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Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

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Abstract

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation1,2. The mature protease is active only as a dimer3,4,5 with each subunit contributing catalytic residues6. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer5,7,8,9,10,11, concomitant with the appearance of mature-like catalytic activity7,9. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-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 precursor5,12. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species13,14,15,16, 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.

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Figure 1: Intermolecular PRE profiles.
Figure 2: Ensemble simulated annealing and the protease mini-precursor encounter complex ensemble.
Figure 3: PRE profiles with spin labels attached at the N- and C termini of the SFNF PR(D25N) mini-precursor.

Change history

  • 14 November 2008

    In the online-only extended Methods, a sentence in the second paragraph of the 'NMR experiments' section has been corrected.

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Acknowledgements

We thank R. Ishima for providing initial backbone assignments for the SFNFPR(D25N) protease construct; C. Schwieters for many discussions; Y. Sheng for help with the CS-Rosetta calculations; Y. Kim for providing the code for structure clustering and d.r.m.s. calculations; and J. Sayer for MALDI measurements. This work was supported by funds from the Intramural Program of the NIH, NIDDK and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

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Correspondence to G. Marius Clore.

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Supplementary Information

This file contains Supplementary Figures and Legends S1-S3 and Supplementary Table S1 In the original Supplementary Fig. 1d, the values of the translational diffusion coefficients (Ds) for the monomeric precursor and mature HIV-1 protease dimer were inadvertently transposed; these have now been corrected. (PDF 287 kb)

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Tang, C., Louis, J., Aniana, A. et al. Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease. Nature 455, 693–696 (2008). https://doi.org/10.1038/nature07342

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