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Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors

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  • Statement: We report the structure (to 2.0 Å resolution) of yeast-expressed human transmembrane CaaX protease ZMPSTE24 responsible for dual proteolytic cleavages of nuclear prelamin A subsequent to farnesylation. This allowed identification of features of the catalytic site, the nature of the large internal cavity, and routes of access to the active site in the cavity. We also show that HIV protease inhibitors used in antiviral therapy directly inhibit the purified human enzyme.

  • Data Deposition: The ZMPSTE24 structure has been deposited in the Protein Database (accession code: 5SYT).

  • Crystallographic studies described in this manuscript made use of structural biology services at the University of Rochester supported by NIH grants S10 RR026501 and P30 AI078498. Crystallographic data collection was conducted at the Advanced Photon Source beamlines of the Northeastern Collaborative Access Team funded by NIH grants P41 GM103403 and S10 RR029205 and of GM/CA@APS, funded in whole or in part with funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). The Advanced Photon Source is a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

Abstract

The function and localization of proteins and peptides containing C-terminal “CaaX” (Cys-aliphatic-aliphatic-anything) sequence motifs are modulated by post-translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature-aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off-target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C-terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.

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