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

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.

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

“…Therefore, a structure‐based search of the PDB database using the DALI server was used to identify those gluzincin families most homologous to Ste24. Currently available structures of Ste24 comprise fungal (SmSte24) and human (HsSte24) orthologs. While SmSte24 and HsSte24 structures are highly similar, Ste24 mammalian orthologs contain a variable length insert between the α3‐helix of the L5D and TM α‐helix VI (37 residues in HsSte24) (Figure S1); this insert is disordered in all HsSte24 crystal structures.…”

“…Additionally, functional complementation between human and yeast Ste24 orthologs has been established in vivo where HsSte24 rescues defects in a ‐factor biogenesis associated with Ste24p knockout yeast ( ste24 Δ) . Crystal structures of yeast ( Saccharomyces mikatae ; SmSte24) and human Ste24 (HsSte24) reveal these two Ste24 structures to be highly similar (RMSD of C α atoms ~1.7 Å) . The structure of Ste24, as exemplified by SmSte24 (PDB: 4IL3), possesses a striking seven‐transmembrane (TM) helical “α‐barrel” structure encapsulating a voluminous reaction cavity (~14 000 Å 3 ; Figure A).…”

“…Converting CAAX Endopeptidase 1 (Rce1), an unrelated cysteine protease, can also perform CAAX box cleavage of both a-factor 7 and prelamin A. 17 Deficiencies in HsSte24 activity, whether by mutation 18,21,22 or inhibition, 23,24 restrict prelamin A maturation and lead to disease states known as laminopathies, which range from progerias (premature aging syndrome) to lipodystrophies. 25,26 Inhibition of HsSte24 has particular clinical relevance, as AIDS patients receiving viral protease inhibitors as part of their drug regimen develop lipodystrophies because of off-target interaction of HIV protease inhibitor drugs with HsSte24.…”

Ste24: An Integral Membrane Protein Zinc Metalloprotease with Provocative Structure and Emergent Biology

“…Improper prelamin A processing is also correlated with deleterious alterations to adipose tissue localization and accumulation, known as lipodystrophy, due to the toxic effects of prelamin A accumulation leading to altered expression of genes responsible for adipocyte proliferation and differentiation [33,34]. Additionally, the inability of some AIDS patients to produce and maintain adipose tissue, acquired from antiretroviral therapy, likely results from off-target interactions of HIV (aspartyl) protease inhibitor drugs with HsSte24 [35][36][37][38]. Because of HsSte24's roles in these syndromes, the Zmpste24 −/− mouse has been suggested as a model for senescent wound healing [39] and lipodystrophy [40].…”

“…Because prenylation is not required for substrate recognition, modern mass spectrometry-based methods utilizing standard peptide libraries, such as multiplex substrate profiling by mass spectrometry [67], could be applied to determine Ste24 substrate specificity (this method has been applied successfully to the rhomboid protease [68,69]). Additionally, while multiple researchers (for examples, see [9,13,28,37,70,71]) have performed steady-state kinetic characterization of Ste24, no absolute characterization of turnover and catalytic efficiency has yet been reported. Several technical barriers currently impede development of a standard "platform" for accurate and precise Ste24 steadystate kinetics characterization.…”