Redox exchange of the disulfides of human two-domain CD4 regulates the conformational dynamics of each domain, providing insight into its mechanisms of control.

CD4, a membrane glycoprotein expressed by specific leukocytes, plays a vital role in the human immune response and acts as a primary receptor for HIV entry. Of its four ecto-domains (D1-D4), D1, D2, and D4 each contain a distinctive disulfide bond. Whereas the disulfides of D1 and D4 are more traditional in nature, providing structural functions, that of D2 is referred to as an "allosteric" disulfide due to its high dihedral strain energy and relative ease of reduction that is thought to regulate CD4 structure and function by shuffling its redox state. While we have shown previously that elimination of the pre-stressed D2 disulfide results in a favorable structural collapse that increases the stability of a CD4 variant comprising only D1 and D2 (2dCD4), we sought to further localize and determine the nature of the biophysical modifications that take place upon redox exchange of the D1 and D2 disulfides by using amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to measure induced changes in conformational dynamics. By analyzing various redox isomers of 2dCD4, we demonstrate that ablation of the D1 disulfide enhances the dynamics of the domain considerably, with little effect on that of D2. Reduction of the D2 disulfide however decreases the conformational dynamics of many of the ß-strands of the domain that enclose the bond, suggesting a model in which inward collapse of secondary structure occurs around the allosteric disulfide upon its eradication, resulting in a marked decrease in hydrodynamic volume and increase in stability as previously described. Increases in the dynamics of regions important for HIV gp120 and MHCII binding in D1 also result allosterically after reducing the D2 disulfide, which are likely a consequence of the structural changes that take place in D2, findings that advance our understanding of the mechanisms by which redox exchange of the CD4 disulfides regulates its function.

Antigp41 membrane proximal external region antibodies and the art of using the membrane for neutralization.

PURPOSE OF REVIEW: We summarize the latest research on the progress to understand the neutralizing epitopes present within the membrane proximal external region (MPER) of the HIV-1 fusion protein subunit gp41. RECENT FINDINGS: The HIV-1 fusion protein subunit gp41 contains a highly conserved sequence that is essential for membrane fusion and targeted by broadly neutralizing antibodies such as 2F5, 4E10, Z13e1, and 10E8. These antibodies recognize a linear gp41 epitope with high affinity, but require additional hydrophobic sequences present in their heavy chain CDR3 for neutralization. Recent structural studies on mAbs 4E10 and 10E8 provide molecular details for specific interactions with lipids and implicate part of the transmembrane region as the relevant 10E8 epitope. Although many different approaches have been applied to engineer gp41 immunogens that can induce broadly neutralizing antibodies directed toward MPER, only modest success has yet been reported. SUMMARY: The new structural details on the complex gp41-lipidic epitope will spur new approaches to design gp41-MPER immunogens that might induce broadly neutralizing antibody responses.

Human CD4 Metastability Is a Function of the Allosteric Disulfide Bond in Domain 2.

CD4 is expressed on the surface of specific leukocytes where it plays a key role in the activation of immunostimulatory T-cells and acts as a primary receptor for HIV-1 entry. CD4 has four ecto-domains (D1-D4) of which D1, D2, and D4 contain disulfide bonds. Although disulfide bonds commonly serve structural or catalytic functions, a rare class of disulfide bonds possessing unusually high dihedral strain energy and a relative ease of reduction can impact protein function by shuffling their redox state. D2 of CD4 possesses one such "allosteric" disulfide. While it is becoming accepted that redox exchange of the D2 allosteric disulfide plays an essential role in regulating CD4 activity, the biophysical consequences of its reduction remain incompletely understood. By analyzing the hydrodynamic volume, secondary structure, and thermal stability of the reduced and nonreduced forms of the single D1 and D2 domains, as well as the various redox isomers of two domain CD4, we have shown that ablation of the allosteric disulfide bond in domain 2 results in both a favorable structural collapse and an increase in the stability of CD4. Conversely, ablating the structural disulfide of D1 results in destabilizing structural rearrangements in CD4. These findings expand our understanding of the mechanisms by which oxidoreduction of the D2 allosteric disulfide regulates CD4 function; they reveal the intrinsic disulfide-dependent metastability of D2 and illustrate that redox shuffling of the allosteric disulfide results in previously undescribed conformational changes in CD4 that are likely important for its interaction with its protein partners.

Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.

Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization.

Human CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes, where it plays a key role in the activation of immunostimulatory T cells and acts as the primary receptor for human immunodeficiency virus (HIV) glycoprotein (gp120). Although growing evidence suggests that redox exchange reactions involving CD4 disulfides, potentially catalyzed by cell surface-secreted oxidoreductases such as thioredoxin (Trx) and protein disulfide isomerase, play an essential role in regulating the activity of CD4, their mechanism(s) and biological utility remain incompletely understood. To gain more insights in this regard, we generated a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, and used these to show that while protein disulfide isomerase has little capacity for 2dCD4 reduction, Trx reduces 2dCD4 highly efficiently, catalyzing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulfide-linked 2dCD4 dimer. Moreover, we show that HIV gp120 is incapable of binding a fully oxidized, monomeric 2dCD4 in which both domain 1 and 2 disulfides are intact, but binds robustly to reduced counterparts that are the ostensible products of Trx-mediated isomerization. Finally, we demonstrate that Trx-driven dimerization of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is impaired when CD4 is bound to gp120. These observations reinforce the importance of cell surface redox activity for HIV entry and posit the intriguing possibility that one of the many pathogenic effects of HIV may be related to gp120-mediated inhibition of oxidoreductive CD4 isomerization.

Stabilization of HIV-1 gp120-CD4 receptor complex through targeted interchain disulfide exchange.

HIV-1 enters cells via interaction between the trimeric envelope (Env) glycoprotein gp120/gp41 and the host cell surface receptor molecule CD4. The requirement of CD4 for viral entry has rationalized the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. In this study, we describe a novel recombinant CD4 protein designed to bind gp120 through a targeted disulfide-exchange mechanism. According to structural models of the gp120-CD4 receptor complex, substitution of Ser(60) on the CD4 domain 1 alpha-helix with Cys positions a thiol in proximity of the gp120 V1/V2 loop disulfide (Cys(126)-Cys(196)), satisfying the stereochemical and geometric conditions for redox exchange between CD4 Cys(60) and gp120 Cys(126), and the consequent formation of an interchain disulfide bond. In this study, we provide experimental evidence for this effect by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). We show that 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulfide exchange and that this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. We propose that targeted redox exchange between conserved gp120 disulfides and nucleophilic moieties positioned strategically on CD4 (or CD4-like scaffolds) conceptualizes a new strategy in the development of high affinity HIV-1 Env ligands, with important implications for therapy and vaccine development. More generally, this chalcogen substitution approach provides a general means of stabilizing receptor-ligand complexes where the structural and biophysical conditions for disulfide exchange are satisfied.