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Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding
Published on: September 16, 2010
Mapping the CD4 binding site for human immunodeficiency virus by alanine-scanning mutagenesis
A Ashkenazi1, L G Presta, S A Marsters
1Department of Molecular Biology, Genentech, South San Francisco, CA 94080.
Summary
Researchers identified key areas outside the CDR2-like sequence in CD4's V1 domain crucial for human immunodeficiency virus (HIV) gp120 binding. This finding expands understanding of the HIV binding site and suggests strategies for improved HIV-blocking molecules.
Area of Science:
- Virology
- Immunology
- Structural Biology
Background:
- Human immunodeficiency virus (HIV) infects mononuclear cells by binding its gp120 envelope glycoprotein to the CD4 receptor.
- The first extracellular domain of CD4 (V1) is sufficient for HIV binding and contains sequences similar to immunoglobulin complementarity-determining regions (CDRs).
- Previous studies indicated only the CDR2-like region and flanking amino acids of CD4 V1 were involved in gp120 binding.
Purpose of the Study:
- To investigate regions within CD4 V1 beyond the CDR2-like sequence that participate in gp120 binding.
- To identify amino acids critical for CD4 V1 conformation.
- To explore potential for enhancing CD4-based HIV-blocking molecules.
Main Methods:
- Alanine scanning mutagenesis of 64 amino acids in the CD4 V1 domain, including hydrophilic residues.
- Assessment of gp120 binding affinity to mutated CD4 V1 variants.
- Probing V1 structure using conformationally sensitive monoclonal antibodies.
Main Results:
- Mutations at four specific locations outside the CDR2-like sequence (amino acids 29, 59-64, 77-81, and 85) significantly impacted gp120 binding without altering V1 structure.
- The gp120-binding site on CD4 V1 is composed of discontinuous segments, not solely the CDR2-like sequence.
- Three amino acid substitutions were identified that individually increased gp120 binding affinity 1.7- to 2-fold, and 4.2-fold when combined.
Conclusions:
- The gp120-binding site on CD4 V1 involves multiple discontinuous regions, extending beyond the previously identified CDR2-like sequence.
- Specific amino acids are critical for maintaining the conformation of CD4 V1, with implications for domain folding.
- Enhancing the gp120 binding affinity of CD4-based molecules through targeted amino acid substitutions is a viable strategy for improving HIV-blocking capabilities.
