Research

Designing peptide inhibitors of HIV entry

HIV membrane fusion (mediated by the HIV gp41/gp120 complex) has recently been identified as a promising target for inhibition. Fusion is initiated by contact with a CD4+ target cell, which triggers a conformational change in gp41. gp41 extends to lance the target cell before collapsing into a six-helix "trimer of hairpins" that pulls the viral and target membranes together, leading to fusion. During this conformational transition, gp41 forms a transient pre-hairpin intermediate composed of a trimeric coiled coil. We are developing peptide and protein inhibitors that bind to this intermediate and prevent the progression of HIV fusion and entry. In particular, we are interested in discovering small D-amino acid peptides that inhibit HIV membrane fusion and entry using mirror-image phage display. In this technique the target of interest is synthesized from D-amino acids. L-peptides displayed on phage are selected for binding to the D-target. By symmetry, D-versions of the discovered peptides will bind to the natural L-target. D-amino acids have many potential advantages as therapeutics including low immunogenicity, protease resistance, and possible oral bioavailability. These peptides also allow us to study for the first time the nature of high affinity interactions between L and D-peptides.

Understanding filamentous bacteriophage entry

Phage entry is a complex and poorly characterized process. In filamentous bacteriophage, bacterial entry is mediated by the gIII protein on phage and the TolA/Q/R complex in the bacterial inner membrane. Extensive biochemical and structural studies of the gIII protein have shown that it is divided into 3 functional domains (N1, N2, and CT). The primary recognition event for infection is the binding of the gIII-N2 domain to the tip of the bacterial F pilus. After binding, the pilus retracts, pulling the phage close to the surface of the outer membrane. The gIII-N1 domain then recognizes the C-terminal domain of TolA (TolA-CT), which triggers entry of the phage genome by a completely mysterious mechanism.
The N1 and N2 domains of gIII form a complex that is disrupted by the binding of N2 to the F pilus, freeing N1 to bind TolA-CT. This 2-step entry process shares a striking resemblance with other tightly regulated viral entry systems (e.g., HIV). The virus is not competent to enter a cell until the suitability of the target cell has been verified (by the presence of an F pilus for phage or CD4 for HIV). The fusion active protein (N1 in phage, gp41 in HIV) is covered by a protective partner (N2 in phage, gp120 in HIV) until an appropriate target cell is encountered, preventing non-specific viral entry. We hope to extend the reach of phage display technology by understanding the interactions required for infectivity and the extent to which these interactions can be engineered in a selection.

References
1. Root MJ, Kay MS, Kim PS (2001) Protein Design of an HIV-1 Entry Inhibitor. Science 291:884-888
2. Kay MS, Ramos CH, Baldwin RL (1999) Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate. Proceedings of the National Academy of Sciences of the United States of America 96(5):2007-12
3. Kay MS, Baldwin, RL (1998) Alternative Models for Describing the Acid Unfolding of the Apomyoglobin Folding Intermediate. Biochemistry 37(21):7859-7868