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Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR.

ABSTRACT: The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr-Purcell-Meiboom-Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease-Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.

SUBMITTER: Deshmukh L

PROVIDER: S-EPMC5699096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Exchange dynamics between molecules free in solution and bound to the surface of a large supramolecular structure, a polymer, a membrane or solid support are important in many phenomena in biology and materials science. Here we present a novel and generally applicable solution NMR technique, known as dark-state exchange saturation transfer (DEST), to probe such exchange phenomena with atomic resolution. This is illustrated by the exchange reaction between amyloid-? (A?) monomers and polydisperse, NMR-invisible ('dark') protofibrils, a process of significant interest because the accumulation of toxic, aggregated forms of A?, from small oligomers to very large assemblies, has been implicated in the aetiology of Alzheimer's disease. The (15)N-DEST experiment imprints with single-residue-resolution dynamic information on the protofibril-bound species in the form of (15)N transverse relaxation rates ((15)N-R(2)) and exchange kinetics between monomers and protofibrils onto the easily observed two-dimensional (1)H-(15)N correlation spectrum of the monomer. The exchanging species on the protofibril surface comprise an ensemble of sparsely populated states where each residue is either tethered to (through other residues) or in direct contact with the surface. The first eight residues exist predominantly in a mobile tethered state, whereas the largely hydrophobic central region and part of the carboxy (C)-terminal hydrophobic region are in direct contact with the protofibril surface for a significant proportion of the time. The C-terminal residues of both A?40 and A?42 display lower affinity for the protofibril surface, indicating that they are likely to be surface exposed rather than buried as in structures of A? fibrils, and might therefore comprise the critical nucleus for fibril formation. The values, however, are significantly larger for the C-terminal residues of A?42 than A?40, which might explain the former's higher propensity for rapid aggregation and fibril formation.

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Binding kinetics and substrate selectivity in HIV-1 protease-Gag interactions probed at atomic resolution by chemical exchange NMR.

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