Structure, folding and engineering of 5-helix, a designed HIV-1 entry inhibitor

Kelly Champagne, Thomas Jefferson University

Abstract

HIV-1 entry requires fusion of viral and cellular membranes, a process mediated by structural changes in the envelope glycoprotein complex gp120/gp41. Ultimately, a trimer-of-hairpins forms in which the N- and C-terminal regions of three gp41 ectodomains associate, forming a bundle of six α-helices. Previously, a protein that binds the C-terminal region of the gp41 ectodomain was designed, disrupting trimer-ofhairpins formation and blocking HIV-1 entry. This protein, denoted 5-Helix, contains five of the six helices that constitute the gp41 trimer-of-hairpins linked into a single polypeptide. To confirm our design strategy and to assist efforts in optimizing this antiviral approach, we have determined the structure of 5-Helix and two of its variants, 5-Helix-N3D4 and 6-Helix. X-ray crystallographic analysis has pointed to an overall structure similar to that predicted from the design. However, the packing between inner and outer helices, especially within one tryptophan-pocket region, is highly distorted, demonstrating an unanticipated level of plasticity in the interaction surface. Moreover, the length and flexibility of the five amino-acid linkers connecting the N- and C-terminal helices permit 5-Helix to adopt numerous folding topologies. These structures provide a model with which we can begin to design improvements that enhance the stability and potency of this designed HIV-1 entry inhibitor. The immense stability of 5-Helix provides a scaffold for introducing less stable residues into the core of the protein, enabling the production of a protein capable of binding to a C-peptide, T20 that targets a shifted region of the gp41 N-terminal heptad repeat (N-HR). T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (KD = 30 nM), close to its IC50 concentration (∼3 nM) but much weaker than the affinity of a related inhibitory C-peptide C37 (KD = 0.0007 nM). T20 effectively competed with lower-affinity C37 variants for binding to 5H-ex. In addition, the T20 affinity was not reduced by mutations in the N-HR hydrophobic pocket region that substantially disrupted C37 binding. Together, these data confirmed that T20 binds 5H-ex in the hydrophobic groove on the surface of the N-HR coiled coil outside of the pocket region. We used 5H-ex to investigate how the T20 N- and C-terminal contributed to the inhibitor's binding activity. Mutating the three aromatic residues at the T20 C-terminus (WNWF → ANAA) had no effect on affinity, suggesting that they do not contribute in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich et al 2003, Liu et al 2007). By contrast, mutations near the T20 N-terminus substantially influenced inhibitor binding strength. An Ile substitution for Thr in the second T20 position resulted in a substantial increase in binding affinity (KD = 0.75 nM). This increased binding strength did not translate into an improved inhibitory potency against wild type HIV-1. However, the C-peptide variant was more potent against T20-resistant HIV-1. The findings suggest that T20 inhibitory activity may be restricted by kinetic factors that limit exposure of the gp41 N-HR region, but that inhibitor escape can arise at least partially through a mechanism of affinity disruption. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.

Subject Area

Microbiology|Pharmacology|Virology

Recommended Citation

Champagne, Kelly, "Structure, folding and engineering of 5-helix, a designed HIV-1 entry inhibitor" (2008). ETD Collection for Thomas Jefferson University. AAI3390417.
https://jdc.jefferson.edu/dissertations/AAI3390417

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