Journal: PLoS Pathog / Year: 2013 Title: Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. Authors: Jean-Philippe Julien / Devin Sok / Reza Khayat / Jeong Hyun Lee / Katie J Doores / Laura M Walker / Alejandra Ramos / Devan C Diwanji / Robert Pejchal / Albert Cupo / Umesh Katpally / Rafael ...Authors: Jean-Philippe Julien / Devin Sok / Reza Khayat / Jeong Hyun Lee / Katie J Doores / Laura M Walker / Alejandra Ramos / Devan C Diwanji / Robert Pejchal / Albert Cupo / Umesh Katpally / Rafael S Depetris / Robyn L Stanfield / Ryan McBride / Andre J Marozsan / James C Paulson / Rogier W Sanders / John P Moore / Dennis R Burton / Pascal Poignard / Andrew B Ward / Ian A Wilson / Abstract: New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, ...New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1). Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.
History
Deposition
Mar 27, 2013
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Header (metadata) release
Apr 10, 2013
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Map release
Jul 3, 2013
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Update
Jul 3, 2013
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Current status
Jul 3, 2013
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_5624.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Reconstruction of SOSIP.664 HIV-1 envelope trimer in complex with broadly neutralizing PGT122 Fab
Voxel size
X=Y=Z: 2.18 Å
Density
Contour Level
By AUTHOR: 3.87 / Movie #1: 3.87
Minimum - Maximum
-1.24171531 - 12.395413400000001
Average (Standard dev.)
0.0 (±0.99999994)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
160
160
160
Spacing
160
160
160
Cell
A=B=C: 348.80002 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.18
2.18
2.18
M x/y/z
160
160
160
origin x/y/z
0.000
0.000
0.000
length x/y/z
348.800
348.800
348.800
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-132
-122
-147
NX/NY/NZ
250
274
261
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
160
160
160
D min/max/mean
-1.242
12.395
0.000
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Supplemental data
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Sample components
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Entire : Fab fragment of broadly neutralizing antibody PGT122 in complex w...
Entire
Name: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
Components
Sample: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505
Protein or peptide: BG505 HIV-1 Env SOSIP.664
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Supramolecule #1000: Fab fragment of broadly neutralizing antibody PGT122 in complex w...
Supramolecule
Name: Fab fragment of broadly neutralizing antibody PGT122 in complex with HIV-1 SOSIP.664 from BG505 type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: one SOSIP.664 trimer binds 3 PGT122 Fabs / Number unique components: 2
pH: 7 / Details: 20 mM Tris-HCl, pH 7.0, 50 mM NaCl
Staining
Type: NEGATIVE Details: Grids were briefly adsorbed with protein, wicked, and stained with 2% Nano-W for 30 seconds.
Grid
Details: 400 Cu mesh grid with thin carob support, glow discharged in natural atmosphere.
Vitrification
Cryogen name: NONE / Instrument: OTHER
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Electron microscopy
Microscope
FEI TECNAI F20
Temperature
Min: 293 K / Max: 294 K / Average: 293 K
Alignment procedure
Legacy - Astigmatism: Astigmatism of objective lens was corrected at 100,000x Legacy - Electron beam tilt params: -2
Date
Apr 30, 2012
Image recording
Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 0.109 µm / Number real images: 340 / Average electron dose: 16 e/Å2
Tilt angle min
0
Electron beam
Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
All particles were automatically selected from micrographs with DoG Picker [63]. Contrast Transfer function (CTF) estimation for the untilted and tilted micrographs was determined with ctffind3 and ctftilt [64]. Particles were binned by 4 (80x80 sized boxes) and reference-free 2D class averages were calculated using the Sparx package (Fig. S4) [65]. Forty ab initio models were generated from the final reference-free 2D class averages using the EMAN2 package. Each model was then refined against the reference-free 2D class averages using Sparx [65,66]. The model exhibiting Fab-like density was used as the initial model for iterative image reconstruction against the CTF-corrected particles using Sparx [65].
CTF correction
Details: each image
Final reconstruction
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx Details: Final map was calculated from a single data set. Multiple data sets produced indistinguishable maps, but data were not combined. Number images used: 10413
Final angle assignment
Details: SPIDER: theta 45 degrees, phi 45 degrees
Final two d classification
Number classes: 64
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