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In vitro evolution of an HIV integrase binding protein from a library of C-terminal domain ?S-crystallin variants.

ABSTRACT: A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human ?S-crystallin. Multiple loop regions were altered to encode libraries with ?3.6 × 10(11) different variants. A crystallin variant, termed integrase binding protein-10 (IBP-10), inhibits integrase catalysis with nanomolar K(i) values. IBP-10 interacts with the integrase C-terminal domain and inhibits integrase substrate affinity. This allosteric mechanism allows IBP-10 to inhibit drug-resistant integrase variants. The results demonstrate the applicability of the crystallin scaffold for the discovery of binding partners and enzyme inhibitors.

SUBMITTER: Moody IS

PROVIDER: S-EPMC3956296 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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In vitro evolution of an HIV integrase binding protein from a library of C-terminal domain γS-crystallin variants.

Moody Issa S IS Verde Shawn C SC Overstreet Cathie M CM Edward Robinson W W Weiss Gregory A GA

Bioorganic & medicinal chemistry letters 20120710 17

A protein without natural binding functions was engineered to bind HIV-1 integrase. Phage display selections applied a library of variants based on the C-terminal domain of the eye lens protein human γS-crystallin. Multiple loop regions were altered to encode libraries with ≈3.6 × 10(11) different variants. A crystallin variant, termed integrase binding protein-10 (IBP-10), inhibits integrase catalysis with nanomolar K(i) values. IBP-10 interacts with the integrase C-terminal domain and inhibits ...[more]

PMID: 22858140

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Project description:HIV integrase (HIV-IN), one of three HIV enzymes, is a target for the treatment of AIDS, but the full biological assembly has been difficult to characterize, hampering inhibitor design. The recent crystallographic structures of integrase from prototype foamy virus (PFV-IN) with bound DNA were a breakthrough, revealing how viral DNA organizes two integrase dimers into a tetramer that has the two active sites appropriately spaced for insertion of the viral DNA into host DNA. The organization of domains within each PFV-IN protein chain, however, varies significantly from that found in HIV-IN structures. With the goal of identifying shared structural characteristics, the interactions among components of the PFV-IN and HIV-IN assemblies were investigated with the macromolecular docking program DOT. DOT performs an exhaustive, rigid-body search between two macromolecules. Computational docking reproduced the crystallographic interactions of the PFV-IN catalytic and N-terminal domains with viral DNA and found similar viral DNA interactions for HIV-IN. Computational docking did not reproduce the crystallographic interactions of the PFV-IN C-terminal domain (CTD). Instead, two symmetry-related positions were found for the PFV-IN CTD that indicate formation of a CTD dimer between the two active sites. Our predicted CTD dimer is consistent with cross-linking studies showing interactions of the CTD with viral DNA that appear to be blocked in the PFV-IN structures. The CTD dimer can insert two arginine-rich loops between the two bound vDNA molecules and the host DNA, a region that is unoccupied in the PFV-IN crystallographic structures. The positive potential from these two loops would alleviate the large negative potential created by the close proximity of two viral vDNA ends, helping to bring together the two active sites and assisting host DNA binding. This study demonstrates the ability of computational docking to evaluate complex crystallographic assemblies, identify interactions that are influenced by the crystal environment, and provide plausible alternatives.

Dataset Information

In vitro evolution of an HIV integrase binding protein from a library of C-terminal domain ?S-crystallin variants.

Publications

In vitro evolution of an HIV integrase binding protein from a library of C-terminal domain γS-crystallin variants.

Similar Datasets

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