Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

Wu, Hao; Myszka, David G.; Tendian, Susan W.; Brouillette, Christie G.; Sweet, Ray; Chaiken, Irwin M.; Hendrickson, Wayne A.

doi:10.1073/pnas.93.26.15030

Cited by 60 publications

(53 citation statements)

References 41 publications

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“…Analysis of the resulting binding curves ( Fig. 2A) returned an affinity of 8 nM, in very good agreement with previously reported data using the same technique (38,39). To check whether our protein undergoes conformational changes upon CD4 binding, we monitored the binding of gp120 to mAb 17b, both in the presence and in the absence of sCD4.…”

Section: Time-course Expression and Purification Of Recombinant Gp120supporting

confidence: 88%

The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site

Crublet¹,

Andrieu

Vivès³

et al. 2008

Journal of Biological Chemistry

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It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cellassociated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg 419 , Lys 421 , and Lys 432 , which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition. Human immunodeficiency virus (HIV)4 gains entry into permissive CD4 ϩ cells by sequentially interacting with CD4, the primary receptor, and a co-receptor, usually either CCR5 or CXCR4 (1). Both receptor and co-receptors are recognized by gp120, the glycoprotein that constitutes the surface unit of HIV envelope spikes. This protein consists of five relatively conserved regions (C1 to C5) that fold into a "core" comprising two distinct domains termed "inner" and "outer" and five variable regions (V1 to V5). This protein is prone to structural changes and is believed to sample a number of different conformations. A wealth of evidence showed that upon binding to CD4, roughly half of the gp120 core structure undergoes structural rearrangements, in particular within the inner domain. In the CD4-bound form, the base of the V1/V2 region of the inner domain (␤2 and ␤3 strands) is brought to close proximity to a ␤-hairpin of the outer domain (␤20 and ␤21 strands) and forms a four-stranded ␤-sheet located within the bridging sheet that connects the inner and the outer domain of the glycoprotein. Importantly, this highly conserved structure forms, in conjunction with the V3 loop, the binding site for CCR5 or CXCR4 (2-7).It has been well known that HIV also binds to CD4 Ϫ cells in a gp120-dependent manner through interactions with cell surface molecules including heparan sulfates (HSs) (8). HSs and the closely related heparin belong to a large family of anionic polysaccharides, collectively known as glycosaminoglycans. They occur covalently bou...

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Section: Time-course Expression and Purification Of Recombinant Gp120supporting

confidence: 88%

The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site

Crublet¹,

Andrieu

Vivès³

et al. 2008

Journal of Biological Chemistry

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“…Deviations of the CD4 structure in the complex from the free state were measured as described 39 . Deviations were taken as significant when the root-mean-square (r.m.s.)…”

Section: Structure Analysismentioning

confidence: 99%

“…If so, to what state and, in reverse, how does CD4 binding lead to the state seen in this ternary complex? CD4-gp120 binding kinetics are complex 39 , and microcalorimetry reveals unusually large ΔH and compensating TΔS values for soluble CD4 binding to gp120 (M. L. Doyle, personal communication). These exceptional CD4-binding thermodynamics are suggestive of a large conformational change and are similar for both full-length and core gp120, supporting the relevance of our structural observations on core gp120.…”

Section: Conformational Change In Core Gp120mentioning

confidence: 99%

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Kwong

Wyatt

Robinson

et al. 1998

Nature

2,742

108

3,142

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The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.The human immunodeficiency viruses HIV-1 and HIV-2 and the related simian immunodeficiency viruses (SIV) cause the destruction of CD4 + lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndromeCorrespondence and requests for materials should be addressed to W. A.H. (wayne@convex.hhmi.columbia.edu).Coordinates have been deposited in the Brookhaven Protein Data Bank (accession code 1gc1) and maybe obtained from the authors. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (AIDS) 1,2 . The entry of HIV into host cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric spikes displayed on the surface of the virion. These envelope complexes are anchored in the viral membrane by the gp41 transmembrane envelope glycoprotein. The surface of the spike is composed primarily of the exterior envelope glycoprotein, gp120, associated by non-covalent interactions with each subunit of the trimeric gp41 glycoprotein complex 3,4 . Comparison of the gp120 sequences of different primate immunodeficiency viruses identified five variable regions (V1-V5) (ref. 5 ). The first four variable regions form surface-exposed loops that contain disulphide bonds at their bases 6 . The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gp120 regions are extensively glycosylated 6 . The variability and glycosylation of the gp120 surface probably modulate the immunogenicity and antigenicity of the gp120 glycoprotein, which is the main target for neutralizing antibodies elicited during natural infection 7 .Entry of primate immunodeficiency viruses into the host cell involves the binding of the gp120 envelope glycoprotein to the CD4 glycoprotein, which serves as the primary receptor. The gp120 glycoprotein binds to the most amino-terminal of the four immunoglobulin-like domains of CD4. S...

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“…Furthermore, sCD4 has been reported to bind to envelope proteins, including IIIB gp120, with K D values of 22 to 35 nM (12,59,88). Another study reported K D values in the range of 2.2 to 16 nM for a range of anti-CD4bs Fab fragments (including Fab b12) to MN gp120 (62).…”

Section: Fig 2 Vhh and Mab B12 Icmentioning

confidence: 99%

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Forsman

Beirnaert

Aasa-Chapman

et al. 2008

J Virol

112

138

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Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B /C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC 50 ) and IC 90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

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Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

Cited by 60 publications

References 41 publications

The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site

The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

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