The structural basis for catalysis and substrate specificity of a rhomboid protease
2010; Springer Nature; Volume: 29; Issue: 22 Linguagem: Inglês
10.1038/emboj.2010.243
ISSN1460-2075
AutoresKutti R. Vinothkumar, Kvido Střı́šovský, Antonina Andreeva, Yonka Christova, Steven H. L. Verhelst, Matthew Freeman,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle1 October 2010free access The structural basis for catalysis and substrate specificity of a rhomboid protease Kutti R Vinothkumar Corresponding Author Kutti R Vinothkumar MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kvido Strisovsky Kvido Strisovsky MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Antonina Andreeva Antonina Andreeva MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Yonka Christova Yonka Christova MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Steven Verhelst Steven Verhelst Center for Integrated Protein Science Munich, Lehrstuhl Chemie der Biopolymere, Technische Universität München, Freising, Germany Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kutti R Vinothkumar Corresponding Author Kutti R Vinothkumar MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Kvido Strisovsky Kvido Strisovsky MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Antonina Andreeva Antonina Andreeva MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Yonka Christova Yonka Christova MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Steven Verhelst Steven Verhelst Center for Integrated Protein Science Munich, Lehrstuhl Chemie der Biopolymere, Technische Universität München, Freising, Germany Search for more papers by this author Matthew Freeman Corresponding Author Matthew Freeman MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Kutti R Vinothkumar 1, Kvido Strisovsky1, Antonina Andreeva1, Yonka Christova1, Steven Verhelst2 and Matthew Freeman 1 1MRC Laboratory of Molecular Biology, Cambridge, UK 2Center for Integrated Protein Science Munich, Lehrstuhl Chemie der Biopolymere, Technische Universität München, Freising, Germany *Corresponding authors. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. Tel.: +44 122 340 2351; Fax: +44 122 341 2142; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:3797-3809https://doi.org/10.1038/emboj.2010.243 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism-based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S1- and S2′-binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor-bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design. Introduction Proteases regulate myriad biological and medically important processes—indeed they are the targets of many important drugs (Turk, 2006). Accordingly, they are some of the most-studied and well-characterised enzymes. Much less well understood, however, are the four recently discovered families of intramembrane proteases (site-2 metalloprotease (Rawson et al, 1998), presenilin (De Strooper et al, 1999), signal peptide peptidase (Weihofen et al, 2002), and rhomboids (Urban et al, 2001)), which share the property of cleaving transmembrane regions of substrates, thereby releasing soluble intracellular or luminal/extracellular domains to act as biological effectors (Wolfe and Kopan, 2004; Freeman, 2008). They seem to have evolved as proteases by convergent evolution, bearing no detectable phylogenetic relationship with the classical soluble proteases. Rhomboids were first identified in Drosophila as the cardinal activators of the EGF receptor signalling pathway (Wasserman et al, 2000; Lee et al, 2001; Urban et al, 2001). Rhomboids have since been found in all kingdoms of life (Koonin et al, 2003; Lemberg and Freeman, 2007b). The list of their biological functions is steadily growing but already includes growth factor signalling, quorum sensing, and protein translocation in bacteria, parasitic invasion by protozoa, and regulation of mitochondrial dynamics (Opitz et al, 2002; McQuibban et al, 2003; Urban and Freeman, 2003; Howell et al, 2005; Cipolat et al, 2006; O'Donnell et al, 2006; Stevenson et al, 2007; Baxt et al, 2008). The initial proposal that rhomboids depended on a catalytic serine was indirect, relying on genetic analysis and cell culture-based assays (Lee et al, 2001; Urban et al, 2001). The ability to overexpress and purify rhomboid homologues from bacterial sources paved the way for in vitro assays of proteolysis; this proved that rhomboids by themselves are sufficient to cleave specific substrates without the requirement of additional proteins or subunits (Lemberg et al, 2005; Urban and Wolfe, 2005). These in vitro studies further supported the serine protease classification, but implied that rhomboids were atypical, using a dyad of serine and histidine rather than the catalytic triad found commonly in classical soluble serine proteases (Hedstrom, 2002; Polgar, 2005; Ekici et al, 2008). Further mechanistic insight was inferred, and the final doubts removed regarding whether the active site was truly within the membrane bilayer, with the reports of the crystal structures of the prokaryotic rhomboids, GlpG from Escherichia coli and Haemophilus influenzae. They revealed that the core of the structure consisted of a 6TM helical protein (Wang et al, 2006; Wu et al, 2006; Ben-Shem et al, 2007; Lemieux et al, 2007). Within this core, a short TM helix-4 and an extended loop-3 are surrounded by other helices to create a large hydrophilic indentation that opens to the periplasm. The catalytic serine found at the tip of TM4 and the general base histidine found in TM6 reside within this indentation, and are brought close together by tight packing of the TM helices. This active-site architecture, in conjunction with biochemical analysis, supported the conclusion that rhomboids are indeed a new family of enzyme. Despite these advances in structural and biochemical analysis of rhomboids, the lack of any structure of enzyme–substrate or enzyme–inhibitor complexes leaves important questions to be answered. Understanding the catalytic mechanism of this new family of enzymes is of fundamental theoretical interest, but is also necessary for understanding their biological specificity, and being a key step in the identification and design of inhibitors. Indeed, no potent or specific rhomboid inhibitors have been reported, a gap that becomes more striking as evidence increases regarding their medical significance. In fact, the only inhibitor class reported to have any significant, although weak, activity against rhomboids is isocoumarin; potent and irreversible inhibitor of classical serine proteases (Urban et al, 2001; Lemberg et al, 2005; Urban and Wolfe, 2005). Although isocoumarin inhibition has contributed to the argument that rhomboids use a variation of the classical serine protease catalytic mechanism, neither their specificity for the presumed catalytic residues of rhomboids nor their mechanism of inhibition of rhomboids has been determined. In this study, we describe the structure of a rhomboid protease covalently bonded to an isocoumarin inhibitor through both the active-site serine and histidine. The position and orientation of the inhibitor locates the oxyanion hole, a key structural characteristic of serine proteases. This proves that rhomboids act by a mechanism that closely resembles their soluble counterparts, thereby setting them in a clear enzymological context. Binding of inhibitor is accompanied by conformational changes in the active site, the loop-5, and TM helices-5 and -6. The acyl enzyme structure suggests how a substrate might bind at the active site and provides a rationale for how rhomboid specificity can be achieved. Results Structure of a bacterial rhomboid in complex with an inhibitor Isocoumarin inhibition of classical serine proteases begins with nucleophilic attack by the catalytic serine, resulting in opening of the isocoumarin ring, and formation of an acyl enzyme (Powers et al 2002). This can subsequently react with a nucleophile such as the catalytic histidine, to form a doubly covalent-bonded alkylated acyl enzyme, which is extremely stable (Figure 1A). Dichloroisocoumarin (DCI), a commonly used serine protease inhibitor, has modest activity against rhomboids (Lemberg et al, 2005; Urban and Wolfe, 2005). However, its short half-life of 20 min or less in many commonly used buffers (Harper et al, 1985) limits its usefulness for crystallography. Indeed, efforts to react DCI with GlpG crystals have been unsuccessful, probably due to its instability in solution and rapid deacylation (Wang and Ha, 2007; KR Vinothkumar et al, unpublished data). In a screen of an isocoumarin collection generously provided by Dr Matt Bogyo (Stanford University), we identified a more stable compound, 7-amino-4-chloro-3-methoxy isocoumarin (amino-methoxy-isocoumarin; originally synthesised by Dr James C Powers, Georgia Institute of Technology), with a half-life of ∼13 h in phosphate buffer (Harper et al, 1985), which inhibits GlpG with a half maximal inhibition of ∼6 μM (Figure 1B). Although there was a strong tendency of amino-methoxy-isocoumarin to disrupt GlpG crystals, we found conditions that allowed structural determination of the enzyme–inhibitor complex. The structure described here (PDB code 2XOW) was determined from a single crystal that diffracted to 2.09 Å (Table I). Figure 1.(A) Isocoumarins are heterocyclic compounds that inhibit only serine proteases. The presence of chlorine and amino group at positions 4 and 7, respectively, increases the stability of isocoumarins and substitutions at position 3 and 7 can be selectively designed to inhibit specific serine proteases. Nucleophilic attack by the active-site serine on isocoumarin results in ring opening and formation of acyl enzyme. A subsequent reaction with a nucleophile such as histidine can result in a doubly covalent bonded alkylated acyl enzyme. Figure redrawn from Powers et al, 2002. (B) In vitro cleavage and inhibition assay of Gurken fusion protein by GlpG. Bands are labelled as follows: FL for full-length fusion protein; E for enzyme; P1 and P2 for the two products released upon cleavage. Complete cleavage of the substrate has not been observed under these conditions. In this assay, the IC50 of amino-methoxy-isocoumarin is ∼6 μM. (C) Stereo view of a 2Fo–Fc map (blue mesh) contoured at 1.5σ showing the active-site residues and the covalently bonded inhibitor in stick representation (with carbon, oxygen and nitrogen atoms coloured as green, red and blue, respectively). Water molecules are shown as red crosses. Download figure Download PowerPoint Table 1. Data collection and refinement statistics GlpG-Native enzyme Acyl enzyme Data collection Beam line X06SA IO2 Space group R32 R32 Cell dimensions a, b, c (Å) 110.4, 110.4, 127.8 110.6, 110.6, 122.1 γ (deg) 120 120 Resolution (Å) 55.20–1.65 (1.74–1.65)a 44.62–2.09 (2.20–2.09)a Rmerge 0.055 (0.575) 0.054 (0.394) I/σI 12.4 (2.4) 16.3 (2.9) Completeness (%) 99.8 (100) 97.0 (85.4) Redundancy 4.5 (4.2) 4.9 (3.5) Refinement Resolution (Å) 34.77–1.65 (1.69–1.65)a 31.16–2.09 (2.22–2.09)a No. of reflections 36 038 16 657 Rwork/Rfreeb 0.192/0.218 0.198/0.242 (0.26/0.275)a (0.248/0.276)a No. of atoms Total 1733 1606 Protein 1442 1426 Heteroatoms 204 142 Water 87 38 B-factors (Å2) Total 29.7 44.7 Protein 26.7 43.5 Detergent 47.57 58.2 Ligand — 38.7 Water 38.8 47.0 RMSD Bond lengths (Å) 0.006 0.007 Bond angles (deg) 1.0 1.1 Maximum likelihood coordinate error (Å) 0.17 0.24 a Values in parentheses are for highest-resolution shell. b Rfree was calculated using a randomly selected subset of reflections (5%), remaining (95%) reflections was used for calculation of Rwork. The change of GlpG crystals from colourless to yellowish orange, and an ∼6-Å reduction in the c-axis upon soaking with the inhibitor, indicated a probable reaction of the inhibitor, and an associated structural change of the enzyme. Indeed, the initial map after molecular replacement shows a region of continuous positive density connected with active-site residues serine 201 and histidine 254. This represents the inhibitor bound to the enzyme active site, forming an alkylated acyl enzyme (Figure 1A and C). We observed the change in the unit cell and the formation of the doubly covalent bonded inhibitor independently in four different crystals. The inhibitor lies on the active site, ∼4.5 Å below the plane of the lipid head groups in the membrane (Figure 2A). The amino group (see diagram of the different parts of the inhibitor molecule in Figure 2B) of the inhibitor points towards the GlpG loop-3, whereas the methoxy group of the inhibitor points towards the gap between TM2 and TM5 and the lipid bilayer. The inhibitor amino group hydrogen bonds to a water molecule, and is in close contact to the main chain carbonyl oxygen of glycine 198. The oxygen atoms of the methyl acetate group form contacts with side chains of H150 and H254 (Figure 2C). Figure 2.(A) Side and top view of the acyl enzyme structure of GlpG. Structural elements of GlpG that perform specific roles are colour-coded individually. TM helices that harbour the active-site serine and histidine (shown in sticks) are coloured light blue. Substrate is thought to interact with the enzyme through the gap between TM helices 2 and 5 (yellow). TM helices 1 and 3, which probably have a supporting structural function, are coloured in grey. Loop-1 (pink) has a unique fold that is partially submerged in the membrane. Its function is not clear yet. Loop-5 (orange) falls over the active site, covering it from the extracellular solution and loop-3 (cyan) could function in substrate binding. The bound inhibitor (in stick representation, with carbon atoms in white) lies in the active site, perpendicular to the membrane plane. (B) The structure of the inhibitor molecule after the ring has been opened by nucleophilic attack by serine; distinct groups are described in the text. (C) Environment of the bound inhibitor: The amino group of the inhibitor hydrogen bonds to a water molecule (red sphere) and is in close contact with the main chain carbonyl of G198. The oxygen atoms of the methyl acetate group interact with side chains of histidine H150 and H254. The methyl group of the inhibitor has no neighbours within 3.5 Å of the macromolecule. The carbon atoms of the inhibitor are coloured in white, whereas those of the protein are green. (D) Oxyanion hole. The benzoyl carbonyl oxygen of the inhibitor is within hydrogen bonding distance of four neighbouring residues. Of these, only the main chain amide of S201 forms a strong hydrogen bond (2.61 Å). The main chain amide of L200, the side chain amide of N154, and the imidazole nitrogen of H150 form weak hydrogen bonds (3.4 Å). Download figure Download PowerPoint Formation of an acyl intermediate is one of the hallmarks of serine proteases, and the first description of serine proteases involved the trapping of an acyl intermediate, even before the sequence or structure was determined (Hartley and Kilby, 1950). With the structure of an alkylated acyl enzyme of GlpG, we now demonstrate unequivocally that rhomboids are serine proteases that use a catalytic mechanism analogous, but not identical, to the classical soluble serine proteases. Moreover, the fact that amino-methoxy-isocoumarin reacts only with S201, confirms that this is indeed the reactive nucleophile in the enzyme. Although this has been suggested by previous biochemical and structural study (for review, see Lemberg and Freeman, 2007a), and was hypothesised when rhomboids were first identified as proteases (Urban et al, 2001), they do not share common ancestry with their soluble counterparts, so their mechanism cannot confidently be inferred by evolutionary arguments. Location of the oxyanion hole The catalytic efficiency, as defined by increased rate constant or decreased transition state energy, of classical serine proteases is enhanced by the oxyanion hole (Henderson, 1970; Robertus et al, 1972; Hedstrom, 2002). In chymotrypsin, the main chain amides of the catalytic serine (S195) and a glycine two residues upstream (G193) stabilise the negatively charged oxyanion, a reaction intermediate that is formed from the carbonyl oxygen of the cleaved peptide bond. Residues contributing to the oxyanion hole in rhomboids have previously been tentatively suggested based on the structures (Ben-Shem et al, 2007; Lemieux et al, 2007). The benzoyl carbonyl oxygen of amino-methoxy-isocoumarin mimics the substrate peptide carbonyl, allowing us now to identify its position as the oxyanion hole in GlpG, and to identify the probable residues involved (Figure 2C and D). The main chain amide of G199 (equivalent to G193 in chymotrypsin) points away from the active site, so, at least in the case of GlpG, cannot hydrogen bond to the carbonyl oxygen. However, there are four other potential hydrogen-bonding donors in the vicinity of the benzoyl carbonyl oxygen. These include the main chain amides of S201 and L200, the side chain amide of N154 and the nitrogen (Nε) from the imidazole ring of H150. In this structure, the main chain amide of S201 forms a strong hydrogen bond with the benzoyl carbonyl oxygen of the inhibitor, whereas the other residues form hydrogen bonds that can be inferred from their lengths to be weaker (Figure 2D). Residues H150 and N154 are highly conserved in rhomboids. In GlpG, mutation of H150 to an alanine results in complete loss of activity, whereas the N154A mutant shows a reduction in the rate of cleavage (Baker et al, 2007). However, in another rhomboid, YqgP from Bacillus subtilis, both these residues seem less important for catalysis (Lemberg et al, 2005). These observations along with the inhibitor-bound structure suggest that there could be some redundancy in residues that stabilise the oxyanion, with the main chain amide of S201 having the major function. Structural changes on inhibitor binding A comparison of the inhibitor-bound acyl enzyme structure with that of the native enzyme without a substrate or inhibitor (PDB code 2XOV, for which an independent data set was collected; see Table I) reveals several significant structural changes (Figure 3A and B). In the native enzyme structure, loop-5, with two bulky methionine side chains (M247 and M249), caps the active site. It was observed that this loop is flexible and easily becomes disordered on soaking the crystals (Wang et al 2007). In the acyl enzyme structure, we observe that loop-5 is ordered (Figure 3C) and lifted away from the active site, consistent with the view that it occludes the catalytic centre in the resting state of the enzyme. Accompanied with this movement of loop-5 is a slight displacement of TM5 and TM6 that flank loop-5. The average displacement of both these helices is ∼1.25 Å with the greatest deviation observed at the C-terminus of TM5 and N-terminus of TM6, in particular residues 250–252 (Figure 3D). We observe little change in the position of loop-1 (Figure 3A) that points away from the body of the enzyme in the plane of the lipid bilayer. This loop was initially speculated to function in substrate recruitment (Wang et al, 2006), but since then has been suggested to have a structural function (Baker et al, 2007; Wang and Ha, 2007; Wang et al, 2007; Bondar et al, 2009). Although our data do not rule out a role for loop-1 in the enzyme function, we see no evidence for its role in catalysis. Figure 3.(A) A structural overlay of the native enzyme and the acyl enzyme showing the differences observed upon inhibitor binding. (B) A graphical representation of the root mean square deviation (RMSD) for the Cα atoms of each residue between the two structures (1.104 Å) determined with the CCP4 program Superpose (1994). Regions where the largest deviations observed are labelled. (C) A 2Fo–Fc map contoured at 1σ showing the density of loop-5; residues 243–250 are shown in sticks. (D) An overlay of TM5 and 6, loop-5 and the active-site residues (G199, S201, Y205 and H254; in sticks) displaying the structural change. Native enzyme is coloured in green and the acyl enzyme in purple. (E, F) Active site of the native and acyl enzyme. A hydrogen-bonding network is observed in the active site of native enzyme with the active-site S201 hydrogen bonded to general base H254 and a water molecule (red sphere). The water molecule is in turn hydrogen bonded to H150, part of oxyanion hole. In the acyl enzyme, this water molecule is displaced due to the binding of inhibitor. The hydroxyl group of Y205 hydrogen bonds to a water molecule in the native enzyme, but due to the movement of H254 upon inhibitor binding, the hydroxyl now points towards the main chain carbonyl of W236. Download figure Download PowerPoint We observe two significant changes at the active site of the acyl enzyme. First, covalent binding of the inhibitor to the active-site serine displaces a water molecule that is hydrogen bonded to S201 and H150 in the native enzyme (Figure 3E and F). The benzoyl carbonyl oxygen occupies the position of this water molecule in the acyl enzyme. Second, the covalent bond between the inhibitor and H254 not only results in the displacement of the H254 side chain but also other residues in TM6 (Figure 3D). This movement also affects Y205 in TM4: a rotation of the side chain positions the aromatic ring to be perpendicular to the imidazole ring of H254 in the acyl enzyme structure as opposed to being parallel in the native enzyme (Figure 3E and F). The hydroxyl group of Y205, which hydrogen bonds to a water molecule in native enzyme structure now points towards the main chain carbonyl of W236 in the acyl enzyme structure. To accommodate this movement of Y205, the side chain of W236 in TM5 adopts a different rotamer in the acyl enzyme. Overall, the acyl enzyme structure thus suggests the structural changes that are likely to occur during catalysis, including the opening of the active site by movement of the loop-5, accompanied by small changes in the nearby TM helices. Substrate-binding subsites in GlpG Most proteases cleave specific substrates, and such specificity is achieved by precise complementarity between the structures of enzyme and substrate. In particular, specific grooves and cavities in the enzyme structure often represent substrate-binding subsites (Neil et al, 1966; Blow, 1974). Surface analysis of the rhomboid acyl enzyme structure reveals, within the hydrophilic indentation, two cavities on either side of the bound inhibitor. First of these cavities is in close proximity to the active site and the amino group of the inhibitor points into it (Figure 4A). This cavity is observed in all described rhomboid structures, and is filled with two or three water molecules (Wang et al, 2006; Wu et al, 2006; Ben-Shem et al, 2007; Lemieux et al, 2007). In the native enzyme structure the side chain of methionine 249 from loop-5 plugs this cavity (Figure 4B). The second cavity observed only in the acyl enzyme structure is located on the opposite side of the catalytic dyad and the methyl acetate group of the inhibitor points towards it. This second cavity is largely defined by residues from TM4 and TM5 (Figure 4A and B) and is hydrophobic in character. It is not fully formed in the native enzyme structure, where it resembles a shallow cleft (Figure 4A). Figure 4.Surface representation of GlpG molecules showing the top view of native and acyl enzyme with loop-5 removed for clarity. (A) Positively and negatively charged amino acids are shown in blue and red, respectively, polar residues in light blue, and hydrophobic residues in grey. The active-site serine is coloured green and histidine is coloured orange. A hydrophilic indentation is evident in both structures, and the inhibitor molecule (in yellow stick representation) lies on it. Within this indentation the cavities observed in both structures are marked with red arrows. Water molecules are shown as cyan spheres. (B) Close-up view of the cavities in the native and acyl enzyme. The loop-5 in the native enzyme falls over the active site and plugs the S1 cavity with the side chain of M249 (shown in magenta and stick representation). In the acyl enzyme, the amino group of the inhibitor points towards the S1 cavity (loop-5 not shown for clarity). In both these structures the S1 cavity is filled with three water molecules (cyan spheres). A second hydrophobic cavity fully formed only in the acyl enzyme could represent the S2′ cavity. Download figure Download PowerPoint What do these cavities in GlpG represent? Substrate residues flanking the scissile bond are a major determinant in directing site-specific cleavage by proteases (Blow, 1974). By convention, they are defined as Pn–P1 for the residues N-terminal to the scissile bond and P1′–Pn′ for the residues C-terminal residues to the cleaved P1–P1′ bond (Schechter and Berger, 1967). Several rhomboid proteases, including E. coli GlpG have recently been shown to recognise a substrate sequence motif that defines the cleavage site (Strisovsky et al, 2009). Small side chains are preferred at the P1 position of the substrate, whereas hydrophobic residues are favoured at P4 and P2′ positions. We assume that corresponding binding sites in the enzyme (S subsites in standard protease nomenclature) are present to accommodate the side chains of these amino acids defined by the recognition motif. As the P1 residue is the most constrained (Strisovsky et al, 2009), the corresponding S1 site is expected to be most prominent, as is the case for several soluble serine proteases (Blow, 1974; Bode et al, 1989; Perona and Craik, 1995). In interpreting the cavities in GlpG, we further rely on two widely accepted views on substrate binding to rhomboids: (1) biological data show that rhomboids release N-terminal substrate domains into extracellular/periplasmic/luminal space (Freeman, 2008), indicating that N-terminus of GlpG substrate points towards the periplasm; and (2) structural and functional evidence suggests that rhomboid substrates interact with the enzyme through the gap between TM2 and TM5 (Baker et al, 2007; Ben-Shem et al, 2007). On the basis of these considerations, our data strongly indicates that the cavity in GlpG in which the amino group of the inhibitor points is the S1 subsite of GlpG. This is consistent with the fact this cavity is observed both in the native and acyl enzyme (Figure 4). In contrast, the second cavity is observed only on inhibitor binding and its relative distance from the catalytic residues leads us to propose that this could represent the S2′ subsite (in which hydrophobic P2′ residue binds). The identity of the S4 subsite, in which the substrate's characteristic P4 residue binds is not clear from either of the structures described here. A model for substrate binding in the active site of GlpG Combining our knowledge of the recognition motifs in rhomboid substrates, the substrate-binding subsites in enzyme, and the preferred mode of isocoumarin binding in soluble serine proteases (Powers et al, 2002; Strisovsky et al, 2009), we can now infer how substrate binds at the active site of GlpG. The orientation and the position of the benzoyl carbonyl oxygen of the inhibitor provides a guide to the probable position of the peptidyl carbonyl of the P1 residue of the substrate—so that it is poised for a nucleophilic attack by the catalytic serine. To be consistent with the second stage of the classical serine protease catalytic mechanism, the amide of the P1′ residue of the substrate (the amino acid immediately C-terminal to the scissile bond) has been constrained in a position to be able to accept a proton from the catalytic histidine. Proteases and substrates typically form a β-stranded interaction at the active site, frequently engaging local hydrogen bonds (Tyndall et al, 2005). With this in mind, we modelled a tetrapeptide of TatA (Thr-Ala-Ala-Phe), a prokaryotic rhomboid substrate (Stevenson et al, 2007), into the GlpG active site, using docking and energy minimisation, manually modifying side chain rotamers, in particular those of methionines from loop-5. In our pre
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