CD4 Human (125-202)

Advanced Research Questions

• How do conformational changes in CD4 facilitate HIV membrane fusion?

  Monoclonal antibody studies provide strong evidence for conformational changes in CD4 following binding of HIV envelope proteins:

  • A Fab clone isolated from an HIV-infected individual recognizes cell surface CD4 only after incubation with recombinant HIV gp120 or HIV virions, indicating a virus-induced conformational change .

  • The monoclonal antibody 5A8 binds to domain 2 of CD4 and blocks HIV infection and syncytium formation without impairing binding of HIV or gp120 to CD4, suggesting that domain 2 plays a role beyond initial binding .

  • Other monoclonal antibodies that recognize the CD4-gp120 complex block envelope-mediated cell-cell fusion without impairing binding, further supporting a gp120-induced conformational change in CD4 .

  • The reduction of the domain 2 disulfide appears to be part of this conformational change, potentially triggered by thioredoxin secreted by activated CD4+ cells .

  These conformational changes likely facilitate the complex process of membrane fusion by repositioning the HIV envelope complex relative to the target cell membrane after initial binding.

• What are the evolutionary implications of CD4 domain 2 structure?

  The evolutionary history of CD4 domain 2 structure has significant implications:

  • Primates and rodents are the only animals that possess the cross-strand disulfide in CD4 domain 2, and they appear to have acquired Cys130 independently .

  • Among 1275 C2-type immunoglobulin superfamily domains with two cysteines, only CD4 domain 2 and carcinoembryonic antigen-related cell adhesion molecules 20 have a potential cross-strand disulfide .

  • This potential disulfide bond is found in human but not rodent carcinoembryonic antigen-related proteins, which contain only one cysteine in the domain .

  • The independent acquisition of Cys130 in primate and rodent CD4 domain 2 suggests a strong selective advantage, supporting the functional significance of this unique structural feature .

  This evolutionary convergence highlights the biological importance of the domain 2 disulfide bond and suggests that it evolved to enhance immune function, potentially providing protection against pathogens.

• How can rational design of CD4 mimics target domain 2 for HIV inhibition?

  Rational design of CD4 mimics targeting domain 2 represents a promising approach for HIV inhibition:

  • Using structural information on a CD4-gp120-17b antibody complex, researchers have designed CD4M33, a 27-amino acid CD4 mimic that presents optimal interactions with gp120 .

  • CD4M33 binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity .

  • This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4 .

  • CD4M33 can unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein, mimicking a key functional property of CD4 .

  • The design approach focuses on presenting the critical interactions with gp120 while maintaining optimal binding geometry and stability .

  Strategies promoting CD4 dimerization could impair HIV infection while favoring co-receptor activity, offering another rational design approach based on domain 2 properties .

• How does CD4 expression on CD8+ T cells affect their antigen recognition capabilities?

  CD4 can be up-regulated on CD8+ T cells generating a CD4dimCD8bright phenotype with remarkable properties:

  • In response to cytomegalovirus (CMV) peptide (pp65) priming, CD4dimCD8bright cells recognized CMV pp65 tetramer approximately 19-fold higher than CD4-CD8+ T cells .

  • This indicates that CD4dimCD8bright T cells are capable of antigen-specific recognition to a far greater extent than their CD4-CD8+ counterparts .

  • These cells express both CXCR4 and CCR5 but show differential susceptibility to HIV infection; they are susceptible to T-tropic but not M-tropic HIV infection .

  • A soluble factor, believed to be β-chemokine, is responsible for the inhibition of M-tropic HIV infection in CD4dimCD8bright T cells .

  • CD4dimCD8bright T cells do not produce significant intracellular levels of IFNγ, IL-2, or IL-10 but express elevated levels of intracellular IL-4 compared to CD8+CD4- and CD4+ T cells .

  This enhanced antigen recognition capability suggests a potentially important role for these double-positive T cells in immune responses.

• What methodological challenges exist in studying conformational changes of CD4 domain 2?

  Studying conformational changes in CD4 domain 2 presents several methodological challenges:

  Challenge	Impact	Potential Solutions
  Transient nature of conformational states	Difficult to capture intermediate states	Time-resolved structural methods, computational simulations
  Multiple conformational equilibria	Complex data interpretation	Single-molecule techniques, conformation-specific probes
  Membrane environment effects	Altered behavior in vitro vs. in vivo	Native membrane mimetics, in-cell studies
  Redox sensitivity	Changes during sample preparation	Rapid fixation techniques, redox-controlled conditions
  Heterogeneity of cell surface CD4	Mixed populations in measurements	Flow cytometry sorting, single-cell analysis

  Overcoming these challenges requires combining multiple complementary techniques, including structural biology, biophysical characterization, and functional assays, ideally performed under conditions that preserve the native conformational distribution of CD4.

• How can domain 2-specific inhibitors be developed for therapeutic applications?

  Development of domain 2-specific inhibitors requires strategic approaches:

  • Structure-based design: Using crystal structures of CD4-gp120 complexes to identify critical interaction points within domain 2 that could be targeted .

  • Disulfide stabilization: Developing compounds that prevent reduction of the domain 2 disulfide bond, which would inhibit HIV preference for the reduced form .

  • Dimerization promotion: Creating molecules that enhance CD4 dimerization through domain 2, favoring the form with lower HIV entry efficiency .

  • Peptide mimics: Designing constrained peptides that mimic key regions of domain 2 involved in post-binding conformational changes, as demonstrated with CD4M33 .

  • Allosteric modulators: Identifying compounds that bind to domain 2 and allosterically prevent the conformational changes needed for HIV entry.

  • Redox modulation: Developing inhibitors of thioredoxin or other redox pathways potentially involved in domain 2 disulfide reduction during HIV entry .

  The development pathway should include in vitro screening, structural validation of binding modes, cell-based functional assays, and eventually animal models to assess pharmacokinetics and efficacy.