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The Structural Basis for Catalysis and Substrate Specificity of a Rhomboid

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The Structural Basis for Catalysis and Substrate Specificity of a Rhomboid The EMBO Journal (2010) 29, 3797–3809 | & 2010 European Molecular Biology Organization | All Rights Reserved 0261-4189/10 www.embojournal.org TTHEH E EEMBOMBO JJOURNALOURN AL The structural basis for catalysis and substrate specificity of a rhomboid protease Kutti R Vinothkumar1,*, Kvido Strisovsky1, releasing soluble intracellular or luminal/extracellular do- Antonina Andreeva1, Yonka Christova1, mains to act as biological effectors (Wolfe and Kopan, 2004; Steven Verhelst2 and Matthew Freeman1,* Freeman, 2008). They seem to have evolved as proteases by convergent evolution, bearing no detectable phylogenetic 1MRC Laboratory of Molecular Biology, Cambridge, UK and 2Center for Integrated Protein Science Munich, Lehrstuhl Chemie der Biopolymere, relationship with the classical soluble proteases. Rhomboids Technische Universita¨tMu¨nchen, Freising, Germany were first identified in Drosophila as the cardinal activators of the EGF receptor signalling pathway (Wasserman et al, 2000; Rhomboids are intramembrane proteases that use a Lee et al, 2001; Urban et al, 2001). Rhomboids have since catalytic dyad of serine and histidine for proteolysis. been found in all kingdoms of life (Koonin et al, 2003; They are conserved in both prokaryotes and eukaryotes Lemberg and Freeman, 2007b). The list of their biological and regulate cellular processes as diverse as intercellular functions is steadily growing but already includes growth signalling, parasitic invasion of host cells, and mitochon- factor signalling, quorum sensing, and protein translocation drial morphology. Their widespread biological signifi- in bacteria, parasitic invasion by protozoa, and regulation of cance and consequent medical potential provides a mitochondrial dynamics (Opitz et al, 2002; McQuibban et al, strong incentive to understand the mechanism of these 2003; Urban and Freeman, 2003; Howell et al, 2005; Cipolat unusual enzymes for identification of specific inhibitors. et al, 2006; O’Donnell et al, 2006; Stevenson et al, 2007; Baxt In this study, we describe the structure of Escherichia coli et al, 2008). The initial proposal that rhomboids depended on rhomboid GlpG covalently bound to a mechanism-based a catalytic serine was indirect, relying on genetic analysis and isocoumarin inhibitor. We identify the position of the cell culture-based assays (Lee et al, 2001; Urban et al, 2001). 0 oxyanion hole, and the S1- and S2 -binding subsites of The ability to overexpress and purify rhomboid homologues GlpG, which are the key determinants of substrate speci- from bacterial sources paved the way for in vitro assays of ficity. The inhibitor-bound structure suggests that subtle proteolysis; this proved that rhomboids by themselves are structural change is sufficient for catalysis, as opposed to sufficient to cleave specific substrates without the require- large changes proposed from previous structures of ment of additional proteins or subunits (Lemberg et al, 2005; unliganded GlpG. Using bound inhibitor as a template, Urban and Wolfe, 2005). These in vitro studies further we present a model for substrate binding at the active site supported the serine protease classification, but implied and biochemically test its validity. This study provides a that rhomboids were atypical, using a dyad of serine and foundation for a structural explanation of rhomboid histidine rather than the catalytic triad found commonly in specificity and mechanism, and for inhibitor design. classical soluble serine proteases (Hedstrom, 2002; Polgar, The EMBO Journal (2010) 29, 3797–3809. doi:10.1038/ 2005; Ekici et al, 2008). emboj.2010.243; Published online 1 October 2010 Further mechanistic insight was inferred, and the final Subject Categories: proteins; structural biology doubts removed regarding whether the active site was truly Keywords: intramembrane protease; isocoumarin; rhomboid; within the membrane bilayer, with the reports of the crystal serine protease; substrate specificity 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, Introduction 2007; Lemieux et al, 2007). Within this core, a short TM helix-4 Proteases regulate myriad biological and medically important and an extended loop-3 are surrounded by other helices to processes—indeed they are the targets of many important create a large hydrophilic indentation that opens to the drugs (Turk, 2006). Accordingly, they are some of the most- periplasm. The catalytic serine found at the tip of TM4 and studied and well-characterised enzymes. Much less well the general base histidine found in TM6 reside within this understood, however, are the four recently discovered indentation, and are brought close together by tight packing families of intramembrane proteases (site-2 metalloprotease of the TM helices. This active-site architecture, in conjunction (Rawson et al, 1998), presenilin (De Strooper et al, 1999), with biochemical analysis, supported the conclusion that signal peptide peptidase (Weihofen et al, 2002), and rhom- rhomboids are indeed a new family of enzyme. boids (Urban et al, 2001)), which share the property Despite these advances in structural and biochemical of cleaving transmembrane regions of substrates, thereby analysis of rhomboids, the lack of any structure of enzyme– substrate or enzyme–inhibitor complexes leaves important *Corresponding author. KR Vinothkumar or M Freeman, MRC questions to be answered. Understanding the catalytic Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK. mechanism of this new family of enzymes is of fundamental Tel.: þ 44 122 340 2351; Fax: þ 44 122 341 2142; theoretical interest, but is also necessary for understanding E-mails: [email protected] or [email protected] their biological specificity, and being a key step in the Received: 3 August 2010; accepted: 6 September 2010; published identification and design of inhibitors. Indeed, no potent or online: 1 October 2010 specific rhomboid inhibitors have been reported, a gap that &2010 European Molecular Biology Organization The EMBO Journal VOL 29 | NO 22 | 2010 3797 An alkylated acyl enzyme of a rhomboid protease KR Vinothkumar et al becomes more striking as evidence increases regarding their active-site residues serine 201 and histidine 254. This repre- medical significance. In fact, the only inhibitor class reported sents the inhibitor bound to the enzyme active site, forming to have any significant, although weak, activity against an alkylated acyl enzyme (Figure 1A and C). We observed the rhomboids is isocoumarin; potent and irreversible inhibitor change in the unit cell and the formation of the doubly of classical serine proteases (Urban et al, 2001; Lemberg et al, covalent bonded inhibitor independently in four different 2005; Urban and Wolfe, 2005). crystals. The inhibitor lies on the active site, B4.5 A˚ below Although isocoumarin inhibition has contributed to the the plane of the lipid head groups in the membrane argument that rhomboids use a variation of the classical (Figure 2A). The amino group (see diagram of the different serine protease catalytic mechanism, neither their specificity parts of the inhibitor molecule in Figure 2B) of the inhibitor for the presumed catalytic residues of rhomboids nor their points towards the GlpG loop-3, whereas the methoxy group mechanism of inhibition of rhomboids has been determined. of the inhibitor points towards the gap between TM2 and In this study, we describe the structure of a rhomboid TM5 and the lipid bilayer. The inhibitor amino group hydro- protease covalently bonded to an isocoumarin inhibitor gen bonds to a water molecule, and is in close contact to the through both the active-site serine and histidine. The position main chain carbonyl oxygen of glycine 198. The oxygen and orientation of the inhibitor locates the oxyanion hole, a atoms of the methyl acetate group form contacts with side key structural characteristic of serine proteases. This proves chains of H150 and H254 (Figure 2C). that rhomboids act by a mechanism that closely resembles Formation of an acyl intermediate is one of the hallmarks their soluble counterparts, thereby setting them in a clear of serine proteases, and the first description of serine enzymological context. Binding of inhibitor is accompanied proteases involved the trapping of an acyl intermediate, by conformational changes in the active site, the loop-5, and even before the sequence or structure was determined TM helices-5 and -6. The acyl enzyme structure suggests how (Hartley and Kilby, 1950). With the structure of an alkylated a substrate might bind at the active site and provides a acyl enzyme of GlpG, we now demonstrate unequivocally rationale for how rhomboid specificity can be achieved. 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- Results methoxy-isocoumarin reacts only with S201, confirms that Structure of a bacterial rhomboid in complex with this is indeed the reactive nucleophile in the enzyme. an inhibitor Although this has been suggested by previous biochemical Isocoumarin inhibition of classical serine proteases begins and structural study (for review, see Lemberg and Freeman, with nucleophilic attack by the catalytic serine, resulting in 2007a), and was hypothesised when rhomboids were first opening of the isocoumarin ring, and formation of an
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