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The configurational space sampled by the finger and thumb subdomains of the p66 subunit of HIV-1 reverse transcriptase was investigated by Q-band double electron-electron resonance pulsed electron paramagnetic resonance spectroscopy, a method for determining long-range distances between pairs of nitroxide spin-labels introduced via surface-engineered cysteine residues. Four constructs were examined, each containing two spin-labels in the p66 subunit, one in the finger subdomain and the other in the thumb subdomain. In the unliganded state, open and closed configurations for the finger and thumb subdomains are observed with the distribution between these states modulated by the spin-labels and associated mutations, in contrast to crystallographic data in which the unliganded state crystallizes in the closed conformation. Upon addition of double-stranded DNA, all constructs adopt open conformations consistent with previous crystallographic data in which the position of the thumb and finger subdomains is determined by contacts with the bound oligonucleotide duplex (DNA or DNA/RNA). Likewise, binary complexes with five different non-nucleoside reverse transcriptase inhibitors are in open or partially open conformations, indicating that binding of the inhibitor to the palm subdomain indirectly restricts the conformational space sampled by the finger and thumb subdomains.

biochemistry

Probing Conformational States of the Finger and Thumb Subdomains of HIV-1 Reverse Transcriptase Using Double Electron–Electron Resonance Electron Paramagnetic Resonance Spectroscopy