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Probing the impact of local structural dynamics of conformational epitopes on antibody recognition Yu Liang, Miklos Guttman, Thaddeus M. Davenport, Shiu-Lok Hu, and Kelly Keisen Lee Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01354 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016
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Biochemistry
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Probing the impact of local structural dynamics of
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conformational epitopes on antibody recognition
4 5 Yu Liang1, Miklos Guttman1, Thaddeus M. Davenport1,2, Shiu-Lok Hu3, Kelly K. Lee1*
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1
2
Department of Medicinal Chemistry, University of Washington, Seattle WA, 98195 Current address: Laboratory of Molecular and Cellular Imaging, National Heart, Lung, and Blood Institute, Bethesda, MD 20814 3 Department of Pharmaceutics, University of Washington, Seattle WA, 98195
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*Address correspondence to Kelly K. Lee,
[email protected]; 206-616-3972; 206-685-3252 (fax)
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Running Title: Modulation of antibody binding by antigen epitope dynamics
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ABBREVIATIONS
19
HIV, Human Immunodeficiency Virus
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HDX-MS, Hydrogen/deuterium-exchange mass spectrometry
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ADCC, Antibody-dependent cell-mediated cytotoxicity
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ELISA, Enzyme-linked immunosorbent assay
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Env, Envelope glycoprotein
24
FL, Full length
25
Coree, Extended core
26
NMR, Nuclear magnetic resonance
27
GNA, Galanthus nivalis lectin
28
BN-PAGE, Blue native Polyacrylamide Gel Electrophoresis
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TCEP, Tris (2-carboxyethyl) phosphine hydrochloride
30
LC-MS, Liquid chromatography- mass spectrometry
31
UPLC, Ultraperformance liquid chromatography
32
TFA, Trifluoroacetic acid
33
ACN, Acetonitrile
34
SAXS, Small angle X-ray scattering
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PBS, Phosphate-buffered saline
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BSA, Bovine serum albumin
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TBS, Tris-buffered saline
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HRP, Horseradish peroxidase
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AHC, Anti-Human IgG Fc Capture
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BLI, Biolayer interferometry
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CD4i, CD4-induced
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KD, Equilibrium dissociation constant
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Ka, Association rate
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Kd, Dissociation rate
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CD4bs, CD4 binding site-specific
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SLS, Static light scattering
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ABSTRACT
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Antibody-antigen interactions are governed by recognition of specific residues and structural
51
complementarity between the antigen epitope and antibody paratope. While X-ray
52
crystallography has provided detailed insights into static conformations of antibody-antigen
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complexes, factors such as conformational flexibility and dynamics, which are not readily
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apparent in the structures, can also have impact on the binding event. Here we investigate the
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contribution of dynamics in the HIV-1 gp120 glycoprotein to antibody recognition of conserved
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conformational epitopes including the CD4- and coreceptor-binding sites, and an inner domain
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site that is targeted by ADCC-active antibodies. Hydrogen/deuterium-exchange mass
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spectrometry (HDX-MS) was used to measure local structural dynamics across a panel of
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variable loop truncation mutants of HIV-1 gp120 including full-length gp120, ∆V3, ∆V1/V2,
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and extended core, which includes ∆V1/V2 and V3 loop truncations. CD4-bound full-length
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gp120 was also examined as a reference state. HDX-MS revealed a clear trend towards increased
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order of the conserved subunit core resulting from loop truncation. Combined with biolayer
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interferometry and ELISA measurements of antibody-antigen binding, we demonstrate that
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increased ordering of the subunit core was associated with better recognition by an array of
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antibodies targeting complex conformational epitopes. These results provide detailed insight into
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the influence of structural dynamics on antibody-antigen interactions and suggest the importance
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of characterizing the structural stability of vaccine candidates in order to improve antibody
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recognition of complex epitopes.
69 70
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Biochemistry
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Interaction of antibody and antigen involves the recognition of residues with a specific
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three-dimensional organization on the antigen (epitope) by a set of residues on the antibody
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(paratope). Understanding the structural basis of antibody recognition and immune response
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against antigens is important for optimization of vaccine immunogens and for understanding
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mechanisms of neutralization. High resolution X-ray crystal structures have illustrated the
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complementary nature of the epitope-paratope coupling. This type of information has enabled
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epitopes to be mapped with atomic detail and suggested modifications to the antibody paratope
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as well as changes on the antigen that can enhance the affinity of antibody-antigen binding in
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optimizing potential vaccine immunogens 1, 2. Structural dynamics and conformational variability
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of immunogens can also influence the presentation and of epitopes, impact the thermodynamics
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of binding as well as the affinity and specificity of antibody binding. These are fundamental
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structural, biophysical properties that are at play essentially in all protein-protein interactions.
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A correlation between conformational dynamics, solvent accessibility, antigenicity and 3-9
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immunogenicity has been proposed for a variety of different antigens
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effect of conformational dynamics of the antigen or antibody on the binding interaction has often
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proved challenging. Most studies that report a linkage between dynamics and antibody-antigen
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affinity have relied upon measurements that provide global thermodynamic parameters but little
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structural dynamic information for epitopes and paratopes
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examined the influence of antibody conformational dynamics on recognition of relatively simple
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target antigens 13, 14. NMR has been used for the study of antibody fragments and small antigens,
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providing information about structural as well as dynamic changes during binding
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remains difficult, however, to apply similar approaches to large, complex antigens, let alone to
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study their association with antibodies.
10-12
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. Directly testing the
. Fluorescence methods have
15-18
. It
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In the case of the majority of broadly neutralizing antibodies targeting the HIV envelope
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glycoprotein (Env) the target epitopes are often formed by discontinuous peptide segments,
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loops, as well as glycan chains
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degree of dynamics or flexibility in the absence of a stabilizing ligand. Indeed, in Env,
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conformational switching and structural flexibility appear to be key mechanisms the virus uses in
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order to evade immune recognition, particularly to hide conserved epitopes such as the
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chemokine co-receptor binding sites from detection. Prior to the primary CD4 receptor binding
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event, the structural elements that will form the conserved coreceptor binding site are
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sequestered and maintained in an organization distinct from that observed in the CD4-bound
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state. CD4 binding induces major structural rearrangements in Env leading to formation of the
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bridging sheet and exposure of V3 that together are needed to engage the coreceptor
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Even at equilibrium, prior to receptor binding, recent studies have demonstrated that Env can
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sample a range of conformational states 24, 25. The frequency with which these states are sampled
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varies depending on specific viral isolate, with more neutralization resistant isolates remaining in
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the “closed” prefusion state in which the coreceptor binding site is masked to a greater extent,
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while neutralization sensitive variants frequently sample more “open” states, transiently
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exposing the binding site as well as additional epitopes throughout Env.
19, 20
. These types of epitopes in some cases may exhibit a high
10, 21-23
.
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The Env gp120 receptor binding subunit provides a highly suitable construct for
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investigating links between epitope structural dynamics and antibody recognition. Gp120 bears a
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number of epitopes that are targets for neutralizing antibodies against HIV and a construct that
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has been intensively investigated as a component in HIV vaccines
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from past thermodynamic measurements that the intrinsic conformational flexibility of the gp120
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subunit can impact antibody recognition. Calorimetry experiments revealed that unliganded
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26-29
. It has been suggested
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gp120 subunits exhibit a high degree of conformational flexibility 11. This led to the hypothesis
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that epitope disorder and conformational dynamics in gp120 may hinder antibody binding and
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serve as a mechanism of immune evasion 10, 30, 31. Subunit flexibility becomes constrained when
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CD4 binds and orders the conserved subunit core
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gp120 variable loop truncation can modulate the antigenicity and structure of the gp120 core to
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the extent that the extended core lacking variable loops (Coree) crystallized in a conformation
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nearly identical to the CD4-bound state 33. Kwon and colleagues reported that the loop truncation
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mutants displayed stronger binding affinity to the antibodies recognizing receptor-bound state
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than the full-length gp120. While the previous study used antibody binding to covalently
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crosslinked gp120 constructs to assess the structure of the panel of truncation mutants, strong
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antibody-antigen interactions and crystallization can themselves impose significant influences on
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antigen structure, rendering the approaches rather invasive probes of structure.
11, 31, 32
. In a recent study, it was shown that
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While a number of studies have examined the effect of overall conformational stability of
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HIV Env on antibody binding 34, 35, to date, it has been difficult to directly demonstrated the link
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between local structural dynamics and the affinity and specificity of antibody binding to those
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local structural features
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antigen and used a combination of solution-phase biopysical methods to investigate the
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underlying the relationship between local epitope structural order, conformational dynamics and
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antigenicity under native solution conditions. Hydrogen/deuterium-exchange mass spectrometry
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(HDX-MS) was used to probe the local structural dynamics in the panel of variable loop
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truncation mutants of gp120, revealing significant changes in structural dynamics of epitopes
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resulting from variable loop truncation. We show that antibody recognition of conserved
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conformational epitopes, such as the CD4 and coreceptor binding sites, correlates inversely with
25, 30
. Here we selected HIV-1 gp120 as a prime example of a complex
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the degree of local dynamics. The findings suggest that it may be beneficial to optimize the
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dynamic profile of antigens in order to enhance the presentation of complex epitopes and
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improve their immune recognition.
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MATERIALS AND METHODS
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Reagents
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The expression vectors for HIV-1 clade BYU2 gp120 glycoproteins, including FL,
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∆V1/V2, ∆V3 and Coree were kindly supplied by Dr. Peter D. Kwong 33. Antibodies 17b
148
48d
149
AIDS Reagents Program. The sCD4 and CD4-IgG2 was obtained from NIH AIDS Reagents
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Program
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George Lewis (University of Maryland) 42, 43.
36
, A32
41
37
, VRC01 38, NIH45-46 1, VRC03
38
, F105
39
, b12
40
21, 36
,
were obtained from the NIH
. The antibodies N5i5 and B18 were generously provided by Yongjun Guan and
152 153
High density transfection and expression of YU2 gp120 envelope glycoproteins
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293F cells were grown in HyClone™ SFM4Transfx-293 medium (Fisher Scientific). The
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various HIV-1 Clade BYU2 gp120 glycoproteins were expressed in the Human Embryonic
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Kidney cell line HEK293F by high density (HD) transient transfection the same as previous
157
description 44. Briefly, 293F cell were re-suspended at 2×107 cells/ml in fresh SFM4Transfx-293
158
medium. The sterile filtered plasmid DNA (20 ug/ml) were added into the cells directly,
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immediately followed by PEI MAX (40 ug/ml, Polysciences, Inc.) (DNA:PEI MAX=1:2). After
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gentle mixing, the cells were incubated at 37 °C for 4 h. The culture volume was expended 20-
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fold by adding fresh medium containing 20 mM Hepes, pH 7.3 (Life Techonogies) and
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1×GlutaMAX (Life Technologies). 72 hours after transfection, the supernatant was collected and
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filtered by a 0.45 µm filter (Fisher Scientific). A cocktail of protease inhibitors (Roche
164
Diagnostics) was added. The supernatant was concentrated 20-fold by passing through KvickLab
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Ultrafiltration flat sheet cassette (30 kD, GE Healthcare) using Masterflex L/S Easy-Load II
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system (Cole-parmer). Then the concentrated supernatant was diluted with Galanthus nivalis
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lectin affinity (GNA) binding buffer (20 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1
168
mM EGTA) to 30 times. Finally the sample was concentrated to 50 ml before loading on GNA
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affinity column.
170 171
Purification of HIV envelope glycoproteins
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The initial purification was conducted by using affinity technique with Galanthus nivalis
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lectin (GNA) (Sigma). Subsequent purification of HIV envelope glycoproteins was by gel
174
filtration using a HiLoad™ 16/60 Superdex 200 column (GE Healthcare) equilibrated with
175
phosphate-buffered saline (1×PBS, pH 7.4, 1 mM EDTA, 0.02% NaN3). Fractions (500 µl) were
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collected and analyzed by using SDS-PAGE and Blue-native PAGE (BN-PAGE). Quantification
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of proteins was carried out by checking the absorbance at 280 nm. The purified HIV envelope
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glycoproteins were pooled and stored in appropriate buffer for further experiments (Fig. S1).
179 180
SDS-PAGE and blue native PAGE (BN-PAGE)
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For SDS-PAGE, the gp120 envelope glycoprotein samples were loaded on a NuPAGE®
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Novex 4–12% Bis-Tris Gel (Invitrogen) and run at 180 V for 50 min. The gel was fixed with
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50% methanol, 7% acetic acid for 10 min, stained with SYPRO Ruby protein gel stain
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(Invitrogen), and washed 10% methanol, 7% acetic acid. Benchmark protein ladder (Invitrogen)
185
was used for SDS-PAGE. For blue native PAGE (BN-PAGE), the protein samples were loaded
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on a 3–12% Bis-Tris Native PAGE gel (Invitrogen). The gel was run at 150 V for 1 h and 250 V
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for 2 h, fixed for 15 min with 50% methanol, 10% acetic acid and destained using dH2O. Native
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Mark protein standard (Invitrogen) was used for BN-PAGE.
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Light scattering measurements
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Dynamic and static diameters of gp120 envelope glycoproteins were measured using a
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Dynapro Nanostar (Wyatt Technology, Santa Barbara, CA) at 20 °C. A total of 30 acquisitions
193
of 5 s were collected for each sample. The data were analyzed by using dynamics analysis
194
software (Wyatt Technology), assuming a spherical model. Theoretical expected molecular mass
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for each gp120 envelope glycoprotein was calculated as the sum of the molecular mass of the
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polypeptide and the mass of the glycans (Man8, 1.7 kDa, for the high-mannose type and
197
fucosylated monosialylated biantennary, 2.1 kDa, for the complex type).
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Hydrogen-deuterium exchange mass spectrometry
200
As previously described 31, hydrogen/deuterium (H/D) exchange was initiated by mixing
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100 µg/ml gp120 envelope glycoprotein in phosphate buffer (10 mM phosphate, 144 mM NaCl,
202
pH 7.4) with 85% D2O (Cambridge Isotope Labs). The solution was maintained at room
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temperature for 3 s, 1 min, 30 min, 20 h and 4 h for the H/D exchange to occur. At each time
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point, the solution was quenched by 1:1 dilution into 1 M Tris (2-carboxyethyl) phosphine
205
hydrochloride (TCEP) in 0.02% formic acid with a final pH of 2.5. The quenched sample was
206
immediately digested with pepsin (Worthington Labs) (2:1 by mass) on ice for 5 min. The
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samples were frozen in liquid nitrogen and stored at -80 °C for future data collection. To correct
208
for H/D exchange during pepsin digestion, a zero time point sample was prepared by mixing the
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gp120 with prequenched PBS plus D2O. To correct for back exchange, a fully deuterated sample
210
was prepared by mixing denatured gp120 in 50 mM Tris-HCl, pH 8.0, 4 M GndHCl, 50 mM
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dithiothreitol with 85% D2O at 40 °C for 4 h. An undertreated sample was also prepared. The
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FL_sCD4 complex was prepared by mixing FL with a 3-fold molar excess of sCD4 at room
213
temperature for 1 h. The final concentration for FL in FL_sCD4 was 100 g/ml. The FL_sCD4
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complex was deuterated under the same conditions as those for the unliganded gp120 samples.
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All spectra were obtained by liquid chromatography-MS (LC-MS) on a Synapt HDMS
216
system (Waters, Milford, MA) integrated with a Waters Acquity ultraperformance LC (UPLC).
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The frozen sample were thawed on ice for 5 min and directly injected into a BEH C18 trap
218
column (1.7 µm; 2.1×5 mm, Waters) in 0.1% trifluoroacetic acid (TFA) at 100 µl/min. After 3
219
min of washing, the peptides were resolved on a Hypersil C18 column (1.9 µm; 50×1 mm;
220
Thermo Scientific) with a linear gradient of 5% to 100% solvent B for 15 min (solvent A, 5%
221
acetonitrile [ACN], 0.05% TFA; solvent B, 80% ACN, 0.05% TFA). Between injections, the
222
syringe, loop and trap column were washed by 10% formic acid, 80% methanol, 2:1 isopropanol-
223
ACN, and 80% ACN sequentially.
224 225
Data analysis
226 227
Mass shifts were analyzed by using HX Express
45
, and the percent deuteration was
calculated by equation 1 46:
228
%D =100 ×
229
(where
is the peptide mass centroid at a given time point,
230
mass centroid, and
is the zero time point peptide
is the fully deuterated peptide mass centroid).
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No obvious differences were observed between different glycoforms of the same peptide.
232
The centroids for the glycoforms of the identical peptide were then averaged. Binomial
233
distribution fitting was applied for overlapped and noisy H/D exchange data 45, 47.
234 235 236
Small angle X-ray scattering (SAXS) SAXS measurements were conducted on Beam Line 4-2 at the Stanford Synchrotron 48
. The data collection procedure was as described before
30, 31
237
Radiation Laboratory
238
each sample (3-5 mg/ml, 100 µl) was injected into a Sepharose 200 column (GE Healthcare) pre-
239
equilibrated with PBSE (1×PBS, 1 mM EDTA, 0.02% NaN3, pH 7.4) at a flow rate of 50 µl/min.
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The eluted sample was captured in a thin-wall quartz capillary cell and irradiated by focused 11-
241
keV X-ray. The signals of one-second exposures were recorded by an MX 225HE detector
242
(Rayonix, Evanston, IL) placed 1.7 m upstream of the quartz cell. The detector pixel numbers
243
were converted into the momentum transfer (
244
angle and
245
capillary position. Protein scattering data were processed by using MarParse, scaled for the
246
transmitted beam intensity integrated for each exposure, and azimuthally averaged
247
scattering data was averaged from the first 100 data points (before the void volume) and
248
subtracted from the corresponding protein scattering data. ATSAS software suite was used to
249
analyze SAXS parameters and patterns
250
determined from the Guinier plot (q x Rg < 1.3)
251
estimate Dmax for the P(r) plot, the pairwise distance distribution histogram 53. The final P(r) plot
252
was generated by GNOM v4.5
253
generated P(r) plot by using DAMMIN v5.3i
where 2
. Briefly,
is the scattering
is the X-ray wavelength of 1.127 Å, calibrated with silver behenate powder at the
50
49, 50
48
. Buffer
. The approximate radius of gyration (Rg) was 51
in Primus
52
. AutoGNOM was used to
. Ab initio shape reconstruction was conducted with the 54
. The bead models were aligned using
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and averaged using DAMAVER
56
254
SUPCOMB13 with enantiomers considered
. Sites of low
255
bead occupancy in the averaged model were filtered using DAMFILT 57. The final models were
256
converted into a volume envelope using pdb2vol in the SITUS2.2 package 58. Molecular weights
257
were calculated program SAXS MoW 59.
258 259
Enzyme-linked Immunosorbent Assays (ELISAs)
260
For ELISAs, the gp120 envelope glycoproteins (50 ng/well) in phosphate-buffered saline
261
(PBS) were immobilized onto an Immulon 2HB plate (Thermo Scientific) at 4 °C overnight. On
262
the following day, the plates were blocked by PBS with 2% bovine serum albumin (BSA) and
263
0.01% Tween-20 for 2 h at room temperature and washed with Tris-buffered saline (TBS) with
264
0.02% Tween-20 subsequently. The primary antibodies with a starting concentration of 10 µg/ml
265
for human antibodies or 5 µg/ml for mouse antibodies were added and then diluted into 4-fold
266
dilution series by antibody dilution buffer (PBS with 1% BSA and 0.01% Tween 20), and
267
incubated for 1 h at room temperature. After several washes, the horseradish peroxidase (HRP)-
268
conjugated goat anti-human or goat anti-mouse secondary antibodies (Jackson ImmunoResearch)
269
(1:5000 diluted) were added into the wells for 1 h at room temperature. After a final set of
270
washes, the plates were developed with SureBlue Reserve trimethylbenzidine (KPL, Inc.) and
271
stopped with 1 M HCl. The absorbance at 450 nm was measured using a Tecan Infinite M200
272
plate reader.
273 274
Kinetic assay by biolayer interferometry (BLI)
275
A FortéBio (Menlo Park, CA) Octet RED biolayer interferometry system was used to
276
measure the binding of each gp120 (FL, ∆V3, ∆V1/V2, Coree) to different antibodies. Anti-
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Human IgG Fc Capture (AHC) biosensors were hydrated for at least 10 minutes prior to antibody
278
loading. Human anti-HIV antibody (10 µg/ml) in HBS-EP+ buffer (10 mM HEPES, 150 mM
279
NaCl, 3 mM EDTA, and 0.05% P-20 at pH 7.4) were then bound to the AHC tips for 4 minutes
280
and washed in buffer for 1 minutes to remove any unbound antibody and reach a stable baseline.
281
The antibody-AHC tips were then dipped into gp120 samples, which were prepared
282
independently with a 2-fold dilution series. After association for several minutes, the tips were
283
then placed back into the baseline buffer to allow dissociation to occur. The time ranges for
284
loading antibody, association and dissociation were optimized according to different antibodies.
285
FortéBio’s Data Analysis 7.0 was used to analyze changes in refractive index. The averaged
286
reference measurements were subtracted prior to data processing. The association and
287
dissociation curves were fit using 1:1 binding model. The final association rate (ka) and
288
dissociation rate (kd) rate constants were used to calculate the ensemble dissociation constants
289
(KD = kd/ka). Standard deviations were calculated from the results of two independent
290
measurements.
291 292
RESULTS
293
Deletion of variable loops does not alter the large-scale morphology of the gp120 core
294
Small-angle X-ray scattering (SAXS) was used to study the overall structural features of
295
unliganded full-length YU2 gp120 monomer (FL) and its truncated variants (∆V3, ∆V1/V2,
296
Coree). Size exclusion chromatography in-line with the sample cell was used to gather SAXS
297
data from monodisperse gp120 constructs (Fig. 1). The Guinier regions showed good linearity
298
for all data sets indicating all the specimens were well-behaved in solution. Radii of gyration
299
(Rg) obtained from Guinier analysis agreed well with those derived from P(r) pairwise distance
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distribution plots 50. Differences in SAXS measurements were consistent with values obtained by
301
static light scattering and with the size of the different YU2 gp120 proteins expected based on
302
their composition (FL>∆V3>∆V1/V2> Coree) (Table 1).
303
To determine whether the variable loop truncations led to large-scale conformational
304
changes, ab initio shape reconstruction was applied to SAXS data for the gp120 constructs (Fig.
305
1). As shown in Fig. 1A, the FL showed the same features of a ventral stalk, central core, and a
306
crest as observed in previous studies
307
including ∆V3, ∆V1/V2 and Coree lost density in the crest region (Fig. 1B-D). Overall, the raw
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SAXS data and the reconstructed models confirmed the reduced size due to truncations of
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different variable loops. However, no obvious conformational changes of the core domain were
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detected from the low-resolution SAXS measurements.
30, 31
. SAXS models of the gp120 truncation mutants
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H/D-exchange mass spectrometry profiles of structural dynamics in gp120 constructs
313
In order to apply a more detailed approach to detect local structural changes resulting
314
from variable loop truncations, HDX-MS was used to examine structural ordering in FL, ∆V3,
315
∆V1/V2, Coree, and FL with sCD4 bound (FL+sCD4). Deuterium exchange could be monitored
316
for peptides covering 73% of the YU2 full length sequence (Fig. S2). The loss of coverage for
317
certain segments, including most of V1/V2, part of V3, part of the CD4 binding loop and a
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portion of V4, is most likely due to the high level of heterogeneous glycosylation in these
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regions. Here we primarily compared deuterium uptake of identical peptides in the conserved
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core domain that are shared by the truncation mutants.
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In order to establish a baseline for the changes in dynamics we might expect to see in the
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set of gp120 constructs, we first compared FL in unliganded and sCD4-bound forms. Consistent
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30, 31
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with previous studies of full-length gp120 monomers
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dramatic decrease in deuterium uptake in the gp120 subunit, reflecting increased structural order
325
in the three layers of the inner domain, bridging sheets and CD4 binding site (Fig. 1F, 3, S3-5).
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Interestingly, a small V2 loop peptide (residues 173-176, sequence YALF) showed some
327
exchange protection in unliganded gp120 but rapid deuterium exchange in the earliest time point
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in CD4-bound gp120 (Fig. 3A, 3J). This hints that a portion of the V2 loop may transition from
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being partially ordered in unliganded gp120 to a disordered state upon CD4 binding echoing
330
changes observed in trimeric forms of Env
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prevented additional portions of the loops from being monitored by HDX-MS.
25
, CD4 binding to FL induces a
. The high degree of glycosylation of V1/V2
332 333
Deletion of variable loops progressively stabilizes unliganded gp120 constructs
334
Truncation of the V3 loop led to detectable increases in ordering at sites distal to the loop
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region itself (Fig. 2, S3-5). Modest ordering was observed in select regions of the inner domain,
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including in layer 2 (residues 101-111) and layer 3 (residues 467-479 and residues 480-483)
337
(Fig. 3C, 3H-I). A peptide covering layer 1 (residues 66-83) showed exchange protection at the 3
338
second time point (Fig. 3B). The β2 peptide (residue 112-127), which forms part of the bridging
339
sheet remained highly dynamic with only a slight measurable increase in protection from solvent
340
exchange in ∆V3 gp120 at 3 seconds (Fig. 3D). β20 and β21 peptides (residues 417-426 and
341
residues 427-433) by contrast showed significant protection in ∆V3 gp120, indicating that loop
342
truncation led to partial ordering of the bridging sheet (Fig. 3F-G). The CD4 binding loop also
343
increased in order compared to that in unliganded FL (Fig. 3E).
344
The ∆V1/V2 truncation mutant, like the V3 truncation mutant, exhibited an increase in
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structural order of the conserved core (Fig. 2, S3-6). The inner domain in ∆V1/V2, including
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layers 1, 2 and 3, showed measurably greater exchange protection compared to the ∆V3 variant
347
(Fig. 2-3, S6C). The same trend was observed in the β20 and β21 peptides of the bridging sheet
348
subdomain in the ∆V1/V2 variant (Fig. 3F-G, S6C). By contrast, in ∆V1/V2, the CD4 binding
349
loop exhibited local dynamics similar to that in unliganded full-length gp120, and did not exhibit
350
the same protection as in ∆V3 (Fig. 3E, S6C).
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The gp120 Coree construct with V1/V2 and V3 loops truncated showed a high degree of
352
protection from solvent exchange and exhibited an H/D exchange profile more similar to that of
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sCD4-bound than unliganded full-length gp120 (Fig. 2, S3-5). All of the available inner domain
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peptides (Fig. 3B-C, 3H-I), bridging sheet peptides (Fig. 3D, 3F-G) and the CD4 binding loop
355
peptide (Fig. 3E) showed increases in protection from deuterium uptake, indicating that deletion
356
of V1/V2 and V3 loops led to substantially increased structural ordering of the inner domain,
357
formation of the bridging sheet and stabilization of the CD4 binding site. These findings mirror
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the observation that unliganded Coree adopts a structure nearly identical to the sCD4-bound core
359
when crystallized 33. Though we note that while some structural elements such as layer 1 of the
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core exhibited the same protection from solvent exchange as that of FL+sCD4, overall the
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unliganded core retained a greater level of deuterium exchange and conformational flexibility in
362
solution than sCD4-bound gp120 (Fig. 2-3, S3-5). Thus, it is likely that crystallization helped to
363
lock in the receptor-bound conformation of the core, while in solution it is more
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conformationally dynamic than suggested by the crystal structure.
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Taken together these data suggest that the greater the extent of variable loops V1/V2 and
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V3 truncation, the greater the ordering of the gp120 core domain elements toward a
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conformation and ordered profile resembling the receptor-bound gp120. However, only sCD4
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binding induces the full extent of structural ordering consistent with the reported crystal
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structures.
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Deletion of variable loops stabilizes CD4-induced epitopes and improves recognition by
372
CD4-induced antibodies
373
To determine whether the differences in the structural ordering of FL, ∆V3, ∆V1/V2 and
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Coree detected by HDX-MS correlated with changes in antigenicity of the glycoproteins, we
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used ELISAs to assess antibody binding to gp120s and Fortébio Octet biolayer interferometry
376
(BLI) to measure the kinetics of antibody-gp120 binding.
377
Bridging sheet-specific antibodies 17b and 48d and inner-domain-specific antibodies A32 36, 42, 60-62
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and N5i5, recognize CD4-induced (CD4i) epitopes
379
MS analysis, the bridging sheet (recognized by 17b and 48d) and gp120 inner domain
380
(recognized by A32 and N5i5) become more ordered with more extensive variable loop
381
truncation (Fig. 4B-F), hence we anticipated these antibodies might exhibit enhanced binding to
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the truncation mutants. First, the monoclonal antibody B18 that recognizes the linear epitope
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VEQMHEDIIS in helix 1, shared by all the gp120 constructs, was used as a control in ELISA
384
experiments
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negligible by ELISA (Fig. 5A). By ELISA, the gp120 FL displayed the weakest binding to 17b
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among the four gp120s and Coree showed the strongest binding. In ELISA, binding of 17b was
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rank ordered FL
