Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry

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1 Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry Zhuoru Wu 1,4, Nieng Yan 1,4, Liang Feng 1,4, Adam Oberstein 1, Hanchi Yan 1, Rosanna P Baker 2, Lichuan Gu 3, Philip D Jeffrey 1, Sinisa Urban 2 & Yigong Shi 1 Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix α4 approximately 10 Å below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix α5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix α5. Intramembrane proteolysis is a widely conserved regulatory mechanism in species ranging from bacteria to humans 1 6. The first description of intramembrane proteolysis came from the investigation of cholesterol homeostasis, where the endoplasmic reticulum membrane bound transcriptional factor SREBP must be cleaved by an integral membrane protease known as site-2 protease (S2P) 7,8. This cleavage results in the release of the N- terminal domain of SREBP, which contains a DNA-binding domain and a transactivation domain 7. The N-terminal domain of SREBP regulates transcription of a number of genes that collectively control biosynthesis of cholesterol and fatty acid. Another prominent example of intramembrane proteolysis is the proteolytic processing of the amyloid precursor protein (APP) by the intramembrane protease γ- secretase, which is central to the development of Alzheimer disease 9,10. The cleavage product of APP, amyloid β peptide, shows pronounced toxicity to neuronal cells and is thought to contribute to Alzheimer disease 9. More recently, study of epidermal growth factor receptor (EGFR) signaling in Drosophila melanogaster identified rhomboid as an essential component in the signal-sending cells Rhomboid, a putative intramembrane protease, cleaves the ligand Spitz, which is inactive in its full-length form, thus regulating EGFR signaling spatially and temporally 13. There are four families of integral membrane proteins that are thought to catalyze intramembrane proteolysis: the serine protease rhomboid family, the metalloprotease S2P family, the aspartyl protease presenilin (catalytic subunit of γ-secretase) family and the signalpeptide peptidase family 2 4. The putative catalytic residues are predicted to be below the membrane surface and within the hydrophobic core of the proteases. If this is the case, as scission of peptide bonds requires the presence of water molecules, how do hydrophilic water molecules enter the active site? More importantly, if the active site is within the hydrophobic core of each protease, how do the substrate proteins gain access to the catalytic residues? Furthermore, are there some common principles that govern all four families of intramembrane proteases? These fundamental questions need to be addressed by a series of structures of the proteases at different stages of their action. The rhomboid family of intramembrane proteases represents a particularly attractive target for structural investigation. In addition to Drosophila rhomboid-1, which has been extensively characterized, representative rhomboid homologs have been reported to have important functions in human 15 17, yeast 18, parasite 19 and bacteria 20. Recently, several bacterially expressed rhomboid proteases have been purified and shown to have catalytic activity in vitro We expressed, purified and crystallized a conserved transmembrane core domain of GlpG, a bacterial rhomboid family member. During structural determination, we became aware of an online manuscript describing the structure of GlpG 24. We proceeded to solve our structure using molecular replacement. Comparison of our GlpG structure with that published online highlights noteworthy variations that reveal insights into the mechanisms of water entry and substrate access. These observations collectively broaden our understanding of the function of rhomboid proteases. RESULTS Characterization and crystallization of GlpG We mounted a systematic effort aimed at crystallizing a bacterial rhomboid family member. We cloned approximately 60 rhomboid homologs from 1 Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, New Jersey 08544, USA. 2 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. 3 State Key Laboratory of Microbial Technology, Shandong University, Jinan , China. 4 These authors contributed equally to this work. Correspondence should be addressed to Y.S. (ygshi@princeton.edu). Received 25 October; accepted 7 November; published online 10 November 2006; doi: /nsmb1179 NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

2 40 bacterial species and examined their expression in bacteria. Small-scale purification was attempted for all clones. On the basis of yield and solution behavior, six homologs were targeted for extensive characterization and crystallization. In all cases, truncation of the nonconserved N- terminal sequences was found to be essential for the generation of diffracting crystals. We primarily focused on crystals of the transmembrane core domain (residues ) of GlpG, a rhomboid family member from E. coli. To facilitate interpretation of structure, we actively sought and succeeded in the crystallization of the GlpG core domain under physiological ph (ph 7.4) and normal ionic strength. To characterize the transmembrane core domain of GlpG, we reconstituted two enzyme activity assays in vitro. First, we showed that the purified transmembrane core domain of GlpG was active toward an artificial protein substrate in detergent micelles and this proteolytic activity was higher at 37 C than at lower temperatures (Fig. 1a). The cleavage of the artificial substrate was due to the catalytic activity of GlpG, because the catalytic mutant S201A did not cleave the substrate (Fig. 1a). Next, we demonstrated that the transmembrane core domain of GlpG (GlpG N) retained a similar level of intramembrane proteolytic activity as the full-length GlpG, using C100Spitz-Flag as the substrate 23 (Fig. 1b, top gel). Notably, although both the full-length GlpG and the GlpG core domain were active toward C100Spitz-Flag, neither was able to cleave C100-Flag (Fig. 1b, bottom gel), suggesting maintenance of the same substrate specificity for Spitz-like transmembrane domains. These biochemical analyses validated the use of the transmembrane core domain of GlpG (hereafter referred to as GlpG) for crystallographic studies. Crystals of GlpG diffracted X-rays to about 2.6-Å resolution at synchrotron sources. We collected a native data set and prepared heavyatom derivatives. As we were trying to determine the crystal structure using MAD, we noticed the online publication of a manuscript describing the crystal structure of GlpG 24. Comparison of our study with that published 24 revealed apparent differences, including the space groups and the crystallization conditions. Notably, there are two molecules per asymmetric unit in our crystals. Encouraged by differences that might give rise to functional insights, we determined the crystal structure of GlpG using molecular replacement. The atomic model has been refined to 2.6-Å resolution. Overall structure There are two molecules of GlpG (molecules A and B) in one asymmetric unit, which form a pseudo dimer in the crystals (Fig. 1c). The overall structure of these two molecules is similar, with an r.m.s. deviation of 0.5 Å over 167 aligned Cα atoms of a a c d Figure 1 Overall structure of the transmembrane core domain of GlpG, an E. coli intramembrane protease of the rhomboid family. (a) The transmembrane core domain of GlpG is catalytically active. The membraneassociated protein CED-4 (ref. 39) was used as an artificial protein substrate for GlpG. Top gel, only the wild-type (WT) protein, and not the catalytic mutant S201A, cleaved the substrate. Bottom gel, the proteolytic activity of GlpG is higher at 37 C. (b) The transmembrane core domain of GlpG retains full intramembrane proteolytic activity and specificity. Top gel, comparison of intramembrane proteolytic activity between the full-length GlpG and the transmembrane core domain (GlpG N) revealed a similar level of enzymatic activity. Cleavage of the substrate, C100Spitz-Flag, was detected by anti-flag western blotting. Bottom gel, activity of GlpG and GlpG N against C100Spitz-Flag and C100-Flag revealed that both GlpG proteins show specificity for Spitz-like transmembrane domains. (c) Overall structure of GlpG in one asymmetric unit. Two molecules of GlpG associate to form a pseudo dimer. These two molecules show marked structural variation in helix α5 and the loop after α5 (known as the L5 loop or the cap 24 ). The putative catalytic-dyad residues Ser201 and His254 are highlighted in red. The extended L1 loop (magenta) comprises three short α-helices (H1, H2 and H3). (d) Structure of molecule A in the asymmetric unit. Left view emphasizes the packing of the L1 loop against the rest of the molecule. Compared with the other GlpG molecule (molecule B), the α5 helix of molecule A is bent away from the rest of the molecule, producing a lateral gap next to the catalytic residue Ser201 (shown in right view). Figure 4a and Figure 5a were prepared using GRASP 40 ; all other structural figures were made using MOLSCRIPT 41. b total of 190 amino acid residues (differences are discussed later). All rhomboid proteases are thought to contain a transmembrane core domain of six putative transmembrane helices 11,21,25,26. As predicted, the structure of the GlpG transmembrane core domain consists of six α-helices that are arranged more or less perpendicular to the surface of the lipid bilayer (Fig. 1d). Helices α1, α2 and α3 contain 20, 22 and 23 residues, respectively, and are likely to traverse the entire lipid bilayer (Figs. 1 and 2). Helices α1 α3 and the 2 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

3 N-terminal portion of α4 stack against one another through extensive van der Waals interactions. These packing interactions are further buttressed by an extended loop (L1 loop) between helices α1 and α2 (Fig. 1d). The L1 loop comprises three short α-helices (H1, H2 and H3) that stack against hydrophobic residues on helix α3. These structural features suggest that the N- terminal half of GlpG, including helices α1 α3 and loop L1, may constitute a structural scaffold on which the rest of the molecule functions (Figs. 1 and 2). Compared to α1 α3, helices α4 α6 are shorter, and none of the latter is expected to traverse the entire lipid bilayer. The putative catalytic residue Ser201 is located at the N terminus of helix α4, which is positioned approximately 10 Å below the membrane surface (Fig. 1d). This feature agrees well with the mapped cleavage sites in physiological rhomboid substrates, which typically occur several residues into the transmembrane segment from the extracellular side 11, Notably, helix α5 shows marked conformational differences in the two GlpG molecules in one asymmetric unit (discussed later). We will primarily focus our discussion on molecule A, the GlpG molecule that has its α5 helix tilted further away from the rest of the helices (Fig. 1d). Helix α6 contains the other putative catalytic-dyad residue, His254, which donates a hydrogen bond to Ser201 in both molecules of GlpG. Together, the conformational flexibility of helix α5, the shorter lengths of the helices and the presence of the catalytic dyad residues suggest that the C-terminal half of GlpG, encompassing helices α4 α6, constitutes a functional entity that catalyzes scission of peptide bonds on the structural scaffold of the N-terminal half of GlpG (Fig. 2). Figure 2 Sequence alignment of rhomboid homologs in diverse species. Secondary structural elements of GlpG are indicated above the sequences and colored as in Figure 1. Yellow highlight, conserved residues; blue highlight, invariant residues in all eight rhomboid homologs; magenta highlight, putative catalytic-dyad residues (Ser201 and His254 in GlpG). EcGlpG, E. coli (GI: ); PaRbd, Pseudomonas aeruginosa (GI: ); VcRbd, Vibrio cholerae (GI: ); YfRbd, Yersinia mollaretii (GI: ); DmRbd, D. melanogaster (GI: ); HsRbd1, Homo sapiens (GI: ); HsRbd2, H. sapiens (GI: ); ScRbd, Saccharomyces cerevisiae (GI: ). The two illustrations below the sequence alignment are designed to facilitate understanding of the sequence alignment information. The active site In molecule A, the putative active site is located at the bottom of a V -shaped cavity, with its opening at the extracellular side (Fig. 3a). This cavity is formed by the N-terminal portion of helix α2, the C-terminal portions of helices α3 and α5, the loop connecting helices α3 and α4 (L3) and the loop linking α5 and α6 (L5). The cavity is considerably larger in molecule A than observed previously 24, largely owing to the opening of transmembrane helix α5 away from the rest of the molecule (discussed later). The cavity is open to solvent and the L5 loop has moved away (Fig. 3a), whereas in the published structure the cavity is closed by the L5 loop (termed the cap) 24. There are three well-ordered water molecules in this cavity, one of which makes a hydrogen bond to the hydroxyl oxygen atom of the putative catalytic residue Ser201 (Fig. 3a). The other putative catalytic-dyad residue, His254, stabilizes Ser201 through a hydrogen bond. In molecule B, the size of the cavity is smaller than that in molecule A; there is little or no electron density for residues in the L5 loop, indicating a high degree of flexibility. There are ten invariant transmembrane residues among most members of the rhomboid family 25, including four glycines (Gly199, Gly202, Gly257 and Gly261), three histidines (His145, His150 and His254), one serine (Ser201), one asparagine (Asn154) and one alanine (Ala253) (Fig. 2). Supporting functional significance of the cavity, all ten invariant residues map to the cavity or its vicinity in the GlpG structure (Fig. 3a). Compared with His254, Asn154, once thought to be a catalytic-triad residue, is located on the opposite side of transmembrane helix α4. This structural organization rules out the possibility that Asn154 would stabilize His254 in catalysis and is consistent with the hypothesis that rhomboid proteases use a Ser-His dyad for catalysis 22. Another invariant residue, His150, is approximately 4.5 Å away from Ser201 (Fig. 3a). The third histidine, His145, is buried in the hydrophobic core, away from the cavity (Fig. 3a). The invariant alanine and glycine residues have at least a structural role, as they are in close contact with neighboring residues. NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

4 Figure 3 Conformation of the active site and the L1 loop. (a) Ribbon diagram of GlpG (molecule A) showing the open cavity leading to the active site. All invariant residues among the eight rhomboid homologs in Figure 2 are shown. Red side chains, putative catalytic-dyad residues Ser201 and His254; gold side chains, all other invariant residues; red spheres, three water molecules in the cavity. Ser201 hydrogen-bonds to His254 as well as a water molecule. (b) Stereo view of interactions surrounding the conserved Trp-Arg motif in GlpG. Trp136 and Arg137 appear to stabilize the conformation of the L1 loop by participating in a network of hydrogen bonds as well as van der Waals interactions with surrounding residues of the L1 loop. (c) Stereo view of packing interactions between residues of the L1 loop and residues in helix α3 and the L3 loop. This interface is dominated by extensive van der Waals interactions. (d) Stereo comparison of packing interactions involving the L1 loop in our structure and in that reported recently 24. Coloring of our structure is as in a except that all side chains are colored yellow. The main chain and side chains of the published structure 24 are in gray. The structure and the packing interactions are nearly identical between these two structures. of Arg137 resulted in more pronounced reduction of protease activity compared with mutation of Trp136 (ref. 11), consistent with their respective structural roles. Supporting the notion that the L1 loop has a structural role, hydrophobic residues in the L1 loop stack against nonpolar residues in the L3 loop and the C-terminal half of helix α3 through many van der Waals interactions (Fig. 3c). Notably, the extensive interactions both within the L1 loop and between the L1 loop and the rest of the GlpG protein are essentially identical between our structure and that published 24. In fact, the main chain as well as a great majority of the side chains in L1 and α3 have identical conformations in both structures (Fig. 3d). In the recent report 24, the L1 loop is proposed to be a lateral gate for substrate entry. This hypothesis may not be consistent with the extensive interactions both within the L1 loop (Fig. 3b and data not shown) and between the L1 loop and the rest of the structure (Fig. 3c). In addition, if the L1 loop were a lateral gate to allow substrate entry, then any mutation destabilizing the L1 loop structure would probably enhance substrate entry and cleavage. This scenario is not supported by the reported effects of mutations of Arg137 and Trp136 in the L1 loop 11,22. Furthermore, compared with the L1 loop, the catalytic residue Ser201 is located on the opposite side of transmembrane helix α4, which suggests that Ser201 is unlikely to be able to catalyze scission of the peptide bond in the substrate unless α4 undergoes pronounced conformational changes. Because helix α4 is at the center of the entire protein, any structural rearrangements in α4 are likely to propagate to the rest of the protein. These analyses support the argument that the L1 loop is unlikely to be a lateral gate controlling substrate entry, as proposed 24. The L1 loop An important element of the active-site cavity is the L3 loop, which stacks against the extended L1 loop. The L1 loop contains three short α-helices that pack against one another and against helix α3 and the L3 loop. There are considerable packing interactions among residues within the L1 loop. Two highly conserved residues, Trp136 and Arg137, serve a structural function by participating in a network of hydrogen bonds (Fig. 3b). At the center of the L1 loop, the guanidium group of Arg137 donates five hydrogen bonds to neighboring residues: two charge-stabilized contacts to Glu134 and three hydrogen bonds to backbone carbonyl oxygen atoms of residues 121 and 122 (Fig. 3b). The carbonyl oxygen atom of residue 122 accepts an additional hydrogen bond from the side chain of Trp136. In addition, Arg137 makes a number of van der Waals contacts with surrounding residues in loop L1 (Fig. 3b). These observations predict that mutation of Arg137 and, to a lesser extent, mutation of Trp136 might compromise the structural stability of rhomboid proteases, hence resulting in compromised function. This prediction is supported by the report that mutation of either residue leads to reduced or abolished protease activity 11,22. Notably, mutation The L5 loop and access to water molecules In our crystal structure, both molecules of GlpG in one asymmetric unit contain a water-accessible cavity and have an opening toward the extracellular side. The opening in molecule A is particularly large (Fig. 4a and Supplementary Videos 1 and 2 online). There are several well-ordered water molecules in the cavity, one of which is hydrogen bonded to Ser201. Comparison with the published structure 24, in which the cavity is tightly closed from the extracellular side by the L5 loop (hereafter referred to as the closed state), shows that the opening of the cavity in molecule A of our structure is provided by an outward movement of the α5 helix, which drags the L5 loop with it, resulting in an open conformation (Fig. 4b). Molecule A is hereafter referred to as the open state. Compared with helix α5 in the closed state 24, the C-terminal half of α5 in the open state is bent by approximately 35 (Fig. 4b,c), resulting in a movement of 5 10 Å for residues at the C terminus of α5 and in the L5 loop. In molecule B, the conformation of the α5 helix is similar to that in the closed state and is considerably different from that in molecule A (open state) (Fig. 4d). However, the entire L5 loop (residues ) 4 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

5 Figure 4 Mechanisms of water access and substrate entry to the active site. (a) Molecule A of GlpG exists in an open conformation. Left and right images show mesh surface representation and electrostatic potential surface of GlpG, respectively. The putative catalytic-dyad residues Ser201 and His254 (red) are at the bottom of the open cavity. The cavity also contains three water molecules (orange spheres), which are thought to come into the cavity from the extracellular side. (b) Stereo comparison of GlpG in the open state (molecule A, blue) and the closed state (orange) 24. Most α-helices are superimposed well, except helix α5, which is bent away from the active site in the open form, producing a large cleft between helices α2 and α5. Helix α5 is proposed to be the gating helix for substrate entry into the active site of GlpG. (c) Stereo comparison of the α5 helices in the open and closed forms of the GlpG protein. The 2F o F c omit electron density, which was calculated with the omission of the entire helix α5 and the L5 loop (residues ), is contoured in gray at 1.5 σ around helices α5 and α4. (d) Stereo comparison of GlpG in the open state (molecule A, blue) and the half-open state (molecule B, green). The half-open GlpG has a structure similar to that of the closed form 24, except that the L5 loop is flexible and disordered in the crystals. has no electron density and is disordered in solution. This observation suggests that the L5 loop is flexible and may readily open to the extracellular side to allow water entry into the cavity. How can this conclusion be reconciled with the observation that the L5 loop is tightly closed onto the cavity in the closed state 24? One explanation is that the L5 loop does not adopt a rigid conformation and different crystallization conditions may favor distinct conformations. The closed GlpG was crystallized in the presence of high ionic strength (3 M sodium chloride) 24, which favors hydrophobic interactions. Thus, it is plausible that the L5 loop may close under this condition and interact with hydrophobic residues at the opening of the cavity. Supporting this explanation, six of nine residues in the L5 loop are hydrophobic amino acids (Fig. 2) that make van der Waals contacts with nonpolar residues at the opening of the cavity in the closed state 24. It should be noted, however, that several water molecules are also present in the active site cavity of the closed GlpG 24, suggesting some degree of conformational flexibility for the L5 loop. Why doesn t the transmembrane helix α5 show conformational flexibility in the published study 24? One likely explanation might be that helix α5 from one GlpG molecule tightly packs against helices α1 and α2 from an adjacent molecule in the crystals 24, ruling out any possibility of noticeable movement for α5. GlpG already contains a lateral opening at its top region that is large enough to accommodate an extended polypeptide chain (Fig. 4a and Supplementary Videos 1 and 2). Presumably such a polypeptide chain is created by unwinding of the top region of the substrate transmembrane helix. This would be consistent with mutagenesis data mapping the crucial region in rhomboid substrates to the top of their transmembrane segments 11, Hence, on the basis of structural observations, we propose a molecular mechanism by which the α5 helix controls substrate entry (Fig. 5a). Structural observations suggest that the rhomboid protease GlpG exists in three distinct conformations: closed 24, half-open (molecule B) and open (molecule A). In our proposal, the L5 loop regulates access of water molecules, which arrive at the active site from the extracellular side through the cavity. Entry of protein substrate is controlled by the α5 helix, which serves as a lateral gate. Bending of the α5 helix away from the rest of the molecule results in an open conformation of the protease. Only in the open conformation can a substrate protein gain access to the active site via the α5 gate (Fig. 5a). Given the considerable sequence conservation (Fig. 2), we further propose that the mechanisms described for GlpG probably apply to other members of the rhomboid family of proteases. Mechanism of substrate entry via helix α5 Our structure suggests a plausible mechanism for substrate entry to the active site of GlpG. The observed conformational shift of the α5 helix makes it an ideal gating device for the regulation of substrate entry (Fig. 4). In fact, because of the outward bending of helix α5, the cavity in the open state of DISCUSSION The strongest support for the proposed mechanism of substrate entry via the α5 gate is provided by the crystal structure itself, which unambiguously shows the existence of such an open conformation in the crystal lattice. We note that our GlpG crystals were generated under ph 7.4 and normal ionic strength, both of which are close to NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

6 a b physiological conditions. In addition, sequence analysis also supports the proposed mechanism. Helices α2, α4 and α6 are unlikely to undergo major conformational rearrangements, because each helix contains at least two invariant residues that are thought to have important structural or catalytic roles, or both. Helices α1 and α3 are farther away from the vicinity of Ser201. In contrast, the α5 helix is one of the least conserved helices in the rhomboid family 25 (Fig. 2). Thus, the α5 helix is an ideal structural element for providing access to entering substrate without causing the entire molecule to rearrange. Furthermore, helix α5 is immediately linked to the L5 loop, which is proposed to regulate water access to the active site. This arrangement ensures that, when the lateral gate (α5) is open, there is unobstructed access of water molecules to the catalytic residue Ser201. One noteworthy structural observation is that the catalytic residue Ser201 is placed at the bottom of a water-filled cavity, approximately 10 Å below the membrane surface. Thus, scission of the peptide bond occurs in a hydrophilic environment that is below the membrane surface but is protected by a local protein environment. In our proposal, the Closed Open (active) Substrate access Figure 6 A proposed general mechanism for intramembrane proteases. In this model, presenilin, S2P and signal-peptide peptidase may each contain a water cavity that opens to the cytoplasm or extracellular region. As in rhomboid GlpG, this water-accessible cavity is probably protected from the hydrophobic lipid bilayer by α-helices and embedded loops. Before catalysis, one or more of the surrounding helices undergoes a structural switch that opens a lateral gate to allow entry of substrate protein. Star denotes active site. Figure 5 A proposed model of action for the rhomboid family of intramembrane proteases. (a) Schematic of the proposed model. In the closed state 24, the L5 loop (the cap) closes onto the cavity, disallowing continued access of water molecules (black dots) to the active site. In the half-open state (molecule B), the L5 loop is flexible and presumably open, allowing access of water molecules to the active site. In the open state (molecule A), the α5 helix is bent and twisted away from the active site, producing a large cleft between helices α2 and α5. This cleft is large enough to allow entry of polypeptide chain into the active site for scission of peptide bond. Surface representations of GlpG in the three states are shown above the schematic diagram. Magenta circle marks active site and water-entry cavity. (b) The L1 loop is unlikely to control substrate entry into the active site. The L1 loop appears to have a structural role in GlpG (Fig. 3) and is unlikely to open up as proposed 24. Even if the L1 loop is completely dislocated from its present position, the substrate polypeptide chain is still unable to reach the catalytic residue Ser201, which resides on the other side of helix α5. In addition, the L3 loop may obstruct access of substrate protein to Ser201. L5 loop is thought to regulate water entry to the cavity. Given the close proximity of the cavity to the extracellular side, it is entirely possible that thermal motions of the L5 loop allow the cavity to be filled with water at all times. Supporting this notion, water molecules are present in the active site cavity of the closed GlpG structure 24. Despite the identification of hundreds of rhomboid family proteases in diverse species, identification of the cleavage sites in rhomboid substrates has been slow to emerge. In Drosophila, Rhomboid-1 recognizes and cleaves its substrate within the sequence ASIASGA 30, which is the N-terminal portion of the transmembrane helix. In Toxoplasma gondii, the rhomboid substrate MIC2 is cleaved several residues into the predicted transmembrane helix at an Ala-Gly bond in the sequence AIA- GGVI (hyphen denotes bond cleaved) 28,29. In Plasmodium falciparum, the substrate protein EBA-175 is cleaved at the N-terminal portion of the transmembrane helix at an Ala-Gly bond in the sequence YYA-GAGV 27. Thus, rhomboid-mediated cleavage of the substrate transmembrane helix, typically after an alanine residue, seems to occur several residues into the transmembrane helix from the extracellular side or Golgi lumen 28,30,31. This feature agrees well with the location of the catalytic residue Ser201 in GlpG. The published report 24 proposes that the L1 loop is a lateral gate for substrate entry and that the substrate enters between helices α1 and α3. This hypothesis does not seem to be fully compatible with structural analysis (Fig. 3b,c) or mutagenesis data 11,22. Notably, compared with the L1 loop, the catalytic residue Ser201 is located on the opposite side of helix α4, which suggests that Ser201, in its present conformation, is unlikely to be able to catalyze scission of the peptide bond if the substrate enters between helices α1 and α3 (Fig. 5b). For catalysis to occur in this way, major structural rearrangements involving turning or melting of helix α4 are probably required (Fig. 5b). However, such structural changes are extremely unlikely, because helix α4 is at the center of the entire protein. Any structural rearrangements involving helix α4 are likely to produce pronounced perturbation to the structure and to disrupt the hydrogen bond between Ser201 and His254. Because GlpG was crystallized in the presence of detergents, it is possible that the observed conformations are in part induced by interactions with the detergent molecules. However, the probability of this is low, as the same detergent, nonyl glucoside, was used in the crystallization of GlpG in our study as was used in the published report 24. Another important factor that can influence protein conformation is crystal lattice contact. The space groups are different in these two studies, each involving quite different crystal contacts. Helix α5 is largely exposed to solvent in our crystals; in the other study 24, however, helix α5 6 VOLUME 13 NUMBER 12 DECEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

7 from one GlpG molecule packs closely against helices α1 and α2 of an adjacent molecule. These different packing interactions probably contribute to the observed conformational differences in helix α5. Although the four families of intramembrane proteases share no apparent sequence similarity, they are all predicted to contain multiple transmembrane helices. It is possible that these four families of intramembrane proteases share a common set of biophysical principles used in regulating water access and protein substrate entry. In this study, we report structural evidence to support the notion that access to water for the rhomboid protein GlpG is provided by a cavity that opens to extracellular side. Moreover, our structural analysis strongly suggests that substrate entry to the active site is gated by the transmembrane helix α5, which shows remarkable conformational flexibility. We further speculate that the mechanisms observed for GlpG may generally apply to other families of intramembrane proteases (Fig. 6). Presenilin, S2P and signal-peptide peptidase may contain a water-filled cavity that opens to the cytoplasm or extracellular region (Fig. 6). As in rhomboid GlpG, this cavity is probably protected from the hydrophobic lipid bilayer by α-helices and embedded loops. Before catalysis, one or more of the surrounding helices may undergo a structural switch that opens a lateral gate to allow entry of substrate protein (Fig. 6). The proposed lateral movement of α-helices within the lipid bilayer to control substrate entry into intramembrane proteases is similar to the mechanism in other systems such as the protein-conducting channel 32. Supporting the notion of a uniform principle, analysis of γ-secretase by electron microscopy has revealed the presence of a cylindrical interior chamber 33,34 that might be used for water entry. Our structural investigation represents only the first step toward a comprehensive understanding of the mechanisms and function of intramembrane proteases. This structure of a rhomboid family protease not only confirms many of the functional characteristics and mechanistic proposals, but also gives rise to two central elements of our conclusion: water molecules gain access to the active site through a central cavity and the transmembrane helix α5 controls substrate entry through lateral gating. These mechanisms, supported by structural evidence and sequence analysis, engender a number of experimentally testable hypotheses. METHODS Protein preparation. The complementary DNAs of 60 bacterial rhomboid homologs were cloned into pet15b (Novagen) and the recombinant proteins were overexpressed in E. coli C43 (DE3). After first-round screening, E. coli rhomboid homolog GlpG became one of our focused targets, given its high yield and good solution behavior during purification. The transformed E. coli cells were induced with 0.5 mm IPTG at an A 600 of 1.5. After growing for 8 h at 37 C, the cells were harvested, homogenized in a buffer containing 25 mm Tris (ph 8.0) and 500 mm NaCl and lysed by French press. Cell debris was removed by centrifugation. The supernatant was collected and subjected to ultracentrifugation at 150,000g for 1 h. Membrane fraction was dissolved with 2% (w/v) n-nonyl-β-d-glucoside (NG, Anatrace) and loaded onto a nickel-nitrilotriacetic affinity column (Qiagen). The protein was eluted with a buffer containing 25 mm Tris (ph 8.0), 150 mm NaCl, 250 mm imidazole and 0.4% (w/v) NG, and the N-terminal His 6 tag was removed by thrombin. The protein was concentrated to about 20 mg ml 1 and applied to Superdex-200 columns (GE Healthcare) in a buffer containing 10 mm Tris (ph 8.0), 150 mm NaCl and 0.4% (w/v) NG. The peak fraction was collected for crystallization. During purification, however, we noticed that there was always a degradation product about 10 kda smaller than the expected size, indicating that the full-length protein was unstable. To identify a stable core domain of GlpG, we digested the protein with different proteases, including chymotrypsin, trypsin, subtilisin and elastase. Most of the proteolysis trials generated a similar stable core domain of about 20 kda. In particular, chymotrypsin digestion yielded a very stable doublet. N-terminal sequencing of the chymotrypsindigested GlpG showed that both fragments began at residue 87. We engineered a core domain (residues ) of GlpG. This protein was purified using the protocol described above. Crystallization and data collection. Crystals of the GlpG transmembrane core domain were grown at 22 C using hanging drop vapor diffusion. The well buffer contained 0.1 M tricine (ph 7.4), 6% (w/v) PEG 3,000 and mm Li 2 SO 4. Crystals of trigonal plate appeared immediately and grew to full size within 5 d. The crystals belong to the space group P3 1 and contain two molecules per asymmetric unit. The unit cell has dimensions of a = b = Å, c = Å, and α = β = 90, γ = 120. Crystals were equilibrated in a cryo-protectant buffer containing reservoir buffer plus 20% (v/v) glycerol and were flash-frozen in a cold nitrogen stream at 170 C. The native data set was collected at National Synchrotron Light Source beamline X29 and processed using DENZO and SCALEPACK 35. Structure determination. We solved the structure by molecular replacement using the GlpG coordinates 24 (see Acknowledgments) and PHASER 36. The solutions were unambiguous and preliminary refinement dropped the free R-factor to about 37%. Pronounced differences were noticed around residues , including the entire α5 helix and the L5 loop. There are two molecules of GlpG in each asymmetric unit. Noncrystallographic symmetry constraint was applied in the early refinement cycles. The atomic model was examined and modified using O 37 and refined to 2.6-Å resolution using CNS 38. The final refined Table 1 Data collection and refinement statistics GlpG Data collection Space group P 3(1) Cell dimensions a, b, c (Å) 59.46, 59.46, α, β, γ ( ) 90, 90, 120 Resolution (Å) ( ) R sym (0.508) I / σi 32.0 (2.1) Completeness (%) 98.5 (93.9) Redundancy 9.0 (6.3) Refinement Resolution (Å) No. reflections 13,455 R work / R free / No. atoms 2,861 Protein 2,835 Water 26 B-factors Protein Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) 1.84 X-ray diffraction data were collected on one crystal. Values in parentheses are for highestresolution shell. NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 12 DECEMBER

8 atomic model contains amino acid residues for molecule A and residues and for molecule B (Table 1). Consistent with conformational flexibility, residues in molecule A and residues in molecule B have high temperature factors. Residues in molecule B have little or no electron density and are presumably disordered in solution. In the final atomic model, 86.7% and 12.7% of all residues fall in the most favored and additionally allowed regions of the Ramachandran plot, respectively. One residue in each GlpG molecule (0.6%) resides in the disallowed region of the Ramachandran plot. Protease activity assay. The proteolytic activity of GlpG was examined in two assays. In one assay, an artificial protein substrate, CED-4, which is a membrane-associated protein 39, was used as the substrate. The assay was performed at 37 C (unless otherwise indicated) for 1 h in a buffer containing 150 mm sodium chloride, 0.5% (w/v) nonylglucoside and 25 mm Tris-HCl (ph 8.0). The concentration of the substrate protein was approximately 2 mg ml 1. The concentration of GlpG was 0.1 mg ml 1. The reaction was stopped by addition of SDS sample buffer and the cleavage products were analyzed by SDS-PAGE. In the other assay, C100Spitz-Flag or C100-Flag was used as the substrate, as described 23. Reactions were conducted at 37 C for 1 h. Accession codes. Protein Data Bank: Coordinates have been deposited with accession code 2NRF. ACKNOWLEDGMENTS We thank A. Saxena at Brookhaven National Laboratory National Synchrotron Light Source beamlines for help, J. Chai and Q. Liu for technical discussion and Y. Ha (Yale University) for the atomic coordinates of GlpG. This work was supported by Princeton University (Y.S.). Work in the Urban laboratory is supported by US National Institutes of Health grant 1R01AI and a Career Award in the Biomedical Sciences from the Burroughs-Wellcome Fund (to S.U.). 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