A molecular movie of nucleotide addition by HIV-1 reverse transcriptase.

Abstract

Historically, interactions between substrates and/or inhibitors and enzymes have been viewed in terms of binding of small molecules to relatively rigid protein targets. However, computational and experimental studies have revealed that many proteins, in particular DNA polymerases, undergo molecular motions over a wide range of timescales. Such conformational flexibility is critical for enzymatic activity, drug action and drug resistance. Moreover, contemporary structural biology approaches, such as X-ray crystallography, only have the ability to resolve the structures of thermodynamically stable species, and cannot inform on kinetic intermediates. To address this issue, we have developed a novel methodology to achieve time-resolved cryo-electron microscopy (TR cryo-EM) that, for the first time, facilitates visualization of short-lived protein conformations, including those transiently occurring during catalysis.Using TR cryo-EM, we have for the first time elucidated four intermediate kinetic states during HIV-1 reverse transcriptase (RT) mediated nucleotide (dATP) incorporation, supporting the feasibility and power of these approaches. Interestingly, we observe the presence of three Mg+2 in the active site during an intermediate state of the reaction, contrasting the kinetically stable structures of RT obtained via X-ray crystallography, where only two Mg+2 can be detected. The information derived from these studies improves our understanding of RT function, which in turn may facilitate development of new drugs.