1. Structural Biology and Molecular Biophysics

Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core

  1. Andrew J Borst
  2. Connor E Weidle
  3. Matthew D Gray
  4. Brandon Frenz
  5. Joost Snijder
  6. M Gordon Joyce
  7. Ivelin S Georgiev
  8. Guillaume BE Stewart-Jones
  9. Peter D Kwong
  10. Andrew T McGuire
  11. Frank DiMaio
  12. Leonidas Stamatatos  Is a corresponding author
  13. Marie Pancera  Is a corresponding author
  14. David Veesler  Is a corresponding author
  1. University of Washington, United States
  2. Fred Hutchinson Cancer Research Center, United States
  3. National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
https://doi.org/10.7554/eLife.37688
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Cite this article
  1. Andrew J Borst
  2. Connor E Weidle
  3. Matthew D Gray
  4. Brandon Frenz
  5. Joost Snijder
  6. M Gordon Joyce
  7. Ivelin S Georgiev
  8. Guillaume BE Stewart-Jones
  9. Peter D Kwong
  10. Andrew T McGuire
  11. Frank DiMaio
  12. Leonidas Stamatatos
  13. Marie Pancera
  14. David Veesler
(2018)
Germline VRC01 antibody recognition of a modified clade C HIV-1 envelope trimer and a glycosylated HIV-1 gp120 core
eLife 7:e37688.
https://doi.org/10.7554/eLife.37688
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  3. Download .RIS
6 figures, 3 tables and 4 additional files

Figures

Figure 1 with 6 supplements
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Structural characterization of the 426c DS-SOSIP D3†-VRC01GL complex.

(A–B) BLI binding data of immobilized VRC01GL IgGs binding to WT 426c DS-SOSIP (A) or 426c DS-SOSIP D3 trimers. The concentrations of 426c DS-SOSIP trimers injected are indicated on each panel. Fit …

https://doi.org/10.7554/eLife.37688.002
Figure 1—figure supplement 1
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Multiple sequence alignment of analyzed HIV-1 426c constructs.

HIV-1 constructs derived from the 426c strain used in this study were subjected to multiple sequence alignment using Clustal Omega and rendered using ESPript (Gouet et al., 1999; Sievers and …

https://doi.org/10.7554/eLife.37688.003
Figure 1—figure supplement 2
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Multiple sequence alignment of analyzed antibody and Fab constructs.

VRC01GL-class antibody and Fab constructs used in this study were subjected to multiple sequence alignment using Clustal Omega and rendered using ESPript (Gouet et al., 1999; Sievers and Higgins, …

https://doi.org/10.7554/eLife.37688.004
Figure 1—figure supplement 3
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Structural characterization of the 426c DS-SOSIP D3†-VRC01GL complex.

(A) Strategy implemented to increase the occupancy of VRC01GL Fab for structural studies. VRC01GL can engage 426c DS-SOSIP D3, but its low apparent affinity relative to VRC01GL IgG precludes …

https://doi.org/10.7554/eLife.37688.005
Figure 1—figure supplement 4
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Validation of the 426c DS-SOSIP D3†-VRC01GL cryoEM reconstructions.

(A) Local resolution estimates of 426c DS-SOSIP D3† bound to three copies of VRC01GL-A60CHC as determined using ResMap (Kucukelbir et al., 2014). (B) Graphical plot depicting distribution of …

https://doi.org/10.7554/eLife.37688.006
Figure 1—figure supplement 5
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Comparison of gp120 interface contacts between VRC01GL and VRC01MAT.

Tables highlighting gp120 residues and their associated buried surface area (BSA) which comprise the interface with VRC01-class antibodies, as determined by PISA. BSA values are colored light gray …

https://doi.org/10.7554/eLife.37688.007
Figure 1—figure supplement 6
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Example of glycans resolved in the 426c DS-SOSIP D3†-VRC01GL structure.

CryoEM density (green semi-transparent surface) and atomic model for glycans at positions Asn230, Asn197, Asn386 and Asn262 are shown.

https://doi.org/10.7554/eLife.37688.008
Figure 2 with 1 supplement
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Reintroduction of V5 loop NLGSs does not hinder VRC01GL binding to the 426c core.

(A–D) BLI curves and the corresponding equilibrium dissociation constants for VRC01GL IgG binding to the S278A/T462A/T465A (A), S278A/T462A (B), S278A/T465A (C), and S278A (D) 426c core constructs …

https://doi.org/10.7554/eLife.37688.010
Figure 2—figure supplement 1
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Representative LC-MS/MS glycan identifications of 426c core constructs.

The top-left ribbon diagram corresponds to the sample analyzed. The LC-MS/MS fragmentation pattern is indicated in the top-right inset. A graphical depiction of the Asn276 residue (dotted white …

https://doi.org/10.7554/eLife.37688.011
Figure 3
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The VRC01GL Fab bound to the 426c core in presence of a glycan at position Asn276.

(A–B) BLI binding data of the immobilized VRC01GL Fab with the 426c core expressed in either HEK293F (A) or HEK293 GnTI-/- cells (B). (C) Crystal structure of 426c core†-VRC01GL highlighting glycan …

https://doi.org/10.7554/eLife.37688.013
Figure 4
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VRC01GL binding in the presence of a glycan at position Asn276 and protection against EndoH-mediated digestion.

(A) Summary of identifications for 426c Asn276 glycopeptides. 426c core constructs that were subjected to qualitative LC-MS/MS are indicated on the left. Glycopeptide identifications detected using …

https://doi.org/10.7554/eLife.37688.014
Figure 5 with 2 supplements
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Glycan length impacted VRC01GL IgG recognition of the 426c core.

(A) Semi-quantitative LC-MS/MS analysis depicting the relative signal intensities for identified Asn276 glycoforms in 426c core (blue), 426c S278T core (yellow), and 426c core expressed in the …

https://doi.org/10.7554/eLife.37688.015
Figure 5—figure supplement 1
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LC-MS/MS glycan identifications of kifunensine-treated 426c core constructs.

Representative LC-MS/MS spectra of glycan Asn276 identifications from the kifunensine-treated 426c core sample used for BLI experiments. The top-left ribbon diagram corresponds to the sample …

https://doi.org/10.7554/eLife.37688.016
Figure 5—figure supplement 2
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Trimeric 426c DS-SOSIP has a variable glycan length at position Asn276.

Representative LC-MS/MS spectra of glycan Asn276 identifications from the 426c DS-SOSIP. The top-left ribbon diagram corresponds to the sample analyzed. The LC-MS/MS fragmentation pattern is …

https://doi.org/10.7554/eLife.37688.017
Figure 6 with 1 supplement
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Asn276 glycosylation frequency modulated VRC01GL-class IgGs recognition of the 426c core.

(A–D) BLI binding data and associated KD values of 426c core constructs, expressed in HEK293 GnTI-/- cells, with two immobilized VRC01GL-class IgGs. VRC01GL IgG binding was assessed against the 426c …

https://doi.org/10.7554/eLife.37688.018
Figure 6—figure supplement 1
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VRC01GL binding affinity was not significantly affected by the identity of the residue 278 in the absence of a glycan at position Asn276.

(A) BLI kinetics parameters determined from panels (B–G) and Figure 6. (B–D) BLI binding data and determined equilibrium dissociation constants for VRC01GL IgG binding to 426c S278A core (A), 426c …

https://doi.org/10.7554/eLife.37688.019

Tables

Table 1
CryoEM data collection, refinement, and model validation statistics.
https://doi.org/10.7554/eLife.37688.009
ParameterValue
Data Collection
No. of Micrographs1993
No. of Particles134,443
Pixel size, Å1.36
Defocus range, μM2.0–3.5
Voltage, kV300
Dose Rate, counts/pix/sec8
Electron dose, e-/Å243
Refinement
Resolution, Å3.8
Map-sharpening B factor, Å2−230
Model validation (3 Fab structure)
Favored rotamers, %98.36%
Poor rotamers, %0.30%
Ramachandran outliers, %0.13%
Clash Score0.99
Molprobity score
EM ringer score		1.02
1.97
Table 2
Crystallographic data collection and refinement statistics
https://doi.org/10.7554/eLife.37688.012
426c core†-VRC01GL
Data collection
Space groupC2
Cell dimensions
 a, b, c (Å)197.082, 109.003, 103.225
 α, β, γ (°)90.000, 114.468, 90.000
Resolution (Å)50–2.32 (2.36–2.32)*
Rsym or Rmerge0.076 (0.643)*
I/sI23.4 (1.8)*
Completeness (%)95.6 (66.8)*
Redundancy7.4 (5.7)*
CC1/2(0.823)*
Refinement
Resolution (Å)46.98–2.315 (2.398–2.315)*
No. reflections83086
Rwork/Rfree24.38/29.55 (42.67/49.28)
No. atoms12470
 Protein11746
 Water325
 Ligand399
B-factors (Å2)74.22
 Protein73.57
 Water69.62
 Ligand97.10
R.m.s deviations
 Bond lengths (Å)0.003
 Bond angles (°)0.60
Ramachadran Favored %93.39
Ramachadran Outliers %0.13
MolProbity all-atoms clashscore4.05
Key resources table
Reagent type
(species) or Resource		DesignationSource or referenceIdentifiersAdditional information
Software,
algorithm		Leginondoi: 10.1016/
j.jsb.2005.03.010		http://emg.nysbc.org/redmine/projects/leginon/wiki/Leginon_Homepage
Software,
algorithm		RELION-2doi: 10.1016/
j.jsb.2012.09.006		RRID:SCR_016274http://www2.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page
Software,
algorithm		MotionCor2doi:10.1038/
nmeth.4193		http://emg.nysbc.org/redmine/projects/appion/wiki/Appion_Home
Software,
algorithm		GCTFdoi: 10.1016/
j.jsb.2015.11.003		RRID:SCR_016500https://www.mrc-lmb.cam.ac.uk/kzhang/
Software,
algorithm		CTFFIND4doi:10.1016/
j.jsb.2015.08.008		http://grigoriefflab.janelia.org/ctffind4
Software,
algorithm		Frealigndoi: 10.1016/
bs.mie.2016.04.013		http://grigoriefflab.janelia.org/frealign
Software,
algorithm		Appion Packagedoi: 10.1016/
j.jsb.2009.01.002		http://emg.nysbc.org/redmine/projects/appion/wiki/Appion_Home
Software,
algorithm		DoG Pickerdoi:10.1016/
j.jsb.2009.01.004		http://emg.nysbc.org/redmine/projects/appion/wiki/Appion_Home
Software,
algorithm		Cootdoi: 10.1107/
S0907444910007493		RRID:SCR_014222http://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/devel/build-info.html
Software,
algorithm		Rosettadoi: 10.1146/annurev.
biochem.77.062906
.171838		RRID:SCR_015701https://www.rosettacommons.org/software
Software,
algorithm		UCSF Chimeradoi: 10.1002/jcc.20084RRID:SCR_004097http://plato.cgl.ucsf.edu/chimera/
Software,
algorithm		PMI-Byonicdoi: 10.1002/
0471250953.bi1320s40		https://www.proteinmetrics.com/products/byonic/
Software,
algorithm		Skylinedoi: 10.1093/
bioinformatics/btq054		RRID:SCR_014080https://skyline.ms/project/home/software/Skyline/begin.view
Software,
algorithm		Octet Data
Acquisition		Pall ForteBioCFR 10.0.3.12dhttps://www.fortebio.com/octet-software.html
Software,
algorithm		Octet Data AnalysisPall ForteBioCFR 10.0.3.1https://www.fortebio.com/octet-software.html
Software,
algorithm		Phaserdoi:10.1107/
S0021889807021206		https://www.phenix-online.org/documentation/reference/phaser.html
Software,
algorithm		Phenix.refinedoi:10.1107/
S0907444912001308		https://www.phenix-online.org/documentation/reference/refinement.html
Software,
algorithm		GraphPad PrismGraphPadRRID:SCR_002798https://www.graphpad.com/scientific-software/prism/
Software,
algorithm		PymolDelano, 2002https://pymol.org/2/
Cell Line
(Homo sapiens)		HEK293S GnTI-/-ATCCATCC: CRL-3022;
RRID:CVCL_A785		https://www.atcc.org/Products/All/CRL-3022.aspx
Cell Line
(Homo sapiens)		HEK293FThermoFisher
Scientifc		Cat# R79007https://www.thermofisher.com/order/catalog/product/R79007
Gene
(Homo sapiens)		gl VRC01 Igk(3-11)doi: 10.1126/
science.1192819
Gene
(Homo sapiens)		gl VRC01 Igg Fabdoi: 10.1038/
ncomms10618
Gene
(Homo sapiens)		gl VRC01 Igg
Fab A60C/C98S		This paper
Gene
(Homo sapiens)		gl VRC01 Igg10.1126/science.1192819
Gene
(Homo sapiens)		gl 12A21 Igk(1-33)10.1084/jem.20122824
Gene
(Homo sapiens)		gl 12A21 Igg Fab10.1038/ncomms10618
Gene
(Homo sapiens)		gl 12A21 Igg10.1084/jem.20122824
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		WT_426 c_DS-SOSIP10.1016/j.cell
.2016.07.029
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_G459C
_DS-SOSIP_D3		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_WT_
gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_G459C
_gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_S278A
_gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_S278A_T462A
_gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_S278A_T465A
_gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_S278A_T462A_
T465A_gp120c_core		This paper
Strain, strain
background (Human
Immunodeficiency
Virus-1, Strain: 426 c)		426 c_S278T_gp120cThis paper
Recombinant
DNA reagent		pTT3PMID: 11788735https://biochimie.umontreal.ca/en/department/professors/yves-durocher/
Recombinant
DNA reagent		pVRC8400NIH
Chemical
compound, drug		KifunensineSigma-AldrichCAS Number
109944-15-2		https://www.sigmaaldrich.com/catalog/product/sigma/k1140?lang=en&region=US&gclid=Cj0KCQjwr53OBRCDARIsAL0vKrNtYwTyRzHU65HyVBwdntcP3kGpZ0ElVwYeSK3OcorLn0wf8U1iMQgaAssSEALw_wcB
Peptide,
recombinant protein		Endoglycosidase-HNew England BiolabsCatalog #P0702Shttps://www.neb.com/products/p0702-endo-h#Product%20Information

Additional files

Supplementary file 1

BLI kinetics parameters of various 426c core constructs expressed using HEK293F cells.

https://doi.org/10.7554/eLife.37688.020
Supplementary file 2

RMSD Table comparing unliganded and liganded VRC01GL CDRL1 loop conformations.

https://doi.org/10.7554/eLife.37688.021
Supplementary file 3

BLI kinetics parameters of various 426c core constructs expressed using GnTI-/- cells.

https://doi.org/10.7554/eLife.37688.022
Transparent reporting form
https://doi.org/10.7554/eLife.37688.023

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