Combinatorial Optimization of a CD4-Mimetic Miniprotein and Cocrystal Structures with HIV-1 gp120 Envelope Glycoprotein

Stricher, François; Huang, Chien‐Yi; Descours, Anne; Duquesnoy, Sophie; Combes, Olivier; Decker, Julie M.; Kwon, Young Do; Lusso, Paolo; Shaw, George M.; Vita, Claudio; Kwong, Peter D.; Martin, Loı̈c

doi:10.1016/j.jmb.2008.06.069

Cited by 51 publications

(81 citation statements)

References 63 publications

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“…This low frequency in selecting gp120-specific DARPin binders prompted us to probe three alternative selection strategies to determine whether modified gp120 proteins with reduced structural flexibility or decreased camouflage by glycosylation proved to be better selection targets. In a first approach, we limited the structural flexibility of gp120 by ligation with the CD4-mimetic miniprotein CD4M47, which arrests gp120 in the CD4-bound conformation (30) (Fig. 1B, selection II).…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, DARPin 5m3_D12 was in part capable of bypassing the V1V2 shielding of tier 2 HIV isolates, albeit in a strain-dependent manner, enabling it to block the entry of these virus isolates. (30). Linear and cyclic V3 mimetic peptides from strains JR-FL and MN were synthesized as described previously (31) For the peptide enzyme-linked immunosorbent assay (ELISA), biotinylated peptides were used.…”

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confidence: 99%

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Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Mann

Friedrich

Krarup

et al. 2013

J Virol

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Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Mann

Friedrich

Krarup

et al. 2013

J Virol

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“…Furthermore, the antibodies used in the immunochemical characterization of gp140 (12,17) cover the most pertinent gp120 and gp41 epitopes, namely the CD4 binding site (CD4BS-IgGCD4 and sCD4), the coreceptor binding site (CrBS-17b and 447D), a neutralizing epitope on gp41 (2F5), and a glycan-dependent epitope (2G12), thus thoroughly covering the topology of gp140 so as to display its similarity to native Env trimers. The CD4 miniprotein (CD4m) used to elicit the liganded conformation displays nanomolar affinity to gp120 (19), and elicits a nearly identical gp120 conformation as does soluble CD4 (sCD4) (20). The small size of CD4m allowed the structural analysis in this work to focus on the quaternary changes in gp140 and circumvent possible steric aggregation induced by stoichiometric sCD4-gp140 binding.…”

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confidence: 99%

Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

Moscoso

Sun²,

Poon

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120-gp41 interactions, whereas a "flat open" concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N-and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.T he human immunodeficiency virus envelope proteins gp120 and gp41 associate in a trimeric arrangement (1-3) and mediate membrane fusion through conformational rearrangement and fusion peptide insertion into the host membrane. The gp120 tertiary conformational change observed via X-ray crystallography has not been definitively characterized in the context of a quaternary structure. Several observations have been gleaned from tomography studies, though with inconclusive and sometimes contradictory results. Quaternary structural features reported previously include trimer morphology, with some groups reporting a mushroom-like shape and others a more rod-like arrangement, as well as the relative location of the primary epitope, hypervariable loops, and N-and C-termini, which have some variation among the different tomograms. Another point of conflict is the presence of a cavity at the threefold axis, capped by a density. One final feature is the arrangement of gp41, with some groups reporting a thin, stalk-like rod arrangement of gp41 and another group reporting a more splayed out, tripod-like conformation of gp41 proximal to the viral membrane. Taken together, these contrasting features portray a heterogeneous set of registers that seem to hinder a cohesive and thorough model of ligand-induced quaternary arrangement...

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“…For example, the ß-hairpin motif in the toxin has been exploited to generate mimics of an epitope on the cellular receptor CD4 [22][23][24] , which binds to the HIV-1 glycoprotein gp120. HIV-1 viral entry is initiated by the binding of gp120 to CD4 on host cells.…”

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confidence: 99%

“…Crystallographic studies have shown that the key epitope on CD4, used for binding to gp120, is based largely on a surface ß-hairpin loop [25] . Transplanting this hairpin epitope from CD4 onto the scorpion toxin scaffold afforded, after optimization, mimetics that bind tightly to gp120 and inhibit HIV-1 entry to cells (see Figure) [22][23][24] .…”

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confidence: 99%

Design of Protein–Protein Interaction Inhibitors Based on Protein Epitope Mimetics

Robinson

2009

ChemBioChem

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Combinatorial Optimization of a CD4-Mimetic Miniprotein and Cocrystal Structures with HIV-1 gp120 Envelope Glycoprotein

Cited by 51 publications

References 63 publications

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

Quaternary structures of HIV Env immunogen exhibit conformational vicissitudes and interface diminution elicited by ligand binding

Design of Protein–Protein Interaction Inhibitors Based on Protein Epitope Mimetics

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