Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid

Abstract

The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.

SCHEME 1.

Schematic representation of the NYAD-13 peptide structure. The non-standard amino acid, (S)-2-(2′-pentenyl) alanine, is indicated by X in the amino acid sequence of the peptide.

FIGURE 1.

A, the helical representation of a single structure of mCA-CTD (148–221) and NYAD-13 (–11). The secondary structure consists of an N-terminal 3₁₀ helix, a type 1 β-turn, and a four-helix bundle. B, the side chains (blue) from residues in helix I and helix II are represented in the ensemble of NMR structures. For clarity, the peptide has been removed from the structure. Conserved residues from the MHR motif are colored in magenta. The structural representations were generated in MOLMOL 2.1 (30).

FIGURE 2.

Structural details of intermolecular contacts with ribbon representation of the protein (blue) and peptide (pink) backbone. A, the top view of the binding surface displays the interactions between the side chains of Phe-3 and Tyr-10 from the peptide and helix I and II of mCA-CTD. B, the side view of the complex displays the interactions that anchor Leu-6 and Tyr-9 from the peptide using Leu-211 and Met-215 from helix IV. C, the top view of the x-ray structure of CAI in complex with CA-CTD (2BUO). D, superposition of the backbone Cα atoms of CA-CTD (pink) and mCA-CTD (green) based on alignment generated from residues in helix I, helix III, and helix IV (r.m.s.d. = 0.8 Å). When helix II is included, the r.m.s.d. increases to 1.3 Å. Residues that are important for binding the target peptide and rearranged through the helix movement are indicated in the figure. The PDB code for CA-CTD structure used in the alignment is 1A8O. The figures were generated in MOLMOL 2.1 (30).