Membrane-embedded protease poses for photoshoot.
نویسندگان
چکیده
I ntramembrane-cleaving proteases (I-CLiPs) are a class of hydrolytic enzymes that cleave hydrophobic substrates within the lipid bilayer (1). Unlike their well understood soluble or membrane-tethered counterparts, I-CLiPs are integral membrane proteins, with catalytic residues thought to lie within the confines of the membrane. Although the identities of these residues have suggested membrane-embedded active sites and mechanistic convergence with other proteases, I-CLiPs bear little or no sequence similarity to their soluble cousins, and their assignment into mechanistic categories has been tentative. Three new articles (2–4), including one by Ben-Shem et al. in this issue of PNAS (4), describe the first crystal structures of an I-CLiP, the Escherichia coli serine protease GlpG. Together, these reports provide complementary pictures that offer exciting insights into how an I-CLiP handles transmembrane substrates and carries out hydrolysis in a hydrophobic environment. The known families of I-CLiPs include the site-2 protease (S2P) zinc metalloproteases, presenilin and presenilin-like aspartyl proteases, and the rhomboid family of serine proteases. These enzymes are found throughout the major kingdoms of life, are highly conserved, and play key roles in biology and disease. S2P-type proteases release membrane-tethered transcription factors controlling sterol and fatty acid biosynthesis in animals and sporulation in bacteria (5, 6). Presenilin is the catalytic engine of the four-component secretase complex critical for cell-fate determinations and the pathogenesis of Alzheimer’s disease (7). Signal peptide peptidase, the prototype presenilin-like protease, releases remnant signal peptides from the membrane but, unlike presenilin, does not require other protein cofactors (8). E. coli GlpG is a homolog of Drosophila rhomboid-1, which was the first identified intramembrane serine protease (9). Rhomboid-1 releases the extracellular domain of the membranetethered epidermal growth factor-like protein Spitz as part of a key developmental signaling pathway in flies. The specific physiological roles of most rhomboids, including the seven homologs identified in the human genome and the many of bacterial and parasitic origin, are largely unknown, but research to date points to critical roles in mitochondrial membrane remodeling (10), apoptosis (11), quorum sensing (12), and cell invasion (13). Interestingly, even though GlpG has been shown to cleave Spitz (14), its normal function in E. coli remains elusive. Careful biochemical and cell biological studies have contributed to our understanding of hydrolysis by intramembrane serine proteases since their discovery in 2001. Such studies have suggested a sixor seven-transmembrane architecture, a Ser–His catalytic dyad (14), and a critical Trp–Arg (WR) motif (9). Substrate cleavage is predicted to occur several residues into the transmembrane helix on the periplasmic/extracellular side and requires helix-destabilizing residues at or near the cleavage site (15). The crystal structure confirms and helps clarify several of these predicted features in molecular detail and also enables new structure-based hypotheses for catalysis by this family of enzymes. As predicted, the crystal structure of GlpG reveals a core molecular architecture composed of six transmembrane helices (TM) plus two helices, H1 and H2, that are part of the unusual loop 1 connecting TM1 and TM2 (Fig. 1a). Although membrane proteins are typically thought to form a bundle of uniformlength helices that cross the membrane and are parallel to each other, the TM helices in GlpG are of varying lengths and angles with respect to the membrane normal. The particular spatial arrangement creates a hydrophilic cavity within the protein and emphasizes the importance of the overall protein scaffold in forming the unique chemical environment amenable to hydrolysis. A major contribution of the GlpG structures is in establishing that residues implicated in catalysis by site-directed mutagenesis are indeed located within the boundaries of the membrane, 10 Å below the surface within a water-filled cavity on the periplasmic side of the membrane. These residues, Ser-201 and His-254, which form the proposed catalytic dyad, are hydrogen-bonded with a distance of 3.1 Å and are located at
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عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 104 2 شماره
صفحات -
تاریخ انتشار 2007