Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.
X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.
Kensch
O
O
Max-Planck-Institut für molekulare Physiologie Abteilung Physikalische Biochemie, Otto-Hahn-Strasse 11, Dortmund, 44227, Germany.
Restle
T
T
Wöhrl
B M
BM
Goody
R S
RS
Steinhoff
H J
HJ
D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005615:Freezing; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism; D012996:Solutions; D013113:Spin Labels; D013696:Temperature; D013816:Thermodynamics
D008024:Ligands; D018894:Reverse Transcriptase Inhibitors; D012996:Solutions; D013113:Spin Labels; D012313:RNA; D004247:DNA; D054303:HIV Reverse Transcriptase; D003545:Cysteine