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.

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.