Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease.
HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.
Tang
Chun
C
Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Louis
John M
JM
Aniana
Annie
A
Suh
Jeong-Yong
JY
Clore
G Marius
GM
D019281:Dimerization; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism
D011498:Protein Precursors; D013113:Spin Labels; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1