doi:10.1006/jmbi.2000.4291 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 304, 941±951
Structure of the FHA1 Domain of Yeast Rad53 and
Identification of Binding Sites for both FHA1 and its
Target Protein Rad9
Hua Liao, Chunhua Yuan, Mei-I Su, Suganya Yongkiettrakul,
Dongyan Qin, Hongyuan Li, In-Ja L. Byeon, Dehua Pei* and
Ming-Daw Tsai*
Departments of Chemistry and
Biochemistry, The Ohio State
Biochemistry Program, and
Campus Chemical Instrument
Center, The Ohio State
University, Columbus
OH 43210, USA
Forkhead-associated (FHA) domains have been shown to recognize both
pThr and pTyr-peptides. The solution structures of the FHA2 domain of
Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide,
have been reported recently. We now report the solution structure of the
other FHA domain of Rad53, FHA1 (residues 14-164), and identi®cation
of binding sites of FHA1 and its target protein Rad9. The FHA1 structure
consists of 11 b-strands, which form two large twisted anti-parallel bsheets folding into a b-sandwich. Three short a-helices were also identi®ed. The b-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there
are signi®cant differences in the loops. Screening of a peptide library
[XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp
at the ‡3 position and a preference for Ala at the ‡2 position. These two
criteria are met by a pThr motif 192TEAD195 in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif,
188
SLEV(pT)EADATFVQ200 from Rad9, binds to FHA1 with a Kd value of
0.36 mM. Other peptides containing pTXXD sequences also bound to
FHA1, but less tightly (Kd ˆ 4-70 mM). These results suggest that Thr192
of Rad9 is the likely phosphorylation site recognized by the FHA1
domain of Rad53. The tight-binding peptide was then used to identify
residues of FHA1 involved in the interaction with the pThr peptide. The
results are compared with the interactions between the FHA2 domain
and a pTyr peptide derived from Rad9 reported previously.
# 2000 Academic Press
*Corresponding authors
Keywords: FHA domain; Rad53; Rad9; phosphopeptide; NMR
Introduction
The forkhead-associated (FHA) domain was ®rst
identi®ed as a 55-75 amino acid module found in
many proteins from yeast to human, including
nuclear protein kinases and transcriptional factors
(Hofmann & Bucher, 1995). The conserved regions
of FHA domains are not continuous, as three highly
This is the third paper in the series about the structure-function relationship of FHA domains. For paper II, see
Wang et al. (2000).
{The ®rst four authors contributed equally to this work.
Abbreviations used: AP, alkaline phosphatase; B, b-alanine; BCIP, 5-bromo-4-chloro-30 -indolylphosphate p-toluidine
salt; FHA, forkhead-associated domain; GST, glutathione S-transferase; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexa¯uorophosphate; HBS, Hepes buffer saline; HBS-EP, Hepes buffer saline with EDTA and
P20; HOBT, 1-hydroxybenzotriazole; HSQC, heteronuclear single-quantum coherence; MALDI-TOF, matrix-assisted
laser desorption ionization-time of ¯ight; NHS, N-hydroxysulfosuccinimide; Nle, norleucine; NOE, nuclear
Overhauser effect; NOESY, nuclear Overhauser enhancement correlated spectroscopy; ppm, parts per million; Rad9p,
phosphorylated Rad9; TOCSY, total correlation spectroscopy; X, 19 amino acids including norleucine, except cystiene
and methionine; Z, norleucine.
E-mail addresses of corresponding authors: Pei.3@osu.edu; Tsai.7@osu.edu
0022-2836/00/050941±11 $35.00/0
# 2000 Academic Press
942
conserved blocks are separated with two segments
with different lengths and sequences. Recently it
has been shown that an additional ¯anking segment at each terminus is needed for the domain to
be functionally active and structurally stable (Li
et al., 1999; Liao et al., 1999; Hammet et al., 2000).
Increasing evidence suggests that this domain participates in signal transduction pathways by promoting protein-protein interactions through
recognition of phosphorylation sites. The degree of
sequence homology is relatively low among different FHA domains (ca 20-30 %).
Yeast Rad53, a checkpoint protein that prevents
cell division when DNA is damaged or incompletely replicated, contains two highly divergent FHA
domains: FHA1 in the N-terminal region and
FHA2 in the C-terminal region. Sun et al. (1998)
reported that the FHA2 domain of Rad53 from Saccharomyces cerevisiae interacts with phosphorylated
Rad9 (abbreviated as Rad9p hereafter) in response
to DNA damage at G2/M cell cycle phase, while
Durocher et al. (1999) showed that both FHA
domains of Rad53 interact directly with Rad9p.
The latter paper also reported that a phosphothreonine peptide APPLSQE(pT)FSDLWKL derived
from tumor-suppressor p53 can compete effectively
for binding between FHA1 and Rad9p, but not
binding between FHA2 and Rad9p. Using alanine
scanning for the residues from 3 to ‡3, they
demonstrated that the residue most critical for recognition is an Asp at ‡3 position. Together, these
observations suggest that both FHA domains of
Rad53 could interact with Rad9p but with distinct
phosphopeptide-binding speci®city.
We recently reported the ®rst structure of an
FHA domain, the FHA2 domain of Rad53 (Liao
et al., 1999). We also showed that a pTyr-containing
peptide derived from Rad9, 826EDI(pY)YLD832,
binds to FHA2 with modest af®nity (Kd ca 100 mM).
The structure of the complex between FHA2 and
this pTyr peptide has been subsequently solved
(Wang et al., 2000). Furthermore, we have screened
a pTyr peptide library, identi®ed consensus
sequences for binding of pTyr peptides to FHA2,
and demonstrated that two speci®c pTyr peptides
bind to FHA2 tightly (Kd ˆ 1-5 mM). Taking the
results from our laboratory and others together, we
proposed that FHA domains could have pThr/Ser
and pTyr dual speci®city, or that different FHA
domains could have different phosphopeptide
speci®city (Liao et al., 1999; Wang et al., 2000).
It is important to note that none of the reports
mentioned above have established the actual sites
of Rad9 that are recognized by FHA1 or FHA2.
The tight-binding pThr peptide identi®ed by
Durocher et al. (1999) for binding to FHA1 was not
related to Rad9. In our studies, FHA2 was only
tested for binding of pTyr peptides. Thus, our
results only suggested that, if the recognition site
involves pTyr, the Tyr829 of Rad9 is the most
likely candidate. As pointed out previously (Wang
et al., 2000), our results could not rule out pThr or
pSer as the natural recognition site of FHA2. Our
NMR Structure and Speci®city of FHA1
preliminary results indicate that the FHA2 domain
does bind to pThr peptides, but the binding af®nity and speci®city are still under investigation.
The present paper focuses on the studies of
structure and speci®city of the interaction between
the FHA1 domain of Rad53 and phosphothreonine
peptides. In what follows, we ®rst report the solution structure of FHA1 (residues 14-164), then
present the use of mass spectrometry to identify
selected pThr peptides from the screening of a
pThr peptide library. On the basis of the consensus
sequences obtained, we identi®ed a pThr peptide
from Rad9 that binds tightly to FHA1. This peptide
represents the most likely site of Rad9 that is
recognized by the FHA1 domain of Rad53. This
pThr peptide was then used to probe the residues
of FHA1 that are involved in binding of the pThr
peptide.
Results and Discussion
Construction and characterization of the FHA1
domain of Rad53
We started with the expression and puri®cation
of fragment 2-175. The domain was expressed as a
GST-fusion protein and then digested with thrombin. However, the product contained a mixture of
two fragments of slightly different size due to a
minor thrombin-cleavage site near residue 165.
Subsequently, the fragment 2-164 was constructed.
However, the ®rst 15 residues were shown to exist
in random coil conformations by NMR. These residues were best deleted in order to avoid interfering
with assignments. The fragment of residues 14-164
was ®nally constructed, which has been used for
structural determination in this work. This version
displayed an almost identical HSQC spectrum to
those of longer versions, indicating retention of the
overall structure. Figure 1 shows the amino acid
sequences of FHA1 and FHA2 domains used in
our structural studies. The sequence alignment
shown in Figure 1 was assisted by structural alignment as described below.
Determination of the structure of free FHA1
by NMR
The chemical shift assignments were based on
multidimensional NMR experiments such as 3D
HNCACB, CBCA(CO)NH, HNCA, HCCH-TOCSY,
and 15N-edited TOCSY and NOESY. All but six
(K63, G79, N86, G94, D96, and G97) backbone
amide groups have been assigned, and the chemical shift assignments of side-chains were more
than 92 % complete. It is interesting to note that
more than 38 % of Ha protons experience a
>0.2 ppm down®eld shift from the respective random coil values, indicating rich b-strands in this
protein. NOE assignments were performed on 3D
15
N-edited NOESY recorded in H2O, 3D 13C-edited
NOESY recorded in 2H2O, and 2D homonuclear
NOESY experiments recorded in H2O and 2H2O.
943
NMR Structure and Speci®city of FHA1
Figure 1. Sequences of the FHA1 domain used in this work and the FHA2 domain used in previous studies (Liao
et al., 1999; Wang et al., 2000). The alignment is based on sequence homology as well as structural homology as
explained in the text. Secondary structural elements are shown in blue for b-strands and pink for a-helices. Identical
residues are indicated by stars.
Hydrogen-deuterium exchange at 293 K reveals a
set of slow exchanging amides, which together
with NOE assignments helped to identify the secondary structure. An ensemble of 50 structures
was generated with X-PLOR from 1962 distance
constraints including 78 hydrogen bond restraints.
A total of 192 backbone torsional angles f and were also included in the structural calculation,
which were predicted from chemical shifts of 15N,
1 a 13 a 13 b
H , C , C , and 13C0 with the program TALOS
(Cornilescu et al., 1999). Some 20 resulting structures possessing low X-PLOR energy, as well as
satisfying the experimental restraints (e.g. no NOE
violations over 0.5 angstrom), were selected to represent the structure of FHA1 as shown in
Figure 2(a). The average RMSD for this ensemble is
Ê for backbone heavy atoms (N, Ca,
0.51(0.05) A
Ê for all heavy atoms of
and C) and 0.96(0.05) A
residues 28-157 excluding the loop 52-62. The conformations of the N-terminal (residues 14-27) and
the C-terminal tails (158-164) are not de®ned as
these regions do not show long-range NOEs. The
loop 52-62 is also not well de®ned compared with
other regions, as a result of lacking long-range
NOE. Overall, the structures display good stereochemical quality as evaluated by PROCHECK
(Laskowski et al., 1993). The structural statistics are
summarized in Table 1.
Structural description of FHA1
As shown in Figure 2(b), the FHA1 structure
consists of mainly anti-parallel b-strands, numbered from b1-b10 sequentially and b30 for the
short strand that follows and parallels with b3.
They form two large twisted anti-parallel b-sheets,
which further fold into a b-sandwich. Three short
a-helices were also identi®ed based on NOE
assignments, one on each terminus (residues 16-24
and residues 149-157), and the third one in the
loop that links b2 and b3 (residues 52-57). The
b-strands are linked by several irregular loops and
turns. The ®ve absolutely conserved residues in
FHA domains are located in these loops and turns:
G69 and R70 follows b3 immediately, S85 and H88
reside in the loop linking b30 and b4, and N107 is
positioned in a turn immediately following b5.
Similar to the case of FHA2 (Liao et al., 1999), the
``FHA1 domain'' de®ned by sequence analysis
(residues 66-116) (Hofmann & Bucher, 1995) does
not constitute a minimal structural domain. The
¯anking sequences are required to form a stable bsandwich structure. With the exception of several
residues at N and C-termini, the current version of
residues 14-164 appears to be suf®cient to maintain
the structural integrity.
Structural comparison between FHA1 and FHA2
Three structures in the Protein Data Bank were
found to possess similar modular structure
Table 1. Structural statistics of 20 FHA1 structures
Restraint
Long range NOE (ji jj 5 5)
Medium range NOE (1 < ji jj < 5)
Sequential NOE ((ji jj ˆ 1)
Intraresidue NOE
Hydrogen bond constraints
Dihedral angles fa
Dihedral angles a
Ê)
RMSD for distance restraints (A
RMSD from idealized covalent geometry
Ê)
Bonds (A
Angles (deg.)
Impropers (deg.)
PROCHECK (Ramachandran plot)
Most favored regions (%)
Additionally allowed region (%)
Generously allowed region (%)
Disallowed region (%)
RMSD with respect to mean structure
Backbone (residues 27-51, 63-157)
Heavy atom (residues 27-51, 63-157)
Backbone (residues 27-157)
Heavy atom (residues 27-157)
Number of
Restraints
671
193
490
532
78
96
96
0.045 0.001
0.0035 0.0000
0.51 0.01
0.50 0.01
77.3 1.7
16.5 2.0
4.7 1.0
1.5 0.6
0.51 0.05
0.96 0.05
0.74 0.16
1.26 0.15
None of the 20 structures exhibited distance violations
Ê.
greater than 0.5 A
a
The torsion angle restraints were derived by using TALOS
(Cornilescu et al., 1999).
944
NMR Structure and Speci®city of FHA1
Figure 2. (a) Stereoview showing superposition of peptide backbone traces (N, Ca C0 ) of 20 FHA1 structures (residues 27-158). Five residues are numbered for convenience of tracing the backbone. The N-terminal (14-26) and Cterminal (159-164) segments are disordered and thus not displayed. (b) Schematic ribbon diagram of a representative
FHA1 structure. The 11 b-strands are numbered from 1 to 10 for the well de®ned anti-parallel strands and 30 for the
marginally de®ned parallel b-strand (residues 76-77). This numbering system corresponds to the b-strands in FHA2
(Wang et al., 2000). The Figure was created with MOLMOL (Koradi et al., 1996).
(b-sandwich) to FHA1 protein (Z-scores 57.2 for
1QU5, 1DEV, and 1YGS, whereas Z-score 43.0 for
the rest) using the program DALI (Holm &
Sanders, 1993), among which FHA2 (accession
code 1QU5) has the closest fold (Z-score ˆ 8.5).
Figure 3 shows the comparison of the ribbon diagrams between FHA1 and FHA2. Both domains
consist of mainly b-strands (11 for FHA1 and 12
for FHA2). The 11 b-strands counted from the N
terminus are structurally equivalent. The best
superposition was achieved in the following segments: (1) residues 29-39 of FHA1 and residues
573-583 of FHA2, which include the b1 strand; (2)
residues 40-50 of FHA1 and residues 586-596 of
FHA2, which include the b2 strand; (3) residues
64-77 of FHA1 and residues 599-612 of FHA2,
which include b3 and b30 strands; (4) residues 8296 of FHA1 and residues 616-630 of FHA2, which
Figure 3. Stereoview of the superposition of the ribbon diagram structures of FHA1 (green, residues 26-155) and
FHA2 (red, residues 570-702). The structures are superimposed according to the feedback from DALI (Holm & Sander, 1993). The segments 29-39, 40-50, 64-77, 82-96, 97-131, and 132-148 of FHA1 superpose well with the segments
573-583, 586-596, 599-612, 616-630, 645-679, 681-697, respectively, of FHA2. The residues that have large chemical
shift perturbations upon peptide #2 binding (see Figure 7) are highlighted with green balls. The side-chains of R605,
R617, and R620 in FHA2, which have been shown to interact directly with the phosphate group of a pTyr peptide,
are shown in pink. The side-chains of the corresponding residues in FHA1, R70, R83, and K87, are shown in blue.
The Figure was generated using MOLMOL (Koradi et al., 1996).
945
NMR Structure and Speci®city of FHA1
include the b4 strand; (5) residues 97-131 of FHA1
and residues 645-679 of FHA2, which include b5,
b6, b7, b8, and b9 strands; (6) residues 132-148 of
FHA1 and residues 681-697 of FHA2, which
include the b10 strand. The RMSD of backbone
atoms (N, Ca, C0 ) for these superimposed 103 resiÊ . Such structural alignments were
dues is 2.45 A
used along with sequence homology to achieve the
sequence alignment between FHA1 and FHA2, as
shown in Figure 1.
As also shown in Figures 1 and 3, there are
several noticeable differences in non-conserved
regions. First, the lengths of the loops are different;
the loop between b2 and b3 is short in FHA2 but
longer in FHA1 (forming an a-helix), while the
opposite is true for that between b4 and b5.
Second, b11 and a long loop following b10 in
FHA2 do not exist in FHA1. Consequently, the
packing of the C-terminal helix against the top
b-sheet does not occur in FHA1. In FHA1, a helix
immediately follows b10.
Comparison between the structures of FHA1
and FHA2 also allows us to de®ne further the
``minimal structural unit'' of an FHA domain. By
eliminating the N-terminal and C-terminal segments that have random conformations or assume
different secondary structures, it is possible that
the minimal structural units for FHA1 and FHA2
domains are residues 29-148 and residues 573-697,
respectively. However, whether these minimal
units will be able to form a stable structure with
full binding capability remains to be tested.
Ligand specificity of FHA1
As the ®rst step toward identifying the natural
ligand of FHA1, we performed systematic
speci®city analyses for the FHA1 domain using a
combinatorial pThr peptide library constructed on
TentaGel S resin. The overall approach is similar to
the screening of a pTyr peptide library for binding
to FHA2 reported previously (Wang et al., 2000).
The pThr library with random amino acid residues
at ‡1 to ‡3 and 1 to 3 positions was designed:
acetyl-AXXX(pT)XXXABBRM-resin. A peptide
linker, ABBRM (B ˆ b-alanine), was synthesized to
the C terminus of the random region (Yu & Chu,
1997). Each of the random positions has an equal
representation by 18 natural amino acid residues
(except for Cys and Met) plus norleucine (Nle),
which is used as a substitute for Met. Therefore,
the theoretical diversity of the library is 196 or
4.7 107. Each bead carries 100 pmol of a unique
peptide sequence. The biotinylated FHA1 was
used for pThr-library screening. The association of
biotinylated FHA1 to resin-bound pThr peptide
was detected by observing the blue color formation
on the resin bead as a result of recruitment of
SA-AP and subsequent enzymatic cleavage of
BCIP. A negative control in which the reaction was
performed without the biotinylated FHA1 yielded
no color.
Similar experiments were also performed for
pSer and pTyr libraries (unpublished results). All
three types of libraries gave blue beads, but the
pThr library gave signi®cantly more beads with
more intense color. Hence, in this work we focused
on the identi®cation of consensus sequences for
pThr peptides.
Screening of 100 mg of resin (approximately
300,000 beads) resulted in 383 colored beads. Complete sequences were obtained for 117 beads by
MALDI-TOF mass spectrometry. Figure 4 summarizes the frequencies of appearance for each residue
at each of the randomized positions. The most
striking feature of the results is that every selected
sequence contains an Asp at the ‡3 position.
Although Durocher et al. (1999) reported that
replacement of Asp by Ala in the peptide
APPLSQE(pT)FSDLWKL resulted in markedly
decreased binding af®nity to FHA1, our results
unequivocally show that the (pT)XXD is an absolute consensus sequence for high-af®nity binding to
FHA1. Furthermore, our results further show that
the ‡2 position prefers Ala (the highest abundance) and Ile (the second). It is important, however, to note that the (pT)XXD is not a universal
recognition sequence for all FHA domains. Our
unpublished results showed that the FHA2 domain
of Rad53 also binds pT peptides, but with different
consensus sequences (D is not preferred at the
‡3 position).
Although the number of beads screened
(3 105) represents only 0.6 % of the theoretical
diversity of the library (4.7 107), we believe the
results shown in Figure 4 are suf®cient to represent
the ligand speci®city of FHA1 because the FHA
domain-pThr peptide interaction clearly does not
involve all six positions. The number of beads
screened is suf®cient to cover any random tetrapeptide systems (194 ˆ 1.3 105). This argument is
supported by the results shown in Figure 4: some
positions show only weak preferences, and
position 3 shows little or no preference. For the
positions that show strong preferences (positions
‡3 and ‡2), the results are very clear. Furthermore, our goal is not to identify a peptide that
binds FHA1 most tightly in an absolute sense;
instead, we intend to use the information to help
us identify the site of Rad9 that is recognized by
FHA1, described below.
Identification of possible site(s) of Rad9
involved in binding with FHA1
Since the two most important features in the
results of library screening is that the ‡3 position
has an absolute requirement for Asp and the ‡2
position has a strong preference toward Ala, we
searched the sequence of Rad9 for these features
and found a match for residues 192TEAD195.
We therefore obtained a pThr peptide containing
this site, 188SLEV(pT)EADATFVQ200 (designated as
peptide #2) for analyses of its binding to FHA1. In
addition to the best match for positions ‡3 and
946
NMR Structure and Speci®city of FHA1
Figure 4. Results of pThr peptide sequence speci®city for binding to the FHA1 domain. Displayed are the amino
acid residues selected at 3 to ‡3 positions relative to the pThr residue. The x-axis indicates the identity of the 19
amino acid residues in single-letter codes, whereas abundance on the y-axis represents the number of occurrence of
an amino acid residue at a certain position. M, norleucine.
‡2, this peptide also has a reasonably good match
at other positions: position 2 (E) is also the best
®t, while position 1 (V) and position ‡1 (E) are
among the most favorable residues. Thus, on the
basis of sequence analyses, we predicted that this
peptide of Rad9 is likely to contain the main residues recognized by FHA1 and thus binds tightly
to FHA1.
For the purpose of comparison, the binding assays also included the four other
peptides
containing
TXXD
sites
from
Rad9: 148KKMTFQ(pT)PTDPLE160 (peptide #1),
537
SNAA(pT)RDDIIIAG549 (peptide #3), and
803
LEVD(pT)NQDGCWIY815 (peptide #4), and
1293
HDDI(pT)DNDI1301 (peptide #5). In addition,
peptide #6, SGSYSQE(pT)FSDLG, was designed on
the basis of the results by Durocher et al. (1999).
The residues from 3 to ‡4 in this peptide are
identical to a peptide reported by Durocher et al.,
APPLSQE(pT)FSDLWKL, which was shown to
bind tightly to GST-FHA1 with Kd ˆ 1.68 mM as
determined by isothermal titration calorimetry.
The dissociation constants of the FHA1-pThr
peptide complexes were determined by surface
plasmon resonance (SPR), which is a very useful
technique in characterizing biomolecular interactions (Karlsson & Stahlberg, 1995; Lookene et al.,
2000; Schuck, 1997). Figure 5(a) shows the sensorgrams of FHA1 binding to peptide #2. The Kd
value (0.36 ( 0.03) mM) was obtained by ®tting
the maximal response at each FHA1 concentration
to a hyperbola curve (Figure 5(b)). The Kd values
of all pTXXD peptides are summarized in Table 2,
which indicate that all six peptides bind to FHA1
with reasonably good af®nity, since they all satisfy
the absolute requirement of pTXXD. However, the
binding af®nity of peptide #2 is substantially higher than that of the rest of the peptides. These
results support that the most likely residues of
Rad9 involved in the binding of FHA1 are Thr192
(in the phosphorylated state) and the surrounding
residues. However, it cannot be ruled out that
FHA1 binds to one or more of the other pTXXD
sites of Rad9. We further hypothesize that, while
the absolutely required Asp residue at ‡3 probably
contributes signi®cantly to the binding energy, the
residues at other positions could contribute to the
speci®city of binding between different FHA
domains and their speci®c targets. This hypothesis
will be tested by further structural and functional
analyses.
Although the Kd value of peptide #6 (13.6 mM) is
higher than the value of 1.68 mM (measured by
isothermal titration calorimetry) reported by
Table 2. Dissociation constants of FHA1-pThr peptide
complexes
Peptide #
#1
#2
#3
#4
#5
#6
Sequence
148
Kd (mM)
160
KKMTFQ(pT)PTDPLE
SLEV(pT)EADATFVQ200
537
SNAA(pT)RDDIIIAG549
803
LEVD(pT)NQDGCWIY815
1293
HDDI(pT)DNDI1301
SGSYSQE(pT)FSDLG
188
10.2 0.3
0.36 0.03
71.1 2.2
3.5 0.3
5.03 0.19
13.6 0.8
947
NMR Structure and Speci®city of FHA1
Identification of possible residues of FHA1
involved in binding with Rad9
Figure 5. (a) Surface plasmon resonance sensorgrams
for the binding of FHA1 protein to immobilized pThr
peptide #2, 148KKMTFQ(pT)PTDPLE160, derived from
Rad9. The control signals, which were generated by
¯owing FHA1 protein through a blank channel without
peptide bound on the surface, have already been subtracted. (b) Plots of the maximal responses in the sensorgrams versus the concentration of FHA1. The Kd value
was extracted by ®tting the maximal response at each
FHA1 concentration to a hyperbola curve y ˆ a*x/
(Kd ‡ x), where y is the response in RU (resonance unit,
in arbitrary value), x is the protein concentration, and a
is the maximum response.
Durocher et al. (1999), these two peptides are not
identical, nor are the FHA1 domains used in our
study (ours is shorter and theirs is longer and with
a GST tag). Another point which should be noted
in comparing our results with their results is that
they have obtained Kd values in the order of 50100 nM using BIAcore, but as explained in their
paper, these lower Kd values were caused by GSTmediated protein dimerization effects in the SPR
analysis. Since our FHA1 domain was not fused to
GST, such a problem did not exist in our analysis.
In any case, the Kd values obtained from SPR analyses are best used in comparing relative binding
af®nities between different ligands, which is the
main purpose of our studies.
Above we identi®ed possible residues of Rad9
involved in binding with FHA1. The goal of this
section is to obtain complementary information:
possible residues of FHA1 involved in binding
with Rad9. Our approach is to use 15N-HSQC
experiments to identify the residues of FHA1 that
are perturbed upon binding of peptide #2. For the
purpose of comparison, pThr peptide #1 was also
included in such studies. The results of titration
experiments, as shown in Figure 6, indicate that
FHA1-peptide #1 are in ``fast exchange'' on the
NMR time scale, whereas FHA1-peptide #2 are in
``slow exchange'', which is clear evidence that peptide #2 binds to FHA1 more tightly than peptide
#1. NMR is not a sensitive method for quantitative
Kd measurements. However, NMR results support
the results of binding analyses by SPR.
The following FHA1 residues are perturbed by
>0.05 ppm for protons or >0.4 ppm for nitrogen
atoms upon binding peptide #2: T67, F68, G69,
R70, N71, I81, S82, S85, K87, T106, N107, K118,
G133, V134, G135, V136, and E137. A subset of
these residues (those with >0.1 ppm shifts in proton or >0.6 ppm shifts in 15N) are shown in
Figure 7 and highlighted in Figure 3. The ®rst
three residues are located in the b3-strand. The rest
are in the loops or turns: R70 and N71 in the b3-b30
loop, I81, S82, S85, and K94 in the b30 -b4 loop,
T106 and N107 in the b5-b6 loop, K118 in the b7b8 loop, and G133-E137 in the b9-b10 loop. These
residues are the likely (but not the only) candidates
for interacting with the pThr peptide. As shown in
Figure 3, these residues reside in the front-bottom
pocket formed by the loops. It is worthwhile to
point out that the ¯anking sequences could also
contribute to binding of the phosphopeptide, in
addition to their contribution to the structural
integrity.
Similar results were obtained for the pThr peptide #1 (Figure 7) and peptide #5 and #6 (not
shown), though the binding af®nities and overall
chemical shift perturbations are generally smaller
relative to those of peptide #2. Detailed structural
analyses of these complexes are in progress, which
is required to understand fully the structural basis
of ligand speci®city.
Comparison between FHA1-pThr peptide and
FHA2-pTyr peptide
Here we compare the result of FHA1-pThr peptide complex presented in this paper with that of
FHA2-pTyr peptide complex reported previously
(Liao et al., 1999; Wang et al., 2000). Our results
suggest that the same set of structurally equivalent
residues are involved in the phosphopeptide binding in both complexes. For example, N71, S85 and
G133 in FHA1 are aligned structurally with S606,
S619 and W682, respectively, of FHA2 (Figure 1),
and all of these residues display substantial chemi-
948
NMR Structure and Speci®city of FHA1
Figure 6. Partial 2D 1H-15N HSQC spectra showing fast exchange and slow exchange when FHA1 is titrated with
peptide #1 and #2, respectively. (a) Overlay of partial HSQC spectra of free FHA1 (black) and FHA1 in the presence
of 0.5, 1.0, 2.0 equivalents (red, green, and blue, respectively) of peptide #1, with arrows showing the direction of
shifts. (b) Single HSQC spectrum of FHA1 in the presence of ca 0.3 equivalents of peptide #2, which reveals two sets
of FHA1 amide peaks in slow exchange. Binding experiments were performed by adding aliquots of the peptide to a
sample of uniformly 15N-labeled FHA1 in 10 mM phosphate buffer, pH 6.5 at 293 K. Experiments were performed on
a Bruker DRX-800 NMR spectrometer at 293 K.
cal shift perturbations in both complexes. However, the relative magnitudes of perturbations differ, suggesting differences in detailed interactions.
In the FHA2-pTyr peptide complex, Arg605,
Arg617, and Arg620 are the three key residues
interacting with the phosphate group. These three
residues correspond to Arg70, Arg83, and Asn86
of FHA1 in the sequence alignment shown in
Figure 1. The two arginine residues have been perturbed seriously in side-chain resonances, although
side-chain assignments are tentative at this stage.
Asn 86, or its neighboring residue Lys87, could be
the third residue involved in phosphate binding.
As shown in Figure 7, Lys87 is one of the residues
with largest chemical shift perturbations. The sidechains of Arg70, Arg83, and Lys87 of FHA1 are
highlighted in Figure 3. Although they do not
superpose well with the side-chains of Arg605,
Arg617, and Arg620 of FHA2, they are well positioned to bind the phosphate group. This comparison suggests that the pThr-peptide binding site of
FHA1 is generally similar to the pTyr-peptide
binding site of FHA2, but there are clear differences in detailed interactions that can only be elucidated when high-resolution structures of FHA1pThr peptide complexes become available. However, the results and discussions presented in this
paper, along with our unpublished ®nding that the
FHA2 domain of Rad53 also binds some pThr peptides tightly (but with different consensus
sequences), lends further support to our previous
proposal that FHA domains can bind to both pThr
and pTyr peptides chemically (Liao et al., 1999;
Wang et al., 2000). Biologically, whether the FHA2
domain of Rad53 binds to a pThr site or a pTyr
site of Rad9 remains to be established, but the
results presented here are suf®cient to suggest that
the FHA1 domain of Rad53 very probably binds to
Thr192 and surrounding residues of Rad9.
Materials and Methods
Materials
Figure 7. Summary of FHA1 residues whose 15N or
H chemical shifts are perturbed by binding of peptide
#1 (two equivalents) and peptide #2. The y-axis indicates
the magnitudes of shifts. Only the residues perturbed by
>0.10 ppm for 1H chemical shifts or >0.6 ppm for 15N
chemical shifts upon binding of peptide #2 are shown.
Three other residues (R70, T106 and E137), for which
only lower limit can be estimated due to incomplete
assignments, could also belong to this category.
1
TentaGel S NH2 resin, N-9-¯uorenylmethoxycarbonyl
(Fmoc)-amino acids, 1-hydroxybenzotriazole (HOBt)
and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexa¯uorophosphate (HBTU) were purchased from
Advanced ChemTech (Louisville, KY). N-Hydroxysuccinimidobiotin, acetylglycine, N-acetyl-D,L-alanine, 5bromo-4-chloro-3-indolyl phosphate, and streptavidinalkaline phosphatase were obtained from Sigma Chemical Co. All other chemicals were purchased from Aldrich
Chemical unless stated otherwise.
Protein expression and purification
Three versions of FHA1 domain were constructed as
fragments 2-175, 2-164, and 14-164 of Rad53. They were
cloned into a pGEX-4T vector (Pharmacia Biotech) for
expression and puri®cation as GST fusion proteins.
949
NMR Structure and Speci®city of FHA1
Escherichia coli BL21(DE3) cells transformed with the
plasmids were grown to mid-log phase in rich media.
Expression was induced at 30 C by adding 1.0 mM
IPTG. Cells were harvested eight hours later and lysed
using lysozyme and sonication. GST fusion proteins
were puri®ed using glutathione agarose (Sigma). The
GST tag was removed by thrombin (Sigma) digestion
and gel ®ltration chromatography. The 15N and 15N/13C
isotopically labeled proteins were expressed in M9 minimal media with 15NH4Cl as the only nitrogen source and
13
D-glucose- C6 as the only carbon source. Protein puri®cation was checked by SDS-PAGE, and protein concentrations were determined spectrophotometrically at
280 nm using extinction coef®cient of 12900 M 1 cm 1.
NMR spectroscopy and assignments
NMR samples typically contained 0.5 mM protein,
10 mM sodium phosphate buffer, 1 mM DTT, and 1 mM
EDTA in 90 % H2O/10 % 2H2O or 100 % 2H2O at pH 6.5.
Data were acquired at 293 K on Bruker DRX-800 and
DMX-600 spectrometers, both equipped with tripleresonance probes and three-axis gradients. The triple
resonance experiments HNCACB (Muhandiram & Kay,
1994), CBCA(CO)NH (Grzesiek & Bax, 1992b), HNCA
(Grzesiek & Bax, 1992a; Ikura et al., 1990), and HNCO
(Grzesiek & Bax, 1992a; Ikura et al., 1990) as well as
HCCH-TOCSY were performed on the DMX-600, while
the NOESY-type experiments (e.g. 13C-edited NOESY
and 15N-edited NOESY) and 3D 15N-edited TOCSYHSQC were carried out on the DRX-800. Mixing times
were 100 ms and 30-40 ms for NOESY and TOCSY
experiments. Data were processed using XWINNMR
(Bruker) or Felix (Molecular Simulations Inc.) installed
on Silicon Graphics workstations. Generally, one time
zero-®lling was employed in all indirectly detected
dimensions. Appropriate window function was applied
on each dimension followed by Fourier transformation
and baseline correction. Spectra were referenced
indirectly to TSP at 0.00 ppm.
Structure calculation
The interproton NOE cross-peaks of 2D and 3D
NOESY spectra were classi®ed as 1.8-2.8, 1.8-3.5, 1.8-5.0,
Ê corresponding to strong, medium, weak,
and 1.8-6.0 A
and very weak NOEs. Upper limits for distances involving methyl protons and non-stereospeci®cally assigned
protons were corrected appropriately for center averaging (Wuthrich et al., 1983). Hydrogen bond restraints
Ê and 1.5-2.8 A
Ê for N-O and H-O
were added as 2.4-3.5 A
internuclear distances, respectively, in regions where
regular secondary structure elements were identi®ed by
NOE as well as hydrogen-deuterium exchange results.
The structures were calculated by using simulated
annealing protocol implemented in the X-PLOR program, version 3.843 (Brunger, 1993; Nilges et al., 1988),
installed on a Silicon Graphics computer. The quality of
the NMR structure was externally viewed by MOLMOL
(Koradi et al., 1996) and Insight II (Molecular Simulations
Inc.) and evaluated by the PROCHECK program
(Laskowski et al., 1993). The result was gradually
improved by using an iterative strategy of structurebased assignment. The ®nal 20 structures with lowest
energy among the converged structures were selected.
Synthesis of peptide libraries and peptides
The pTyr library used was the same as that described
(Wang et al., 2000). The library of acetyl-AXXX(pT)XXXABBRM-resin and the corresponding pS library were
constructed on an in-house built peptide synthesis
apparatus. The synthesis was performed at room temperature on a 3.0 g scale of TentaGel S-NH2 resin
(0.3 mmol/g loading, 2.86 106 beads/g, 80-100 mm) by
using Fmoc/HBTu/HOBt chemistry (Bodanszky, 1993).
The amino acid linker, ABBRM, was ®rst synthesized on
the resin, followed by acetyl-AXXX(pT)XXX in which a
split synthesis approach was used for randomization
(Lam et al., 1991; Yu & Chu, 1997), resulting in a onebead, one-peptide library. Amino acid residues with the
same molecular mass were differentiated by capping
reagents: 5 % acetylalanine was added to the coupling
reaction of Leu, while 5 % acetylglycine was added in
the coupling reactions of Lys and norleucine. The complete coupling reaction of each round was monitored by
ninhydrin test. Deprotection was performed in the ®nal
stage by reacting for two hours with large excess of the
solution made up of crystalline phenol, tri¯uoroacetic
acid (TFA), anisole, thioanisole, and 1,2-ethanedithiol.
The beads were then thoroughly washed with alternating methanol and dichloromethane, dried under vacuum, and kept at 20 C.
Screening of resin-bound peptide libraries
FHA1 was ®rst treated with sulfo-NHS-biotin (Pierce)
to give biotinylated FHA1. Sulfo-NHS-biotin (in 1.2-fold
excess) was mixed with the puri®ed FHA1 in 10 mM
phosphate buffer, pH 7.5 for four hours at room temperature. The unbound biotin was removed from the
reaction by dialysis. The biotinylated FHA1 was used for
screening peptide libraries. TentaGel beads (100 mg) containing peptide libraries (approximately 300,000 beads)
transferred to a polypropylene column connected to vacuum manifold (Bio-Rad) were sequentially washed with
100 ml HBS buffer (25 mM Hepes and 150 mM NaCl,
pH 7.5) and 100 mL HBST buffer (HBS containing 0.1 %
(v/v) Tween-20, pH 7.5). Subsequently, the beads were
incubated with 20 ml HBSTB solution (HBST containing
1 mg/ml bovine serum albumin, pH 7.5) for one hour
with mixing. After the incubation, the beads were
washed twice with 50 ml HBSTB solution. The beads
were then incubated with 0.1 mM biotinylated FHA1 in
20 ml HBSTB at room temperature for six to eight hours
with mixing. The unbound proteins were then removed
by washing with 100 ml HBSTB solution. The binding of
biotinylated FHA1 to a peptide was observed by a staining procedure in which 0.5 mg/ml streptavidin-AP and
20 mM phosphate in 20 mL HBSTB were subsequently
incubated with the beads for 15 minutes at room temperature. After incubation, the unbound streptavidin-AP
was removed and the beads were washed with 100 ml
HBST buffer, 150 ml HBS buffer and 100 ml TBS buffer
(25 mM Tris-HCl and 150 mM NaCl (pH 8.0)). 0.5 mg/
ml BCIP in 20 ml TBS buffer (pH 8.0) was added into
the reaction. The blue color typically developed on positive beads within ten minutes to one hour of incubation.
The color intensity depends on the binding af®nity, with
more intense color developed on beads that carry tighter
binding sequences. The positive beads were selected
from the library and subjected to peptide ladder sequencing as described (Hu et al., 1999; Wang et al., 2000;
Youngquist et al., 1995), except that the peptide ladder
was generated after screening using a novel partial
950
Edman degradation strategy (P. Wang and D. Pei, in
preparation). The mass spectra were acquired in positive
ion mode by MALDI-TOF mass spectrometer (Re¯ex III,
Bruker Daltonics).
Binding analysis by surface plasmon resonance
The peptides were purchased from Genemed Sythesis,
Inc., CA. Each peptide showed a single peak on analytical reversed-phase HPLC and gave a mass identical to
that calculated. The dissociation constants between
FHA1 (residues 2-175 with a V5 tag, which is longer
than the fragment used in NMR studies) and the peptides were determined on a BIAcore 2000 instrument
(Karlsson & Stahlberg, 1995) at 25 C. The target peptides
were ®rst biotinylated by treating with sulfo-NHS-biotin
(Pierce) on ice for two hours, followed by diluting in
HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 3 mM
EDTA, 0.005 % Tween-20 (pH 7.4)) to a concentration of
ca 2 mM. The SA sensor chip, with streptavidin from
Streptomyces adidinii bound on the gold-dextran surface,
was conditioned according to the manufacturer with
one-minute injection of 1 M NaCl in 50 mM NaOH,
three times in succession. The chip surface was regenerated with MgCl2 and NaCl after each individual
measurement. A total of 40 ml peptide solution was
injected each time at a rate of 10 ml/min. After extensive
washing with HBS-EP buffer and a one-minute injection
of 1 M NaCl to remove non-speci®cally bound peptides,
a baseline increase of 100-200 resonance units (RU) is
typically obtained. FHA1 solutions of increasing concentration were then ¯owed across the SA chip at a rate of
10 ml/min for 240 s. As a result of binding between pThr
peptide and FHA1, changes build up with time forming
the ``sensorgram''. The maximal response units at equilibrium binding condition are proportional to the mass of
protein bound to the surface. At least ®ve different protein concentrations were performed and excess protein
solutions were injected each time to reach the ``equilibrium region'' in the sensorgram, de®ned as the maximal response. The latter (after correcting for the control
signals from the blank channel without immobilized
peptide) was then plotted against the protein concentration to determine the equilibrium dissociation constant (Kd).
Accession number
Coordinates for the structure of free FHA1 have been
deposited in the RCSB PDB (accession number 1G3G).
Acknowledgments
The authors thank Ohio Supercomputer Center for
the T94 Cray supercomputer resource grant, Ohio
Board of Regents for funding of the 800 MHz NMR
and the mass spectrometers, and NIH for ®nancial
support (CA87031).
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Edited by M. F. Summers
(Received 20 September 2000; received in revised form 26 October 2000; accepted 26 October 2000)