Structure of the Integral Membrane Protein CAAX Protease Ste24p

Abstract

Post-translational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CaaX sequence motifs. Isoprenylation is followed by cleavage of the aaX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CaaX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active-site and substrate binding groove. The cavity is accessible to the external milieu via gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds via a processive mechanism of substrate insertion, translocation and ejection.

A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases

“…The pocket is very similar in depth and hydrophobicity in both zymogens and framed in projannalysin by A 39 (E 93 ). As for proabylysin, the globular part of projannalysin spanning D 9 -L 109 indicates a competent fold of catalytic moiety, active-site cleft, metal-binding site, and general base/acid glutamate, as usual for active MPs (see also below).…”

“…However, structural biochemistry of integral membrane proteins in general faces formidable conceptual and technical challenges due to the low concentrations at which they are naturally present in organisms, the difficulty in finding adequate recombinant overexpression systems that yield sufficient amounts of native-like protein, and their insolubility in strictly aqueous media, which requires screening for detergents and lipids that mimic the native membrane environment (35). Consequently, the only IMMP structures published to date are that of the intramembrane site-2 protease (36) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (MEROPS family M50), an ortholog of a human enzyme that releases the N-terminal transcription factor domain from membranebound sterol regulatory element-binding proteins (36 -38) and is unrelated to M48 and M56 IMMPs, and of human and yeast FACE1/Ste24p (Protein Data Bank (PDB) codes 4AW6 and 4IL3 (39,40)), the only functional, truly integral-membrane representatives of families M48 and M56. In such a scenario, strategies aimed at obtaining high resolution structural information on soluble, correctly folded fragments of IMMP target proteins, such as globular catalytic domains (CDs) inserted into the overall transmembrane scaffold, may prove helpful in both the study of catalytic mechanisms and the design of drug-like inhibitors.…”

Expression and purification of integral membrane metallopeptidase HtpX

“…Despite showing the equivalent histidines at positions compatible with catalytic zinc binding, the structure exhibited an overall non-functional active site due to the lack of the third residue that is required for proper zinc coordination in competent MPs. This residue, probably a glutamic acid by analogy with FACE1/Ste24p (PDB entries 2YPT/4IL3; [10,11]), is predicted to be located within a "glutamate helix" [6,18] spanning residues 220-230, which is found after the fourth transmembrane helix as inferred from homology modeling and secondary structure predictions (see Fig. 1B and [7]).…”

“…The biological relevance of IMMPs fosters the need to acquire atomic structural information, mainly by means of X-ray crystallography, in order to be able to modulate the activity of these enzymes. However, the only IMMP structures published to date are those of site-2 protease from Methanocaldococcus jannaschii (family M50) and human/yeast FACE1/Ste24p (family M48) [9][10][11]. Another member of this latter family is HtpX, which was initially described as a heat-shock, Cpx-controlled protein [12,13].…”

Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides

“…Rce1p and Ste24p are both ER 2 membrane-localized proteases capable of cleaving isoprenylated CAAX proteins, yet they are neither structurally related nor functionally redundant (3)(4)(5)(6)(7). Rce1p cleaves the isoprenylated CAAX motifs of Ras and Ras-related GTPases (Rho, Rac, etc.)…”