Fremont – 30 Sept 2011
Bio 5357
Protein crystallography in practice
Biology 5357: Chemistry & Physics of Biomolecules
30 Sept 2011
Daved H. Fremont
Department of Pathology and Immunology
Department of Biochemistry and Molecular Biophysics
Washington University School of Medicine
1.
An 8-step program for protein structure
determination by x-ray crystallography
1. Produce monodisperse protein either alone or as relevant complexes
2. Grow and characterize crystals
3. Collect X-ray diffraction data
4. Solve the phase problem either experimentally or computationally
5. Build an atomic model using the electron density map
6. Iteratively refine and rebuild the structural model
7. Validation How do you know if a crystal structure is right?
8. Develop structure-based hypothesis
Produce monodisperse protein
either alone or as relevant complexes
Methods to determine protein purity, heterogeneity, and monodispersity
!
Gel electrophoresis (native isoelectric focusing and SDS-PAGE)
!
Size exclusion chromatography
!
Dynamic light scattering http://www.protein-solutions.com/
!
Circular Dichroism Spectroscopy http: /www-structure llnl.gov/cd/cdtutorial.htm
Characterize your protein using a number of biophysical methods
Establish the binding stoichiometry of interacting partners
2. Grow and characterize crystals
! Hanging Drop vapor diffusion
! Sitting drop, dialysis, or under oil
! Macro-seeding or micro-seeding
! Sparse matrix screening methods
! Random thinking processes, talisman, and luck
The optimum conditions for crystal nucleation are not
necessarily the optimum for diffraction-quality crystal growth
Space Group P21
4 M3 /ASU
diffraction >2.3Å
Protein interaction stoichiometry as estimated by size exclusion chromatography
No Xtals?
Decrease protein heterogeneity
1 . % Peg6K
NaCacodylate pH 7.0
200mM CaCl2
Space Group C2
2 M3 /ASU
diffraction >2 1Å
18% Peg K
Malic Acid/Imidazo e pH 5.1
100mM CaCl2
Commercial sc een ng kits a ailable from
http //www.hamptonresearch com
http //www.emeraldb ostruc ures.com
Space Group P3121
3 M3 3 MCP-1/ASU
diffraction > 2.3Å
18% Peg K
NaAcetate pH .1
100mM MgCl2
3. Collect X-ray diffraction data
! Initiate experiments using home-source x-ray generator and detector
! Determine liquid nitrogen cryo-protection conditions to reduce crystal decay
! While home x-rays are sufficient for some questions, synchrotron radiation is preferred
! Anywhere from one to hundreds of crystals and diffraction experiments may be required
!  Remove purification tags and other artifacts of protein production
!  Remove carbohydrate residues or consensus sites (i.e., N-x-S/T)
!  Determine domain boundaries by limited proteolysis followed by mass
spectrometry or amino-terminal sequencing. Make new expression constructs
if necessary.
Argonne National Laboratory Structural Biology Center beamlineID19
at the Advanced Photon Source ht p //www.sbc.anl.go
!  Think about the biochemistry of the system! Does your protein have cofactors, accessory proteins, or interacting partners to prepare as complexes?
Is their an inhibitor available? Are kinases or phosphatases available that will
allow for the preparation of a homogeneous sample?
!  Get a better talisman
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Fremont – 30 Sept 2011
Bio 5357
3. Collect X-ray diffraction data
Lawrence Berkeley National Laboratory
ALS Beamline 4.2.2
4. Solve the phase problem either
experimentally or computationally
! Structure factor equation
! By Fourier transform we can obtain the electron density.
We know the structure factor amplitudes after successful data collection.
Unfortunately, conventional x-ray diffraction doesn t allow for direct phase measurement.
This is know as the crystallographic phase problem.
! Luckily, there are a few tricks that can be used to obtain estimates of the phase !(h k l)
Experimental Phasing Methods
! MIR - multiple isomorphous replacement - need heavy atom incorporation
!  MAD - multiple anomalous dispersion- typically done with SeMet replacement
! MIRAS - multiple isomorphous replacement with anomalous signal
! SIRAS - single isomorphous replacement with anomalous signal
Computational Methods
! MR - molecular replacement - need related structure
! Direct and Ab Initio methods - not yet useful for most protein crystals
5 Build an atomic model using the electron density map
MAD phasing statistics for the AP-2 !-appendage
P1
SeMet-1
Diffraction Data
Wa elength (Å)
Resolut on (Å)
Number of sites
P1
SeMet-2
P1
SeMet-3
P1
SeMet-4
P21
Na i e
1.07813
0.97956
0 97945
0.94645
0.97945
100-1 60
100-1 60
100-1.60
100-1 60
100-1 40
---
4
4
4
4
measured
(un que)
126 734
(49 265)
146 273
(56 587)
146 405
(56 619)
149 455
(57 749)
495 848
(45 711)
Completeness
o era l
(outer shell)
81 8
(35 3)
93.9
(80.4)
94.0
(80.9)
95 8
(94 8)
96 1
(82 1)
I ! (I)
o era l
(outer shell)
22 5
(7.9)
26.8
(11.6)
21.9
(9.8)
19 1
(7.3)
23 0
(3.0)
Rsym(%)
o era l
(outer shell)
iso/ano
5.9
(19 2)
58
(17.0)
6.0
(19.3)
65
(28 6)
10 0
(67 2)
0 359 0.563
0.283/0 529
0 607/0.729
4.56 3.10
6.19/3 30
0.819/2 31
Reflections
Rcu lis (20-1.6 Å)
Phasing power
iso/ano
---/0.967
---/0.818
Resolu ion
Number of
eflections/
Refinement
Range (Å)
completion
P!"SeMet
20.0-1 60
(1.66-1.60)
29 053/ 97.1%
(2750/95.7% )
P21-native
20.0-1 40
(1.45-1.40)
45 632/ 96.3%
(3965/84.6% )
Number of
protein a oms/
sol ent atoms
Rcrystal/
Rms
Electron densi y for the AP-2 !-appendage
----De iations
Rfree (%)
Bonds (Å)
Angles (o)
1957 266
16 8/21.1
(26 2/27 8)
0.010
19
1957 244
17 8/20.9
(26 0/26 3)
0.011
15
Initial bones trace for he AP-2 !-appendage
Final trace for the AP-2 !-appendage
7 Validation: How do you know if a crystal structure is right?
7 Validation: Mapping of sequence conservation in AP-2 !-subunit appendages
The R-factor
R "(|Fo-Fc|)/"(Fo)
where Fo is the observed structure factor amplitude and Fc is calculated using the atomic model.
R-free
An unbiased, cross-validation of the R-factor. The R-free value is calculated with typically 5-10% of the observed reflections which are
set aside from atomic refinement calculations.
Main-chain torsions: the Ramachandran plot
Geometric Distortions in bond lengths and angles
Favorable van der Waals packing interactions
Chemical environment of individual amino acids
Loca ion of inser ion and deletion positions in rela ed sequences
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of the
!-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.
Natl. Acad. Sci. U.S.A. 96:8907-8912.
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Fremont – 30 Sept 2011
Bio 5357
8 Develop structure-based hypothesis
How do motif peptides bind?
Structure-Based Mutagenesis of the !-appendage
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of the
!-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.
Natl. Acad. Sci. U.S.A. 96:8907-8912.
Brett TJ, Traub LM, Fremont DH.
Accessory protein recruitment motifs in clathrin-mediated endocytosis.
Structure (Camb). 2002 Jun;10(6):797-809.
How do motif peptides bind?
West Nile and Dengue Viruses are Flaviviruses
About 70 members, half of which are associated
with human disease (Yellow fever, Japanese encephalitis)
Enveloped, spherical virion, 40 - 50 nm in size
Three structural proteins: C,M (prM) and E ; seven
non-structural proteins (NS1-5)
ssRNA genome, linear, positive polarity, 11 kb,
infectious
Structural proteins
Production of soluble E proteins and ectodomain fragments
Immunize mice with
soluble E (25 µg x 3)
Fuse splenocytes
with myeloma line
Large panels of flavivirus mAbs
Non-structural proteins
Structure Determination of WNV Envelope Protein
Table 1. Summary of Data Collection and Refinement
Data Collection for West Nile Virus Envelopea
Space Group
P 1212
Unit Cell (Å3)
a=89.6 b=89.6 c=15 0
Wavelength(Å)
0.90
X-ray Source
APS-BM 1
Resolu ion(Å) (outer shell)
20-2.9 (3.08-2.90)
Observations Unique
1 08/62790
ompleteness(%)
98.5 (99 5)
R ym(%)
5.7 (52. )
/!
16.9 (2.05)
Refinement Statisticsb
Resolu ion(Å) (outer shell)
20-3.0 (3.19-3.00)
Reflections Rwo k/Rf e
11506/607
#Protein Atoms/Solvent/He erogen
3031/28 38
Rwo k overal (outer shell) (%)
26.2(35.6)
Rf ee overal (outer shell) (%)
30.8(3 .1)
Rmsd Bond lengths (Å)/angles(o)
0.008/1.6
Rmsd Dihedral/Improper (o)
2 .9/0.8
Ramachandran plot
Most Favored/Additional (%)
78.2/21.8
Generous/Disallowed (%)
0.0/0.0
Average B-va ues
92 0
Est. Coordinate Error (Å)
0. 7
a
Values as def ned in SCALEPACK (Otwinowski and Minor, 1997).
Values as def ned in CNS (Brunger, AT)
b
3
Fremont – 30 Sept 2011
Bio 5357
E16 is a potent neutralizing mAb with therapeutic activity against WNV in mice
Envelope Protein and the Flavivirus virion
X-ray crystal structure of E
Single Dose mAb at Day 5 Post-Infection
E+09
100
DIII
E+07
E16 (2 mg)
60
DII
E24 (2 mg)
PBS
40
20
E+05
E+04
E+03
E+0
0
Mature
E+06
E+02
0
Cryo-EM model of WNV
Immature
E+08
reatment Day 5
80
PFU/g of tissue
DI
5
10
15
20
25
30
E+00
Days Post Infection
Day 9 - E16
Day 9 - PBS
Humanized E16 binds WNV DIII with similar affinities and kinetics as E16
5
U
Summary of Surface Plasmon Resonance (SPR) studies
DIII binding E16
prM Cleavage
2
R
3
U
DIII binding Hm-E16.3
Antibody
E16
Hm-E16.1
Hm-E16.2
Hm-E16.3
k (1/Ms)
1.1 x 106
9.6 x 105
1.0 x 106
9.9 x 105
k (1/s)
0.0118
0.0201
0.0092
0.0070
R
39.5
32.8
2 .7
2 .1
K (nM)
10.8
21.0
9.2
7.1
Chi2
0 33
0 16
0 13
0 16
R
60 trimers of prM/E heterodimers
180 E monomers
Structure determination of DIII-E16 complex by X-ray crystallography
Production and purification of DIII in complex with E16 Fab
Bacterial expression
of WNV E Domain 3
Refolding of DIII
Complex purification by
size exclusion chromatography
DIII
Abs280 (mAU)
I
Data collection for D3-E16 complex
Space Group
P212121
Unit Cell (Å3)
a=52. b=83.3 c=110.6
X-ray Source
ALS
Resolution(Å) (outer shell)
30-2.50 (2.59-2.50)
Observations/Unique
59923/16985
Completeness(%)
97.6 (82.7)
Rsym(%)
8.3 (30.6)
I/
11.3 (2.7)
Atomic refinement statistics
Rwork overall(outer shell) (%)
20.8(25.6)
Rfree overall(outer shell) (%)
28.2(31.8)
Ramachandran plot
Most Favored/Additional (%)
87.5/11.9
Generous/Disallowed (%)
0. /0.2
E ution Volume (ml)
Hybridoma
expression
of E16 mAb
E16 Fab
by papain
cleavage
mAb capture
by Protein A
Structure of the DIII-E16 Fab complex
Selection of E16 specific epitope variants of DIII
E16 Fab
CH
CL
VH
VL
VH
Yeast library of DIII variants
created by error prone PCR
VL
E -DIII
Pooled
DIII
DIII
mAbs
DIII
E16 Fab
E16 staining
L3
DE Loop
DIII mutations at Ser306, Lys307, ThrE330 and Thr332 significantly diminish E16 binding
L1
H2
BC Loop
H1
H3
N-terminal
region
L2
FG Loop
Nybakken et al, Nature 2005
4
Fremont – 30 Sept 2011
Bio 5357
DIII yeast display mutations are centrally located at the E16 interface
E16 Fab
C
CL
C
1A1D-2 Fab C
E16 Fab
H1
CH
CL
VH
VL
CH1
H3
C
E53 Fab
CL
C
CH1
CH1
CL
C
TrpH33
SerH95
N
H2 ArgH58
FG
AB
WNVE
DII
N
Fus on loop
C
LysE307
Ser 306
Thr 332
ThrE330
DIII N-Term
Lys 306
Thr 330
ThrE332
Lys 305
Arg 99
Lys 310
Gly 106
Lys 307
Yeast display
DIII BC loop
E16 Fab could potentially bind 120/180 E protein DIII sites on WNV
# 4 5 Å con acts
Thr 76
Pro 75
Fusion Loop
Cryo-EM reconstruction of
E16 Fab complex with WNV
Cross-section of Cryo-EM reconstruction
E16 binding to 2- and 3-fold clustered DIIIs appears permissive
while 5-fold clustered DIII binding appears sterically non-permissive
Zhang et al Nat Structural Biology 2003
Mukhopadhyay et al Science 2003
Fitting E16 Fab complex into CryoEM reconstruction of WNV
Cryo-EM work done in collaboration with
Rossmann and Kuhn groups at Purdue University
E16 engages the DI-DIII hinge region associated with fusion
2. Virus Entry
Leu 107
E16 Fab exclusion from inner 5-fold clustered DIIIs demonstrated by Cryo-EM
Model of E16 Fab
complex with WNV
1. Virus Binding
N
C
ThrE330
DIII
VL
N
AspH100
SerE306
LysE307
DV2E
DIII
N
VH
N
BC
DE
FG
H3
ThrE332
N
DE BC
WNVE
DIII
DIII N-Term
SerE306
VH
N
LysE307
DIII
VL
VL
VH
E16 internalizes with the virus during infection of vero cells
3. Virus Fusion
Negative
E53
DII-Neutralizing
E16
DIII-Neutralizing
MOI
VH
DIII
0
DIII
DI
DI
ArgH56
TyrE302
VL
DII
Pre-Fusion
Modis et al PNAS 2003
DIII
100
DII
Post-Fusion
Modis et al Nature 200
E16 binding at DI-DIII linker
1 hour post infection
Cholera Toxin
Alexa 647 labeled Ab
5
Bio 5357!
Fremont – 1 October 2010!
E16 internalizes with the virus during infection of vero cells!
E16 Fab decoration appears to trap WNV particles - a fusion intermediate?!
pH 8!
Pre bind virus + Alexa-Ab!
pH 6!
Add to cells at 4 or 37oC!
E53
!
!
!
E16!
15 minutes. Fix, add!
Lyso-tracker!
Confocal microscopy!
DIC / Bright !
Field!
Fluorescent!
Merge!
nucleocapsid core (~ 154Å)
outer lipid layer (~200 Å )
outer glycoprotein layer (~245Å)
Alexa 488-labeled WNV mAbs and lysotracker red (acidified endosomes) !
n ucleocapsid core (~ 158Å)
o uter lipid layer (~205Å)
outer density layer (~340Å)
!"#$%&'()*+(,&$#(&()-.-%*/(0"$,&*1&,2--(,,12%&"++2)*%*'.&#(,($#-3&
Ectromelia"
virus!
mousepox!
The M3 protein encoded by Murine #HV68
Host"
survival"
!
Immune"
evasion"
M3 sequesters chemokines, blocking their ability to activate GPCRs
M3 Purification and Structure Determination !
Baculovirus Expression!
M3 Alone!
Solved by MIR!
phasing to!
2.7 Å resolution!
R-factor=22.8%,!
R-free=27.3%!
electron density map!
6!
Fremont – 30 Sept 2011
Bio 5357
Structure of M3 Alone
Structure of M3/MCP-1 (CC) Complex
M3 CTD
Ig-fold like
MCP 1(P8A)
NTD
CTD
CTD
MCP 1(P8A)
M3 NTD
Similar to Diphtheria Toxin
R-domain
Native PAGE: Experimental support for the
observed M3/MCP-1 Structure
NTD
45 ", $ -*+/(667( ")3"8"9*# *1 -3(+*:")( #(-(/9*# 8")0")'
##,-."#$%&
N-loop
MCP-1 residues implicated in
CCR2 binding
Hemmerich S. et.al. Biochemistry (1999)
"/."#$%&0-1-2
"#$%& '()*+
MCP-1 residues contacting M3
M3 establishes competitive inhibition of chemokine function by
structural mimicry of endogenous chemokine receptors
IL-8/CXCR1 mimic
:(5:
"#$%& 3(4'(45 (46*+789*
;<=
M3/MCP-1
45 /#*+",-2*2,%. 8")0, ;;< ;< $)0 ;=5; -3(+*:")(,
$)0 ,(%(-67(%. 8")0, +(+8(#, *1 93( ;=; 1$+"%.
7
Fremont – 30 Sept 2011
Bio 5357
;#.,9$%%*'#$/3"- $)$%.,", *1 45>-3(+*:")( -*+/%(?(,
45>4;KILC;;D
45>@ABC;D
45>EFGC;=5;D
I212121 space group
MR phasing
2.6 Å resolution
R-factor=23.2%
R-free=29.1%
P41212 space group
MR Phasing
2.9 Å resolution
R-factor=24.4%
R-free=28.2%
M3/chemokine complex structures
2:2 complex
P321 space group
MAD phasing
2.3 Å resolution
R-factor=20.1%
R-free=28.2%
45>HKILMC;=;D
45>H@IJC;=;D
P321 space group
4.0 Å low
resolution data
I4 space group
MR Phasing
5.5 Å low
resolution data
M3/MCP-1(CC)
M3/Frk(CX3C)
Promiscuous chemokine binding is facilitated by conformational plasticity
Electrostatic complementarity with M3 NTD
Electrostatic potential
Ltn
∆Gelec= -67 kJ/mol
M3/IP-10(CXC)
M3/IL-8(CXC)
;*)1*#+$6*)$% K%$,6-"9. *1 45
Role of electrostatics in M3/chemokine interactions:
Salt-dependence of M3/Ltn on-rate
200mM
NaCl
300mM
500mM
1M
LTN
pIexpt = >9.5
m
NaCl-Dependence of M3-Ltn
Kinetics
IP-10
∆Gelec= -60kJ/mol
1 E+ 8
E+ 7
E+ 6
1 E+ 5
0
0 25
05
0 75
1
NaCl (M)
M3
pIexpt = 4.5
NaCl-Dependence of M3-Ltn
Affinity
00
1 E-06
0
1 E-07
kd (s-1)
Frk
∆Gelec= -52 kJ/mol
ka
kd
1 25
15
0 01
1 75
KD (M)
1 E+ 9
ka (M-1s-1)
Long-range
Attractive
forces
MCP-1
∆Gelec= -40 kJ/mol
M3/Ltn(C)
Kd
Keq
1 E-08
1 E-09
1 E-10
0
0 25
05
0 75
1
1 25
15
1 75
NaCl (M)
M3-Ltn kinetics 200mM-1.5M NaCl
on-rate 70-fold (6x107 to 8.3x105 M-1s-1)
off-rate 5-fold (0.1-0.02 s 1)
8
Fremont – 30 Sept 2011
Bio 5357
M3/chemokine interactions in the context of GAGs
Electrostatic interactions mediate rapid
chemokine sequestration by M3
typical
protein-protein
Interactions
(105-106)
Heparin
Disaccharide
Unit
Diffusion (109)
M3BBXB
150mM
(2x106)
M3
150mM
(~108)
*
*
M3
M3
1.5 M NaCl 200mM NaCl
(6x107)
(8.3x105)
*
*
Chemokine
GAG-binding regions
*= shared M3 contacts
*
*
MCP-1 GAG-binding
residues
(Lau et al. 200 )
(Chakravarty et a. 1998)
Ltn GAG-binding residues
(Peterson et al. 200 )
M3 stoichiometrically inhibits chemokine binding to GAGs
Dual GPCR and GAG mimicry by M3
Ltn
MCP-1
R
IP-10
6
5 0µM
5
M
4
50
3
5
R
s
R s
5
3
00
80
60
40
2
25
NL
ReqHepar n bind ng
ReqHepar n bind ng
M
M
08
2
2
1
12
5
3
NL
M3 ( nhib tor) conc M)
5
5
2
5
B? 4"
2.3 µM MCP-1
Heparin MCP-1
M3 competition
M
20
5
8
2
t me s)
&@A µ"
120 nM Ltn
M3 ( nhib tor) conc M)
00nM
75
50
35
4
02
2
s
>? 4"
Heparin-Ltn
M3 competition
5
04
5
m
20
5
0
R sponse
200nM
4
ReqHepar n binding
5
84 nM IP 10
LNL
Heparin- P-10
M3 Compet tion
M3 ( nh bi or) conc (M)
9