on the agarose gel. We conclude that the
materials in the fast and slow-migrating bands are
respectively the unexpanded and expanded forms of
proheads.
Expansion of Prohead II is triggered by a variety
of treatments that we have explored, and these are for
the most part the same treatments that trigger
cross-linking (Table 2). Thus expansion and crosslinking are correlated. This result argues either that
expansion and cross-linking are triggered independently by the same list of reagents, or that the
expansion and cross-linking reactions are linked to
each other in some way
Kinetics of expansion and cross-linking
To measure the temporal relationship between
expansion and cross-linking we measured the
kinetics of both reactions in a sample of Prohead II
Phage HK97 Head Assembly
that was exposed briefly to chloroform. The solution
of proheads was exposed to saturating chloroform at
37°C for 45 seconds and then diluted 20-fold to
reduce the chloroform concentration to one that is
ineffective in causing expansion. Samples were
removed at a series of times and analyzed on an
agarose gel (Figure 7A) to monitor the extent of
expansion and on an SDS/polyacrylamide gel
(Figure 7B) to monitor the extent of cross-linking. As
Figure 7A shows, expansion proceeded rapidly
following the addition of chloroform and was
roughly 70% complete by 45 seconds when the
sample was diluted to lower the chloroform
concentration. Following dilution, expansion essentially stopped, as shown by the constant ratio of the
expanded to the unexpanded bands on the gel at
times after 45 seconds. (In similar experiments in
which the chloroform was not diluted, expansion
continued to completion.) Subunit cross-linking,
seen in Figure 7B, had only begun by the time the
proheads were diluted, but it continued after
dilution until about 30 minutes. Even at late times,
however, a substantial amount of monomer
remained, corresponding to about 30% of the starting
monomer, or the same as the fraction of shells that
did not expand. Separation of this material on a
glycerol velocity sedimentation gradient showed that
the unexpanded (fast sedimenting) proheads contained the uncross-linked protein. Taken together,
these data argue that only proheads that have
expanded (and all those that have expanded)
undergo cross-linking, and we propose that
expansion triggers cross-linking.
Location of the cross-link in the protein
sequence
We previously reported the isolation and characterization of a peptide from cross-linked Prohead II
that is an altered form of a peptide found in
uncross-linked Prohead I (Popa et al., 1991). Analysis
of that experiment was complicated by the then
u n k n o w n difference in the size of the subunits of
Prohead I and Prohead II that results from the
cleavage discussed above. We have now done similar
experiments comparing the uncross-linked and
cross-linked forms of Prohead II, which should differ
chemically only in the absence or presence of
cross-links. Prohead II, either uncross-linked or
cross-linked by treatment with chloroform, was
digested with CNBr followed by trypsin and the
resulting peptides were separated by HPLC on a
reverse phase column. Figure 8A shows superimposed HPLC traces of the peptides from the two
digests and below them a plot of the difference
between the two traces. The two traces are
essentially identical except for a peak at 34.3 minutes
that is present in the uncross-linked peptides and
absent in the cross-linked peptides and a second
peak at 40.2 minutes that is present only in the
cross-linked peptides. Analysis of these two
peptides shows them to be the same as the peptides
625
A
o ~ ~ ~ ~ ~
Time(rain)
I
I n 5 % CHCI 3
!
I
I
I
I
~m='Nm-m
am
~
Diluted
B
I
_
_
-- ~l~=ded
__
-- ~led
..
~
-- Unexl~ded
I I l I I I I I I I I I I I
H ~
Pentamer
Temm~
Trimcr
DL,n ~
Portal
Monomer
4-
4-
In
CHCI 3 :
.
.
.
.
.
.
.
.
.
.
.
.
Diluted Away from CHCI 3
Figure 7. Expansion precedes and induces cross-linking
of HK97 Prohead II. The experiments in Figure 6 and
Table 2 show that expansion of Prohead II can be triggered
in vitro under a variety of conditions and that expansion
can be monitored by native agarose gel electrophoresis,
while the cross-linking status of the same particles can be
monitored by SDS/gel analysis after TCA precipitation. A,
Effect of exposing Prohead II to saturating chloroform for
45 seconds or longer, as analyzed by native agarose gel
electrophoresis, which separates expanded from unexpanded proheads. A 0.25 ml sample of Prohead II in Kdil*
buffer was placed at 37°C in a stirred reaction vial. At time
(t) = 0, 0.025 ml of chloroform was added, and samples
were taken and diluted 20-fold with buffer for later
analysis at the times noted in the Figure. At 45 seconds after
the start of the experiment 0.09 ml was diluted into
0.171 ml of buffer stirring at the same temperature and
samples were also taken and diluted 2-fold with buffer
for later analysis at the times noted. Samples were
electrophoresed in a 1.2% native agarose gel as described
under Materials and Methods. B, Shows the progress of
cross-linking of the proheads that were exposed to
chloroform for 45 seconds and then diluted away At the
times noted appropriate volumes of sample (0.05 ml in
chloroform and 0.1 ml after dilution) were quickly mixed
with ice-cold TCA to stop cross-linking and the resulting
samples were prepared for electrophoresis and analyzed
on an SDS/polyacrylamide gel as described under
Materials and Methods. Although about 70% of the
proheads in the samples removed from chloroform had
expanded, only a small fraction of the head protein in these
samples had undergone cross-linking until considerable
time after they had expanded. The gel contained 10%
(Low-C) acrylamide. The small amounts of pentamer and
hexamer in the zero time sample represent the minor
fraction of proheads that had expanded and cross-linked
during purification and storage prior to the experiment.
626
from our earlier experiment. The peptide from
the uncross-linked protein has the sequence
ALKPESDITFSK (see Table 1), which identifies it
as residues 167 to 178 of the head protein sequence
shown in Figure 4. The peptide from the cross-linked
protein, which we expect to contain the cross-link
bond linking two peptides together, gives the
same sequence as the first peptide when it is
subjected to Edman degradation except that the
lysine signal at position 3 is missing. This is consistent
with the hypothesis that the lysine in position 3 is
linked through its side-chain to a second peptide and
that the second peptide is small enough not to
contribute substantially to the sequence detected in
the Edman degradation. The amino acid compositions of the two peptides (data not shown) suggest
that the second peptide differs from the first in the
presence of homoserine and its lactone from CNBr
cleavage at methionine, and an additional asparagine
or aspartic acid. Examination of the amino acid
sequence in Figure 4 reveals one candidate for the
second peptide which fits these data, namely the
dipeptide NM at position 356-357 in the protein
sequence.
To test this possibilit)4 we repeated the digestion
of the proheads using CNBr followed by Staphylococcus V8 protease. Figure 8B shows the HPLC traces
and the difference trace for these peptides. Again
there is a peptide that is unique to the digest
of the cross-linked protein, and we detect one
(of the expected two) that is found only in the
uncross-linked protein. Edman degradation of
the uncross-linked peptide at 24.5 minutes gives
the sequence VSREDxDNFVKN, which identifies
it as the peptide at position 345 to 357 in the
protein sequence (with the terminal homoserine
not detected). It includes the asparagine (Asn356)
that we propose above is in the downstream part
of the cross-link. The peptide at 32.8 minutes,
which comes from the cross-linked protein, gives
a complex sequence with two amino acids at
several positions (Table 1). These results are
again most simply explained if this peptide
consists of the two peptides from amino acids 166
to 171 and 345 to 357 joined through the side-chains
of lysine169 and asparagine356. The simplest
hypothesis would be that these two side-chains
are joined in an amide linkage. For the patterns
in both Figure 8A and B, there appears to be
complete or nearly complete conversion of the
precursor peptide into the cross-link-containing
peptide. This argues not only that every subunit
of the capsid participates in the cross-linking
reaction, as was evident from the absence of
monomer subunits in fully cross-linked structures,
but that the Asp356 and the Lys169 of every (or nearly
every) subunit both participate in cross-link
formation.
Chemical nature of the cross-link bond
The cross-linked peptides were analyzed with
triple quadrupole mass spectrometry to obtain
Phage HK97 Head Assembly
additional structural information. The use of
mass spectrometry for the analysis of cross-linked
peptides has already been reported (Hegyi et al.,
1992). For the HPLC fraction from 40.2 minutes
in Figure 8A, the experimental molecular weight
recorded on line from a microcapillary HPLC
column was found to be 1534 Da (average mass).
This value agrees with the theoretical value of
1533.7 Da (average mass) for the suggested structure.
The peptide Ala Leu Lys Pro Glu Ser Asp Ile Thr
Phe Ser Lys without the cross-linked part has an
average molecular mass of 1335.5 Da. The mass
difference of 198 Da is compatible with the addition
of an Asp-Hse (lactone). The presence of the
homoserine lactone could be further confirmed. An
aliquot of the HPLC fraction was incubated at
p H = 8 . 5 (30 minutes, room temperature) and
subsequently analyzed as above. As expected a
molecular mass shift of 18 Da was observed due to the
addition of water and opening of the lactone (data not
shown).
To characterize further the nature of the cross
linked peptides, (M + 2H) 2÷ ions (171/z = 767.9 Da,
average mass) were subjected to collision-activated
dissociation (CAD) in the triple quadrupole
instrument (Hunt et al., 1986). The first mass filter,
quadrupole 1, is set to transmit ions within a window
which is centered at m / z = 767.9. The width of the
window is normally chosen to obtain maximum
transmission for the selected ions. In the collision
cell, quadrupole 2, the ions are subjected to low
energy collisions (15 to 40 eV) with argon present
at a pressure of three millitorr. Positively charged
fragments are transmitted to the third mass
filter, quadrupole 3, and are analyzed according
to mass. The spectrum obtained from this analysis
is shown in Figure 9. Shown on top of this Figure are
the predicted average masses for fragment ions of
type b (containing the N terminus) and type y
(containing the C terminus). Fragment ions all contain
the corresponding terminus plus the additional
amino acid residues. The experimental mass
differences between any two fragments that differ
by a single amino acid, NHCH(R)CO, identifies the
corresponding amino acid residue. Ions of type yl
to y9 are indeed seen in the CAD mass spectrum.
Ions of type y9 are at m / z = 1024.1, while ions of
type yl0, with one additional amino acid residue,
are at m / z = 1350.5. The mass difference between
the ions of type y9 and yl0 is incompatible with
the presence of a Lys at this position, however
it is compatible with the presence of a Lys plus
the cross-link. The difference of 326.4 Da results
from Lys (128 Da) plus the cross-link (198.4Da).
The identical mass difference of 326.4 Da between
the corresponding fragment ions of type b confirms this result. Whereas ions of type b2 could be
observed at m / z = 185.2, ions of type b3 were
observed at m/z=511.6. Lys169 is therefore
unambiguously modified through the cross-link.
These results agree with the conclusion that Lys169
forms a cross-link to Asn356 through an amide
linkage.
Phage HK97 Head Assembly
627
A . samples digested with cyanogen bromide
and trypsin
Cross-link--containing
Peptide
|
II
II
Before in vitro cross-linking
II
I]
................ After in vitro cross-linking
II
II
II
tt
It
It
II
II
II
II
II
!
,
-"
-"
i
Difference Map
........
|
A 206 n m
. . . .
' I0
t
i
. . . .
i
. . . .
i
20
. . . .
i
. . . .
i
30
,,
.
.
.
[
40
. . . .
, l , , , . . . .
i
. . . .
i
~la
. . . .
[
. . . .
$0
[.,
.
.
.
i
.
70
, .
i , , , . . . .
I
. . . .
00
i
90
.
l
. . . .
i
. . . .
1051
Time(rain)
B . samples digested with cyanogen bromide
and S. aureus V8 protease
Cross-link--containing
Peptlde
,
Before in vitro cross-linking
.'
II
11.
!
I
"v V
I I
It
I
I
................ After in vitro cross-llnking
Y
'
v
'
V
'~
||
Difference MaD
.
........
||
I|
A 206nm
I I0 ....
i"
•
*>01
V
" ":l
.
.
3. ~o .
.
.
.l
.
.
. 4--"
.~O .
- " i
50r ....
i ....
G01 ....
Time(rain)
Figure 8.
J ....
?01"
i ....
88"! ....
I ....
9 ~@
....
i
. . . I~00
. . .
"
Phage HK97 Head Assembly
628
72.1
185..2
511.6
608.7
737.8
824.9
940.0
1053.2 1154.3 1301.4 1388.5 1534.7
Aln
Leu
Lys-Z
Pro
Glu
Set
Asp
lie
Thr
Phe
Set
Lys
797.9
710,5
595.7
482.6
381.5
234.3
147.2
1436,6 1350.5
1024.1 927.0
I x2.5 I x5
Ix2.Sl x3
b ÷
n
+
Y 11
I
b
PE
t,,
w
O
Z
100
100
60
I
I
t
z
M
I
PESD
\
PES
/
,,
lJ Jli
x30
I
Y7
900
b9
1100
Discussion
assembly
,
Y18
b8 I
/
700
(M+2H)++
Y8
The
I
500
I xl.Sl
F
1,0
/ ys\
300
Ix9
PESDIT
pathway
The experiments described here document three
transitions that take place in the structure of the HK97
head protein as it progresses through the assembly
pathway toward mature heads. These are the
cleavage which removes 102 amino acids from the
amino terminus of the 42 kDa form of the protein to
produce the 31 kDa form, the conformational
transition that results in shell expansion, and the
formation of covalent bonds b e t w e e n subunits in the
cross-linking reaction. We also describe two protein
shells, Prohead I and Prohead II, that differ by the
cleavage of their subunits. By assuming that the two
prohead forms are true intermediates in the assembly
pathwa}~ or close approximations to such intermediates, we are able to arrange the proheads, mature
heads, and the three protein transitions into the pathway shown in Figure 1. The experiment described in
Figure 7 shows that the expansion and cross-linking
reactions are separable and ordered, and we have
added an additional structure, Head I, corresponding
to expanded but not yet cross-linked Prohead II to
accommodate these observations. DNA packaging is
blo /
1300
bll
m/z
Figure 9. Electrospray ionization
collision-activated dissociation mass
spectrum recorded on the (M + 2H) 2.
ions 0n/z =767.9, average mass)
from the peptide Ala167 to Lys178
cross linked to Asn356 to Met357.
Predicted average masses for fragments of types b and y are shown
above and below the structure at the
top of the Figure. Those observed in
the spectrum are underlined. Fragments that result from internal
cleavage at proline are labeled with
the appropriate single-letter codes to
indicate the sequence contained in
those fragments. Z represents the
cross link. The cross-link containing
peptide is the peak identified in
Figure 8A.
shown as initiating just prior to expansion. Although
our data do not address this issue directl~ it is known
for phage ~ (Hohn, 1983) and thought to be true for
other d s D N A phages that DNA packaging triggers
the analogous prohead expansion reaction.
The pathway in Figure 1 is the only simple
arrangement of these elements of the pathway that is
consistent with the data, so we believe the pathway
is an accurate representation of the actual in vivo
pathway if our assumptions are correct that the
prohead intermediates isolated from mutant infections represent true intermediates. These assumptions are not yet tested rigorousl~ However, w e
believe Prohead II is close to and probably identical
to the true intermediate because similar (probably
identical) structures are seen in a wild type infection
and because the Prohead II we have characterized is
capable of expanding to the size of a mature head.
Prohead I is less firmly connected to the in vivo
pathwa34 but the fact that the uncleaved 42 kDa
protein is capable of efficient assembly into a shell
argues that cleavage does not precede shell assembl~
and therefore that an intermediate similar to Prohead
I must exist (as is known to be the case for phage T4,
in which the major head subunit is also cleaved
(Black et al., 1994)). Experiments described in the
Figure 8. Peptide mapping of HK97 Prohead II before and after in vitro expansion and cross-linking--identification and
purification of 2 peptides containing a novel protein cross-link. Samples of Prohead II were expanded and cross-linked
in vitro as described above. Untreated samples and the treated samples were digested with cyanogen bromide followed
by digestion with either trypsin (in A) or Staphylococcus V8 protease (in B) and then separated by reversed phase
chromatography on a Vydac C4 column using Gradient Program 1 (in A) or Gradient Program 2 (in B) as described under
Materials and Methods. In each panel the upper section shows superimposed A ~ traces of cross-linked, or uncross-linked
peptides eluted from the column as noted in the Figure, while the lower section shows the arithmetic difference of the
2 upper traces. In both cases the maps are essentially identical, except for the disappearance of one peak and the appearance
of a new peak. In each case the new peak represents a peptide that contains the novel cross-link. In both cases 2 peptides
should combine into the new peptide, but we were only able to identify one of the donor peptides. The amino acid
compositions and amino acid sequence of each peptide was determined and the cross-link containing peptide in A was
additionally analyzed by mass spectrometry to determine the exact nature of the chemical link (see Figure 9).
Phage HK97 Head Assembly
629
\c_HN /
o// C~H2H
I
CH2
I
CH2
I
CH2
Lysine 169
I
+
NH3
Of
I
CH2 O
H I //
/N--C--C\
CI1_12H
CI-~
CI-h
Ct-~
2
+
+NI-k
NH
C=O
OX-,c~H2
Asparagine 356
Nature and location of the cross-link bond
\,~C--C--N
H
/
.
/N--C--C\
Figure 10. The proposed cross-linking bond. The Figure
shows the overall reaction; intermediate species have not
been identified.
accompanying paper argue that the cleavage is
catalyzed by a phage-coded protease, and we
suggest that the Prohead I we have characterized
differs from the true intermediate in not containing the protease. We have shown elsewhere,
in fact, that ainU4, the mutation that causes
accumulation of Prohead I, maps in the
gene encoding the putative protease (Duda et al.,
1995b).
A~
The data presented above argue strongly that the
head-protein subunits are linked together through
the side-chains of lysine169 and asparagine356,
and that the chemical linkage is an amide bond.
The overall cross-linking reaction, illustrated in
Figure 10, would entail the loss of ammonia. We are
unaware of any examples in the literature of such an
asparagine to lysine cross-link, but there are m a n y
examples of the chemically analogous glutamine to
lysine cross-link: for example, the cross-links in fibrin
and involucrin (Chen & Doolittle, 1971; Simon &
Green, 1988; Folk, 1980). In these latter cases, the
cross-linking reactions are catalyzed by transglutaminase enzymes and therefore differ from the
cross-link under consideration here, which appears
to take place autocatalytically. Although our data do
not speak to the chemical mechanism of cross-link
formation, they do show that cross-linking is
triggered by the conformational change of the
subunits that is manifested as shell expansion. We
propose that it is the conformational change of
expansion that brings the reactive asparagine and
lysine residues into proximity and that, as with an
enzyme catalyzed reaction, positioning the substrates appropriately is an essential component of
this autocatalytic reaction.
How are the cross-links arranged in the capsid?
C~
Figure 11. Schematic representations of cross-linking topologies. The wedge-shaped objects represent head protein
subunits, each of which has its Lys169 and its Asn356 represented by K and N, respectivel)~ and the small shapes
connecting K and N on adjacent subunits represent the covalent cross-link bond discussed in the text. The circled numbers
show the positions of the rotational symmetry axes of the icosahedrally symmetric capsid shell, and the equilateral triangle
outlines one of the 20 faces of the corresponding icosahedron. A, Shows the cross-linking topology based on the 5-fold
and quasi-6-fold symmetries, which we argue is the topology used in the HK97 head. B, Shows an alternative cross-linking
topology based on the 3-fold symmetries of the capsid.'C, The cross-links are arranged as in panel A but about half of
them are missing, as would be the case when the cross-linking reaction in the capsid is about halfway to completion. This
sort of partial cross-linking generates the series of covalent oligomers, from monomer to hexamer, that we believe are
responsible for the six-step ladder of bands produced on an SDS gel by partially cross-linked capsids.
630
Since it appears that all of the subunits of the capsid
participate in the cross-linking reaction and that they
all are linked through the same pair of amino acids,
the distribution of the cross-links in the icosahedral
capsid must follow the same symmetry relationships
as those that describe the positions of the protein
subunits in the capsid. Figure 11 shows the
arrangement of protein subunits on one face of a T = 7
icosadeltahedral capsid (Caspar & Klug, 1962). There
are only three ways of arranging cross-links in such
a structure that allows them all to be geometrically
equivalent (or more accuratel~ quasi-equivalent), and
these are based on the three symmetry elements of an
icosahedron. Figure 11A shows the cross-links
arranged in such a way that they link together the
groups of subunits that surround the 5-fold
symmetry axes of the icosahedron and the related
quasi-6-fold axes. Figure 5C shows how this portion
of such a capsid might look during the course of a
cross-linking reaction, after about half of the possible
bonds had formed. It is apparent from examining this
Figure that the structure is a mixture of different
covalent oligomers of the subunit that would
generate a six step ladder of bands on an SDS gel like
those shown in Figures 5 and 7. Figure 11B shows one
of the other two possible topologies of cross-links, in
which the three subunits surrounding each exact and
quasi-3-fold symmetry axis are linked together. The
end products of this cross-linking scheme are all
trimers, and a capsid partially cross-linked by this
scheme would be expected to generate a three step
ladder on an SDS gel. Not shown is the third possible
bonding scheme, based on the exact and quasi-2-fold
symmetry axes, for which the final products are
dimers joined by two symmetrical bonds.
Based on these considerations, we strongly favor
the cross-linking scheme shown in Figure 11A, in
which each subunit in a five or six subunit grouping
(capsomere) joins its Lys169 to the Asn356 of its
neighbor on one side and its Asn356 to the Lys169 of
its neighbor on the other side. Such a cross-linking
scheme accounts for the.' appearance of the six-step
ladder of bands seen on an SDS gel at times when
cross-linking is incomplete, and it accounts for the
disappearance from the ladder of all but the pentamer
and hexamer bands when cross-linking has gone to
completion.
This scheme, however, does not immediately
account for the fact that in fully cross-linked capsids,
the majority of the protein is in a form that fails to
enter an SDS gel at all. In a mature virion, the amount
of material in the pentamer band corresponds
roughly to the 55 subunits/capsid expected to be in
pentameric capsomeres, but of the 360 subunits/capsid expected in hexameric capsomeres, fewer than
20% are found in the hexamer band, with the
remainder of the mass having moved into the material
that fails to enter the gel (Popa et al., 1991). Thus it
appears that the bulk of the cross-linked hexamers
become further joined to each other to form the very
large material at the top of the SDS gel. It seems
unlikely that the specific Asn356 to Lys169 cross-link
bond that we have identified could directly join two
Phage HK97 Head Assembly
capsomeres together, since it would require certain
subunits to link not to their neighbor but to a subunit
in a different capsomere, presumably with drastically
different geometry than that of the intra-capsomere
cross-link. A more plausible possibility is that there
is a second type of cross-link, involving a different
pair of amino acids, that joins together hexamers
which are themselves internally linked with
geometrically equivalent Asp356 to Lys169 crosslinks. However, experiments such as those shown in
Figure 8 have consistently failed to provide any
evidence for such a second type of cross-link. A third
possibilit34 which we currently favor and will
document elsewhere (R. L. Duda & R. W. Hendrix,
unpublished results) is that the six subunits of a
hexamer are joined together into a topologically
circular covalent structure as shown in Figure 11A,
except that parts of each covalent circle extend out
around the local 2-fold axes and therefore interlink
with the covalent circles of the adjacent capsomeres,
to make a large, multiply catenated structure.
Roles of the protein transitions
A striking feature of all the well characterized
bacteriophage head assembly pathways is that the
protein subunits undergo a series of conformational
and sometimes covalent transitions between their
initial assembly into an icosahedral shell and the
appearance of the mature structure. The data presented here make it clear that the same is true for
HK97 head assembly Various explanations have been
offered for this mode of assembl~ not all of them
mutually exclusive. One proposal is that some steps
in the pathway serve to make the pathway
irreversible. All three of the transitions described
here may serve this function. In the case of the
cleavage, the products of the reaction have a lower
free energy than their precursors and the fragments
that are cleaved off are lost from the structure (J. F.
Conway et al., unpublished results), so this reaction
is not expected to be reversible to any significant
degree. The situation is less clear for the cross-linking
reaction, for which we do not know the details of the
reaction mechanism, but the overall reaction entails
the loss of ammonia, and this should make reversal
of the reaction unlikely The expansion reaction is
probably energetically favorable in the forward
direction, both because it can be triggered to occur
spontaneously by a variety of treatments and by
analogy with the expansion reaction in other dsDNA
phages, where the expansion has been shown by
direct measurement in one case to have a negative
enthalpy (Ross et al., 1985).
In addition to a possible role in making assembly
irreversible, the cleavage may have a role in ensuring
a particular sequence of reactions. Thus although it
is possible to cause Prohead "I to cross-link its
subunits in vitro, this reaction does not go to
completion as it does with Prohead II, and it may be
that in vivo the N-terminal portion of the capsid
protein helps to prevent premature cross-linking. In
any case, it seems likely that one purpose of the
Phage HK97 Head Assembly
cleavage is to remove the N-terminal fragment of the
protein from the capsid structure w h e n it is no longer
needed. Structural studies of HK97 proheads show
that the N-terminal fragment is located on the inside
of the capsid shell, where it might occupy space
needed for DNA, and that it is not retained in the
particle following cleavage (J. F. Conway et al.,
unpublished results).
As we suggest above, one role of the conformational change associated with expansion is likely
to be to trigger the cross-linking reaction. We imagine
it does this by positioning the side-chains of Lys169
and Asn356 next to each other in the appropriate
geometry to form the cross-link bond. More
generall~ the expansion reaction may reposition the
subunits with respect to each other to put them into
conformations or inter-subunit interactions which
could not have formed by direct assembl}~ Examples
of such a situation can be seen in the atomic
resolution structures of several viruses, which show
the polypeptide chains of the subunits to be
intertwined in such a way that it must be concluded
that they rearranged their structures following
their assembly (Liddington et al., 1991; Hogel et al.,
1985; Harrison et al., 1978). Whatever the exact nature
of the HK97 expansion conformational change, it is
also likely that expansion stabilizes the structure.
This has not been tested explicitly for HK97, but is
known to be true for other dsDNA phages (Steven
et al., 1976).
We have no direct evidence concerning the
biological function of the cross-linking. As suggested
above, it may help to make assembly irreversible.
In addition, it seems likely that cross-linking, like
expansion, has a role in stabilizing the capsid
structure. A cross-linking reaction that fuses
several copies of the major head protein of
bacteriophage X to another protein near the
portal vertex has been reported (Hendrix &
Casjens, 1974), but it has not been as fully
characterized as the HK97 case presented here,
nor is its function known. It might be asked, if
HK97 cross-links its head proteins, and particularly
if the cross-linking serves an important biological
function, w h y has similar cross-linking not been
seen in other bacteriophages? In fact, even though
the traditionally studied Escherichia coli phages
do not cross-link their capsid proteins in this wa~
it is now clear that mycobacteriophage L5 cross-links
its head protein subunits in a similar way to HK97
(Hatfull & Sarkis, 1993), and there is indirect
evidence from the SDS/gel patterns of virion
proteins that the same is true for m a n y and probably
most other mycobacteriophages (W. Jacobs, G.
Hat full & R. Hendrix, unpublished results), for
HK97's relative, coliphage HK022, and for occasional
phages of other groups whose gel patterns are
scattered throughout the literature. Thus we
imagine that cross-linking may be just one
mechanism for accomplishing a particular biological
function, possibly stabilization, that has been
accomplished by alternative mechanisms in m a n y
other phages.
631
Materials and Methods
Bacteria and bacteriophage strains
E. coli strains 594 sup ° Smr (Weigle, 1966) and Y-mel mel-1
supF58 (Yanofsky & Ito, 1966) were used as non-suppress-
ing and amber suppressing hosts, respectively Phage
HK97 was isolated from pig dung in Hong Kong by DhiUon
et al. (1980). HK97 amber mutants ainU4, and amC2 were
used for the purification of Prohead I and Prohead If,
respectivel}~ Both of these mutants were isolated from a
spontaneous clear plaque mutant of the wild type phage
and were described previously (Popa et al., 1991). A
Prohead II variant that does not contain the portal protein
was purified from structures assembled in vivo following
the expression of two HK97 head proteins from a plasmid
expression vector in E. coli. The plasmid used for this
purpose, pT7-Hd2.9, is described in the accompanying
paper, and expresses the HK97 gene 4 protein (a 25 kDa
putative protease) and gene 5 protein (the 42 kDa major
head protein). The vector pT7-5 contains a T7 promoter
(see GenBank entry Synpt75a, M21340). The host,
BL21(DE3) (F-ompTr;m;), is described as part of the
phage T7 expression system (Studier et al., 1990) and was
used under the conditions recommended therein.
Enzymes and chemicals
Staphylococcus aureus V8 protease and 1-tosylamido-2phenylethyl chloromethyl ketone (TPCK)-treated trypsin
were purchased from Worthington Diagnostics, Freehold,
NJ. Protease-free DNaseI was purchased from Sigma, St
Louis, MO. Trifluoroacetic acid (HPLC grade in sealed I ml
ampoules), acetonitrile (HPLC/Spectro grade), N-ethylmorpholine (Sequanol grade) were obtained from Pierce,
Rockford, IL. Ammonium bicarbonate buffer was made
from reagent grade ammonium hydroxide and CO2.
Concentrated stock solutions of urea and guanidine HC1
were prepared from high purity reagents and the
concentrations of both were determined from refractive
index measurements. Urea solutions were made fresh and
de-ionized prior to use. All other chemicals were of reagent
grade. Protein concentrations were determined using a
microtiter-plate based assay (Redinbaugh & Turle}~ 1986)
using bicinchoninic acid (BCA) reagents from Pierce.
Media and buffers
Tryptone broth (TB) contains 10 g of Bacto-Tryptone and
5 g of NaC1 per liter, and was often supplemented with
0.4% (w/v) maltose to increase the efficiency of phage
adsorption. Phage were titered on plates containing
Tryptone broth plus 1.0% (w/v) Bacto-Agar in soft agar
overlays containing the same medium plus 0.75% (w/v)
Bacto-Agar. Luria Broth (LB), used for growth of cells for
plasmid expression of Prohead II, contained 10g of
Bacto-Tryptone, 5 g of Bacto-Yeast Extract, and 5 g of NaCI
per liter and was supplemented with ampicillin at
0.05 mg/ml. Buffer Kdil*, used for resuspending and
storing phage stocks, contains 6 mM Tris-HC1 (pH 7.5),
70 mM NaCl, 10 mM putrescineHC1 and 1 mM MgC12.
This same buffer was used for HK97 structure preparations
reported previously (Popa et al., 1991) and for most
experiments reported here, for historical reasons, but
simpler buffers, such as 20 mM Tris-HC1 (pH 7.5) with
40 mM NaC1, were used for more recent experiments. The
phosphate buffer (pH 7) used in Table 2 contained 13.3 g
of Na2HPO4.7H20, 4 g NaC1, and 3 g of KH2PO4 per liter.
632
Preparation of infected cell extracts for purification of
head structures
E. coli strain 594 was grown at 37°C with vigorous
aeration in Tryptone broth supplemented with 0.4%
( w / v ) maltose. For small volumes, 11 of culture was
shaken at 250 r e v s / m i n in one or more 2.8 1 Fernbach
flasks; for larger volumes (20 liters in a 28 1 fermentor,
or 60 liters in a 150 1 fermentor) the culture was aerated
at about 1.51 per minute per 1 of culture in a New
Brunswick industrial fermentor. Cell growth was monitored by measuring light scattering in a spectrophotometer and
when
the cell density reached
3 x l 0 ~ c e l l s / m l , the culture was infected with the
appropriate amber mutant phage at an input ratio of five
phage per cell in 1/20th volume of fresh medium. After
90 minutes the bulk of the culture had usually lysed
(due to the clear plaque mutation carried by each
phage), and the culture medium was clarified to remove
cell debris. CHCI3 was not used to promote cell lysis as
in previous studies (Popa et al., 1991). For large scale
preparations the culture was chilled and cell debris was
removed by continuous flow centrifugation; for smaller
volumes the cultures were chilled on ice and centrifuged at 4°C at 10,000g in a Beckman JA10 rotor for
15 minutes. The clarified supernatants were used for
prohead purification as described below.
Purification of Prohead I from amU4 infections
Prohead I was purified by a combination of PEG
precipitation, differential sedimentation, and velocity
centrifugation in glycerol gradients, as follows. All steps
were carried out on ice or at 4°C, except where noted.
A culture supernatant from amU4-infected cells was
prepared as described above, the volume was measured,
and proheads (plus other large macromolecular structures) were PEG precipitated by adding solid NaC1 to
0.5 M and polyethylene glycol (PEG 8000, as flakes) to
15% ( w / v ) to the suspension while stirring gently on
ice. After incubating the suspension on ice for at least
45 minutes or overnight, the precipitated material was
collected by centrifugation at approx. 10,000g (using a
Beckman JA14 rotor 15 minutes). The supernatant was
discarded and the pellets resuspended gently in cold
~.dil* buffer, but working at room temperature. Material
which did not dissolve was removed by centrifugation
using a Beckman JA20 rotor at 10,000 r e v s / m i n for
15 minutes. The PEG-purified proheads were diluted
at least two to threefold and concentrated by ultracentrifugation in thick walled polycarbonate tubes in a
Beckman Ti45 rotor at 35,000revs/min for 2 hours.
(This step also reduced the amount of PEG and other
low-molecular-mass material present in the sample.)
The supernatant was discarded and the tubes carefully
drained and wiped. The pellets were covered with a
small amount of buffer and allowed to resuspend
overnight in the cold. The following day the pellets were
gently dissolved, then diluted appropriately for the next
step. Following dilution, insoluble material was removed by centrifugation using a Beckman JA20 rotor at
12,000 r e v s / m i n for 20 minutes. Proheads were then
purified by velocity sedimentation in glycerol gradients;
generally one gradient tube was used for each 1 to 2.5 1
of starting culture supernatant. Gradient solutions were
made v / v in ~.dil* buffer and run in the cold. The
gradients were composed of 6 ml 10%, 7 ml 15%, 7 ml
20%, 7 ml 25%, and 7 ml 30% glycerol in clear tubes
appropriate for the SW28 rotor. Samples of 5 ml were
Phage HK97 Head Assembly
layered on top and the samples centrifuged at
27,000revs/min for 2.5 hours. The prohead bands
migrated to near the center of the tubes and were
removed with a syringe and needle from the side.
Pooled gradient peak fractions then were diluted and
concentrated by ultracentrifugation as described above.
The final pellets were usually large and mostly clear
and were left covered with Xdil* buffer overnight, and
then resuspended as above and stored under refrigeration. Preparations of Prohead I gradually dissociated
into capsomeres over time (weeks), under these conditions.
Purification of Prohead II from amC2 infections
Prohead II was purified by a combination of PEG
precipitation, differential sedimentation, and velocity
centrifugation in glycerol gradients, essentially as
described above for Prohead.1. The glycerol gradients often
contained a smaller peak of expanded, cross-linked
Prohead II (empty Head II) as a shoulder above the main
Prohead II peak. This second peak was occasionally also
collected for analysis. Above the prohead peak was a peak
of HK97 tails; all preparations of Prohead II made from
amC2 infections contained some contaminating tails. The
presence of tails was indicated by a characteristic
slow-migrating band in agarose gels, and tails were
observed by electron microscopy Prohead II preparations
slowly accumulated spontaneously expanded and crosslinked particles over a period of months.
To make radioactive Prohead lI, infected cells were
labeled with PSS]methionine as described by Popa et al.
(1991), proheads were purified as above and analyzed on
an SDS/polyacrylamide gel, and the radioactivity in the
head and portal proteins was measured with an AMBIS
Radioanalytic Imager (Scanalytics, Inc., San Diego).
Purification of Prohead II (plasmid) from induced
plasmid containing cells
Host BL21(DE3) containing plasmid pTT-Hd2.9, was
grown in LB to about 4 x 10~cells/ml, induced with
0.4 mM isopropyl-~-D-thiogalactopyranoside (IPTG) for
about 3.5 hours and harvested by centrifugation. Cell
pellets (from 3 1 of culture) were resuspended in 150 ml of
ice cold 50 mM Tris-HC1 (pH 8.5) containing 5 mM EDTA
and slowly stirred during the entire lysis procedure.
Lysozyme (from a fresh stock solution) was added to about
50 ~ g / m l and incubation continued for one hour or longer.
Following lysis, MgSO~ (to 7.5 mM from a 1 M stock) and
DNaseI (to 20 ~ g / m l from a 1 m g / m l stock) were added,
followed by further incubation to reduce the suspension
viscosit~ and cell debris was removed by low speed
centrifugation in a JA14 rotor at 8 5 0 0 r e v s / m i n for
10 minutes. Following cell debris removal, Prohead II
was purified by a combination of PEG precipitation,
differential sedimentation, and velocity centrifugation in
glycerol gradients, essentially as described above for
Proheads I and II, with a few differences. Proheads were
PEG precipitated using 6% instead of 15% PEG 8000, as
used above. Preparations were more highly concentrated
than in the above procedures, so three to four glycerol
gradient tubes were used per liter of starting culture for the
velocity sedimentation step. Preparations of Prohead II
made from plasmid inductions were generally very pure
(they contained no contaminating tails) and in addition
were very stable for long periods of storage; very little
expansion and cross-linking was observed over many
months.
Phage HK97 Head Assembly
Agarose gel analysis of HK97 head-related structures
Native agarose gel electrophoresis provides a convenient
method for analyzing multiple samples for the presence of
large protehl assemblies. The apparatus required is the
same that is used for the electrophoresis of DNA samples;
buffers and running conditions used are nearly identical to
those used for DNA. Samples were electrophoresed in
standard DNA horizontal mini-gel equipment using TAMg
buffer (40 mM Tris base, 20 mM acetic acid (pH 8.1), and
1 mM magnesium sulfate). TAMg is identical to the
standard DNA electrophoresis buffer TAE (Tris-AcetateEDTA), except that the chelating agent EDTA is replaced by
magnesium sulfate. A similar buffer has been used for gel
analysis of ribosomal subunits (Moore & Laughrea, 1977).
Samples were prepared for electrophoresis by mixing nine parts sample with one part of a solution containing
dyes and glycerol (50% (v/v) glycerol, 0.025% (w/v)
bromphenol blue, 0.025% (w/v) xylene cyanol XFF). Staining of protein assemblies electrophoresed in agarose is very
rapid if the gel is fixed and dried before staining. After electrophoresis, a gel was fixed by agitation in gel fixing solvent
(25% (v/v) isopropanol and 10% (v/v) acetic acid) for
20 minutes. The gel was equilibrated with 95% (v/v)
ethanol (three incubations for 15 minutes each) and then
dried using a standard heated vacuum gel drier; the gel was
placed on filter paper, covered with plastic film, dried without heat until it was flattened, and then with heat until dr}~
The thin, dried gel was stained by agitating for 10 minutes in
0.4% (w/v) Coomassie brilliant blue R250 in gel fixing solvent and destained in gel fixing solvent until the background was nearly clear. Non-specific background staining
was reduced by further incubation in 10% acetic acid. Data
was recorded immediately by photography of the wet gel,
or the gel was preserved, either wet, or after drying onto
filter paper in the manner used for polyacrylamide gels.
SDS/polyacrylamide gel analysis
The methods and conditions for SDS/polyacrylamide
gel electrophoresis were modified from Laemmli (1970).
Most gels were prepared with a low-cross-linker stock
formulation containing 33.5% (w/v) acrylamide and 0.3%
(w/v) methylene bis acrylamide (Dreyfuss et al., 1984,
noted as Low-C) instead of the standard (30%:0.8%)
formulation (Laemmli, 1970), noted as Std-C. All samples
were heated in boiling water for 2.5 minutes after mixing
with the appropriate volume of concentrated SDS sample
buffer. The 4 x sample buffer normally used (4 x SB)
contained 0.25 M Tris-HC1 (pH 6.8), containing 8% (w/v)
SDS, 20% mercaptoethanol, 40% glycerol, and approximately 0.025% (w/v) bromphenol blue. A modified
concentrated SDS/gel sample buffer (5 x SB-DTT--containing no glycerol or dye, and with mercaptoethanol
replaced by dithiothreitol), replaced the ~ormal buffer for
some experiments and consisted of 0.3125 M Tris-HC1
(pH 6.8), 10% (w/v) sodium dodecylsulfate, and 0.5 M
dithiothreitol.
In vitro expansion/cross-linking reactions
Most of the expanded and cross-linked proheads
(corresponding to the "Head II" of Figure 1, but without
DNA) were made by treating samples of Prohead II with
chloroform at 37°C. Samples were placed in a 2 ml
stir-bar-equipped Reacti-Vial at 37°C and incubated with
5% (v/v) added chloroform, with continuous stirring at
about 200 revs/min. Incubation was either overnight for
the completely cross-linked material used for peptide
633
mapping, or for various times, as indicated in the Figure
legends. For Figure 3D Prohead II samples were expanded
by treatment with 7 M urea in 50 mM sodium acetate pH 5
for 60 minutes, then diluted 20-fold into 50 mM Tris-HCl
(pH 7.5), and dialyzed to remove the urea (see Table 2,
special note s).
Denaturation of prohead protein for electrophoresis
and peptide analysis
Exposure of HK97 prohead proteins to harsh or
denaturing conditions usually induces transformations that
cause spontaneous, self-catalyzed cross-linking (see the
Results section, Table 2, and Figures 5, 6 and 7). Because of
this instability, routine procedures for preparing denatured
proteins for analysis induce covalent modification of the
sample and could not be used, and special precautions had
to taken to insure that the resulting samples have the same
covalent structure as the starting material. Merely mixing
a prohead sample with SDS/gel sample buffer initiates
spontaneous cross-linking (Figure 5). We found thatvery
rapid mixing into preheated SDS sample buffer was often
adequate to prepare truly representative samples.
Precipitation of samples with 10 to 20 volumes of acetone
or ethanol also induced partial cross-linking (see Figure 5C
and Table 2). Precipitation of prohead samples with 10 to
20% cold trichloroacetic acid (TCA) was later found to be
the ideal procedure for preparing prohead samples for
SDS/polyacrylamide gel electrophoresis (Figure 5C). For
this application, one volume of sample was rapidly mixed
with four volumes of ice cold 10% (w/v) TCA in a 1.5 ml
centrifuge tube, incubated on ice for at least 10 minutes, and
centrifuged for 10 minutes at 14,000 g; the supernatant was
discarded, the pellet washed with 0.5 ml of acetone at
-20°C, re-centrifuged, aspirated to remove the acetone,
dried under vacuum, resuspended in SDS sample buffer
(1 x ) and heated in a boiling water bath for 2.5 minutes.
To prepare bulk denatured samples for peptide analysis
and purification, 3 mg to 5 mg of Prohead II and in vitro
cross-linked Prohead II were denatured by rapid heating to
100°C in an SDS-containing buffer. Each prohead sample,
in approximately 0.5 ml, was rapidly mixed with 1/4
volume of 5 x SB-DTT that was preheated to 100°C in a
magnetic stir bar-equipped 2ml Reacti-Vial (Pierce,
Rockville, IL. to insure fast mixing and heating) and
incubated for 2.5 minutes at 100°C. SDS was removed from
denatured protein samples by methanol-chloroform-water
extraction (Wessel & Fliigge, 1984), carried out in tubes
appropriate for the Sorval HB4 swinging bucket rotor. The
final supernatant was discarded, and the precipitated,
detergent-free protein was dried under vacuum and stored
at -20°C.
Chemical and enzymatic cleavage of denatured
prohead proteins
Detergent-free protein was dissolved in 70% formic acid
at a final concentration of 5 mg/ml and cyanogen bromide
was added to 5 mg/ml. Tryptophan was added to a final
concentration of I mM as a scavenging reagent to protect
tryptophan residues in the sample, and the reaction
mixture was incubated at 22°C in the dark. After 15 hours
the reaction mixture was diluted with 15 volumes of water,
frozen, and dried under vacuum in a SpeedVac (Savant
Instruments, Inc., Farmingdale, NY). The sample was then
resuspended in an additional 15 volumes of water and
re-dried. If necessary, drying was interrupted to re-freeze
each sample so that the sample dried to a powder from the
frozen state, rather than to a film from the liquid state, to
Phage HK97 Head Assembly
634
ensure that it would dissolve in buffer for enzymatic
digestion.
For trypsin digestion cyanogen bromide-digested
prohead peptides were suspended at 1 m g / m l in 0.2 M
ethylmorpholine acetate buffer (pH 8.0) and digested with
10 ~Lg/ml TPCK-treated trypsin at 37°C. After 16 hours, an
additional aliquot of enzyme was added and the incubation
continued for a total of 24 hours. Digestion was terminated
by dilution with water, freezing, and drying as above. For
S. aureus V8 protease digestion, cyanogen bromidedigested peptides were suspended at 1 m g / m l in 0.2 M
ammonium bicarbonate (pH 8.1) and digested with
10 ~tg/ml S. aureus V8 protease at 37°C. After a 15-hour
incubation, an additional aliquot of enzyme was added and
the incubation continued for a total of 19 hours. Digestion
was terminated by dilution with water, freezing, and
drying, as above.
Reversed phase chromatography for peptide
mapping and purification
A Waters (Medford, MA) high performance liquid
chromatography (HPLC) system was used for reversed
phase chromatographic peptide mapping and peptide
purification. The system included two Model 501 pumps,
a Model 580 automated gradient controller, a U6K injector
and a Model 990 photodiode array detector. A 4.6 mm
by 25 cm Vydac 214TP54 analytical C4 column (The
S e p / a / r a / t i o n s Group, Hesperia, CA) was mounted in a
Timberline (Boulder, CO) Model H-500 column heater and
maintained at 34°C for all separations. Solvents were
filtered and degassed under vacuum in a Kontes (Vineland,
NJ) mobile phase filtration-delivery system and equilibrated with helium before and during use. The two
solvents used for chromatography were as follows: solvent
A, 0.06% (v/v) trifluoroacetic acid in water, solvent B,
0.052% (v/v) trifluoroacetic acid in 80% (v/v) acetonitrile.
Two composite gradient programs were used for different
applications. The programs consisted of linear gradient
segments of mixtures of solvents A and B at a flow rate of
0.75 milliliters per minute, as follows: gradient program 1,
starting at 6% B, to 30% B in 64 minutes, to 75% B in
26 minutes, to 98% B in 15 minutes; gradient program 2,
starting at 1% B, to 30% B in 64 minutes, to 75% B in
26 minutes, to 98% B in 15 minutes. Prior to injection the
column was equilibrated at one milliliter per minute for
one hour with the starting solvent. Dry samples containing
30 to 100 ~g (estimated) of peptides were dissolved in 70%
formic acid, diluted to 30% formic acid with the starting
solvent, injected, and run under one of the above programs.
Elution was monitored at 206 nm, and fractions were
collected with the assistance of a Gilson Model 203 fraction
collector (Gilson Medical Electronics, Middleton, WI)
operating in time-delayed peak collection mode with
manual recording of peak locations. Peptides that
contained the putative cross-link were identified by
comparing the absorbance profiles from samples analyzed
before and after in vitro cross-linking (see Figure 8). The
cross-link-containing peptide indicated in Figure 8A was
identified in several sequential runs, the fractions were
pooled, concentrated by evaporation under vacuum, and
re-analyzed, as above, prior to subsequent analysis.
Amino-terminal protein sequencing and amino acid
analyses
Amino acid compositions were determined from
portions of samples hydrolyzed in vacuo at 110°C for
24 hours, using a Beckman Model 6300 Amino Acid
Analyzer (Beckman Instruments, Fullerton, CA). Aminoterminal amino acid sequences of proteins and peptides
were determined using a Porton (Beckman) Model 290E
Sequencing System.
Mass spectrometry
Mass spectra were recorded on a TSQT00 triple
quadrupole instrument (Finnigan-MAT, San Jose, California) equipped for electrospray ionization. Sample aliquots
were injected into a fused silica microcapillary column
with an inside diameter of 75 ~lm and a length of 70 cm. The
last 10 cm of the column was filled with C-18 reversed
phase packing material. Peptides were eluted directly into
the electrospray ionization source with a ten-minute
gradient of 0 to 80% acetic acid (0.5% v/v)/acetonitrile at
a rate of one to two microliters per minute. Experimental
conditions for recording mass spectra have been described
(Hunt et al., 1991).
Electron microscopy
Prohead samples were adsorbed to carbon films and
examined in either a Phillips 300 electron microscope
operating at 60 kV or in a Zeiss EM902 at 80 kV. Two
aqueous negative stains were used, 0.4 to 1% uranyl acetate
(UAc) and 2% potassium phosphotungstic acid pH 7.2
(KPTA). Most samples were prepared by floating a carbon
film (previously evaporated onto freshly cleaved mica)
directly onto the surface of the following: (1) the diluted
liquid sample, (2) water for two minutes, and (3) aqueous
stain for 30 seconds. The carbon was then picked up onto
a copper grid, blotted dr}~ and examined in one of the above
microscopes using a liquid nitrogen-cooled anti-contaminator during operation. Images were recorded on Kodak
4489 film.
Acknowledgements
This work was supported by NIH grants GM47795 and
GM37537 from the U.S. Public Health Service to R.W.H.
and to D.F.H., respectivel}~
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E d i t e d by N. Sternberg
(Received 7 October 1994; accepted 3 January 1995)