"On the Fly" Time Resolved Cryo-EM Studies of Intermediate HIV-1 RT Transition States

SUMMARY
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. Our
group has developed cutting-edge technology - “on-the-fly” time-resolved cryo-electron microscopy (EM) - that,
for the first time, facilitates visualization of novel protein conformations, including those transiently occurring
during catalysis. Specifically, we have developed two TR cryo-EM approaches: (i) rapid chemical mixing of
enzyme and substrates; and (ii) photo-activation that takes advantage of caged substrates and/or amino acid
residues. We have developed substantial preliminary data that support the feasibility and power of these
approaches. In this application, we will apply this technology to address biologically and clinically relevant
knowledge gaps in HIV-1 reverse transcriptase (RT) biology, with specific focus on the incorporation of
nucleosides and nucleoside inhibitors, nucleoside inhibitor resistance, and the relationship between DNA
synthesis and ribonuclease H (RNase H) activity.

Grant Number: 5R01AI175067-04
NIH Institute/Center: NIH
Principal Investigator: Guillermo Calero