Structure

Article A Structure-Based Model of Substrate Discrimination by a Noncanonical PDZ Tandem in the Intramembrane-Cleaving Protease RseP Yohei Hizukuri,1 Takashi Oda,2 Sanae Tabata,3 Keiko Tamura-Kawakami,3 Rika Oi,2 Mamoru Sato,2,4 Junichi Takagi,3 Yoshinori Akiyama,1 and Terukazu Nogi2,* 1Institute

for Virus Research, Kyoto University, Kyoto 606-8507, Japan School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan 3Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan 4RIKEN SPring-8 Center, Kouto 1-1-1, Sayo, Hyogo 679-5148, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.12.003 2Graduate

SUMMARY

During the extracytoplasmic stress response in Escherichia coli, the intramembrane protease RseP cleaves the anti-sE protein RseA only after the membrane-anchored protease DegS truncates the periplasmic part of RseA that suppresses the action of RseP. Here we analyzed the three-dimensional structure of the two tandemly arranged PSD-95/Dlg/ZO-1 (PDZ) domains (PDZ tandem) present in the periplasmic region of RseP and revealed that the two putative ligand-binding grooves constitute a single pocketlike structure that would lie just above the active center sequestrated within the membrane. Complete removal of the PDZ tandem from RseP led to the intramembrane cleavage of RseA without prior truncation by DegS. Furthermore, mutations expected to destabilize the tertiary structure of the PDZ tandem also caused the deregulation of the sequential cleavage. These observations suggest that the PDZ tandem serves as a size-exclusion filter to accommodate the truncated form of RseA into the active center.

INTRODUCTION Regulated intramembrane proteolysis (RIP) is a process wherein membrane proteins are cleaved in their transmembrane (TM) segments by the action of a specific protease family termed intramembrane-cleaving proteases (I-CLiPs) (Weihofen and Martoglio, 2003; Wolfe, 2009a; Wolfe et al., 1999). All of the known I-CLiPs are polytopic membrane proteins and are presumed to possess their active center within the membrane. The first example of RIP was identified in the cleavage of sterol regulatory element binding proteins (SREBPs) that are known precursors of transcription factors for sterol and fatty acid biosynthesis (Sakai et al., 1996). SREBPs are cleaved sequentially—first within the lumen and then in the first TM region—resulting in the liberation of the N-terminal intracellular domain from the membrane. The

first cleavage is catalyzed by site-1 protease (S1P), a membrane-bound protease containing the active center in the luminal domain (Sakai et al., 1998), whereas site-2 protease (S2P), a Zn2+-coordinating metalloprotease whose active center is sequestrated within the membrane, performs the intramembrane cleavage (Rawson et al., 1997). To date, two other I-CLiP families, the rhomboid and g-secretase/SPP families, have been identified, and many of these family members catalyze cleavage of membrane-bound signaling molecules such as transcription factors or receptor ligands (Weihofen and Martoglio, 2003). RIP is therefore now accepted as a novel form of TM signaling (Chen and Zhang, 2010; Wolfe, 2009b). Full-length structures of bacterial or archaeal homologs have already been determined through X-ray crystallography for all of the three I-CLiP families (Feng et al., 2007; Li et al., 2013; Wang et al., 2006), and those studies provided a significant amount of structural information about the catalytic sites and their environment. Nevertheless, it remains to be resolved how the I-CLiPs recognize their target or discriminate between membrane proteins within the lipid bilayer because no substrate-enzyme complex structures are available so far for any of the I-CLiPs. Escherichia coli RseP, known to be involved in the extracytoplasmic stress response, is one of the most biochemically characterized I-CLiPs. Yet even for this I-CLiP, it has not been fully explained how the target membrane protein is recognized on the cell membrane. During stress response, the sE transcription factor is activated through the two-step sequential cleavage of the anti-sE protein RseA (Figure 1). Upon exposure to extracytoplasmic stress, the membrane-anchored protease DegS first cleaves RseA between V148 and S149 in the periplasmic region (Alba et al., 2002; Kanehara et al., 2001, 2002; Walsh et al., 2003), and only this DegS-cleaved product of RseA is subjected to the intramembrane proteolysis by RseP. Hence, there must be some mechanism by which RseP senses the occurrence of the first cleavage. RseP is composed of four TM regions (TM1–TM4) with an N-out, C-out topology, and it possesses two tandemly arranged PSD-95/Dlg/ZO-1 (PDZ) domains, PDZ-N and PDZ-C, in the periplasmic region between TM2 and TM3 (Inaba et al., 2008). Because PDZ domains generally recognize the C-terminal three to five residues of a ligand protein (Harris and Lim, 2001), it was proposed that PDZ-C might interact with the newly exposed

326 Structure 22, 326–336, February 4, 2014 ª2014 Elsevier Ltd All rights reserved

Structure RseP Discriminates Substrates by Size Exclusion

Figure 1. Two-Step Sequential Proteolysis of RseA during the Extracytoplasmic Stress Response in E. coli Extracytoplasmic stress such as exposure to heat and alkali causes accumulation of denatured OMPs in the periplasmic space. The exposed C terminus of the unfolded OMPs (YxF) binds with and activates the bacterial S1P counterpart DegS that is present as trimer on the membrane and contains the catalytic triad (D, H, S) characteristic of the serine protease family in the periplasmic soluble domain. The activated DegS cleaves the periplasmic region of the full-length RseA (RseA FL), resulting in the release of the negative regulator RseB. The truncated RseA [RseA(DP)] is accepted by RseP as its substrate and is cleaved within the membrane. RseP contains two PDZ domains (PDZ-N and -C) in the periplasmic region and the Zn2+-coordinating sequences (HExxH and LDG) within the TM regions. As a consequence of the periplasmic (site-1) and intramembrane (site-2) cleavages, the transcription factor sE is liberated from the membrane and finally induces the expression of the stress-responsive genes. In the present study, the structural analyses have been performed on the PDZ tandem regions (as enclosed with the red dotted box).

C-terminal residue V148 of DegS-cleaved RseA. In vitro cleavage assays using RseP and RseA proteins that were purified and solubilized with detergent demonstrated that the second cleavage was abolished by mutation to dissimilar residues at V148 in RseA (Li et al., 2009). Mutation of a residue located at the center of the putative ligand-binding groove of PDZ-C yielded similar results. Contradictory to these reports, we have shown that mutations of V148 little affected cleavage of the RseA derivatives by RseP when the cleavage assays were performed in vivo (Hizukuri and Akiyama, 2012). Similarly, we found that missense mutations to the putative ligand-binding grooves of the PDZ domains or even complete deletion of the PDZ domains does not abolish the proteolytic activity of RseP toward RseA in vivo. Our recent results strongly indicate that binding of the newly exposed RseA C terminus to PDZ-C is not the crucial step that leads to the second cleavage by RseP on the membrane. In contrast, several regulatory factors that suppress the second cleavage by RseP before the DegS cleavage have been identified. The periplasmic region of RseA contains two glutamine-rich regions downstream of the DegS cleavage site and binds to a periplasmic soluble protein RseB. It is known that deletion of the glutamine-rich regions results in the cleavage of the full-length RseA even without the first cleavage by DegS (Kanehara et al., 2003). A structural study has elucidated that RseB intimately interacts with the region close to the two gluta-

mine-rich regions of RseA (Kim et al., 2010), suggesting that RseA-RseB complex formation is involved in cleavage suppression. In fact, full-length RseA is cleaved at the TM region in a DegS-independent manner without RseB (Grigorova et al., 2004). Furthermore, it is presumed that the PDZ domains of RseP also contribute to the suppression of the second cleavage based on the observation that mutations to or partial deletion of the PDZ domains also led to the DegS-independent cleavage of the full-length RseA (Inaba et al., 2008; Kanehara et al., 2003). The molecular mechanism of the negative regulation, however, has not yet been fully revealed. As suggested by the discrepancies between the in vitro and in vivo mutational analyses, the folding and relative positioning of RseP and RseA in the lipid bilayer appear to be important for regulating the sequential cleavage properly. Hence, it must be determined how the two PDZ domains are arranged in the context of the full-length RseP and, in particular, with respect to the cell membrane, for a precise understanding of their functional role. Although a full-length structure has been determined for an S2P homolog from Methanocaldococcus janaschii (MjS2P) by X-ray crystallography (Feng et al., 2007), that homolog contains no PDZ domains in the periplasmic region. The crystal structures of the individual PDZ-N and -C domains of RseP are available (Inaba et al., 2008; Li et al., 2009), but even the relative arrangement of the two domains remains unknown. In the present study, we analyzed the three-dimensional (3D) structure of the entire PDZ tandem at an atomic level using a bacterial homolog of RseP. Then, we investigated how the PDZ domains are involved in the two-step sequential cleavage of target membrane proteins with mutational analyses on RseP. Based on the results, we propose a model wherein the PDZ domains create a pocket-like structure that discriminates substrates through a steric size-exclusion mechanism. Our model readily explains the biochemical data for this system from previously published reports and our own investigations in this study. RESULTS AND DISCUSSION Crystal Structure of the PDZ Tandem of the Bacterial RseP Homolog In the previous structural study of the PDZ domains, we attempted to crystallize the entire PDZ tandem fragment of the E. coli RseP (EcRseP). However, we obtained no crystals and only performed small-angle X-ray scattering (SAXS) analysis (Inaba et al., 2008). Because the two PDZ domains of EcRseP, EcPDZ-N and EcPDZ-C, are connected with a six-residue linker, the entire PDZ tandem might be too flexible to crystallize. We therefore tried to purify and crystallize a PDZ tandem fragment of an RseP homolog from the hyperthermophilic bacterium Aquifex aeolicus (AaRseP). Because sequence alignment suggested that the inter-PDZ linker is composed of only three residues, the PDZ tandem of AaRseP (AaPDZ tandem) was expected to be more rigid and suitable for crystallization. In addition, we generated a monoclonal antibody against the AaPDZ tandem to use its Fab fragment as a crystallization chaperone. Consequently, we have solved the crystal structures of AaPDZ tandem both in solitary (‘‘Fab-free’’ form) and in complex with the Fab fragment (‘‘Fab-complex’’ form) at 2.8 and 2.2 A˚ resolution, respectively (Table 1).

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Structure RseP Discriminates Substrates by Size Exclusion

Table 1. Data Collection and Refinement Statistics Crystal Form

Fab-Free Form

Fab-Complex Form

P6522

P1

Data collection Space group Cell dimensions a, b, c (A˚)

78.86, 78.86, 211.82 51.24, 86.04, 92.54

a, b, g (
)

90, 90, 120

100.84, 96.92, 101.32

No. of monomers or complexes/asu

1

2

X-ray source

PF/BL-17A

PF/BL-17A

Wavelength (A˚)

1.00000

1.00000

Resolution limits (A˚)

39.4–2.80 (2.95–2.80)

47.4–2.20 (2.32–2.20)

No. of unique reflections 10,257 (1,435)

74,923 (10,882)

Completeness (%)

99.8 (99.6)

98.2 (97.2)

Redundancy

13.8 (14.4)

2.2 (2.1)

I/s(I)

32.5 (6.2)

10.2 (1.8)

Rmergea

0.068 (0.404)

0.074 (0.450)

Resolution limits (A˚)

39.4–2.80 (2.95–2.80)

47.4–2.20 (2.26–2.20)

Rworkb

0.255 (0.346)

0.224 (0.302)

Rfreec

0.295 (0.410)

0.272 (0.362)

No. of non-H atoms

1,409

9,615

Protein/solvent

1,409/0

9,226/389

Refinement

Averaged temperature factors (A˚2) Protein

55.40

40.27

PDZ tandem (1)

30.26

PDZ tandem (2)

32.65

Fab (1)

37.70

Fab (2)

49.96

Solvent

34.37

Rmsd from ideality Bond length (A˚)/bond

0.007/1.25

0.007/1.17

92.70/0.56

96.26/0.17

angle (
) Ramachandran plot (MolProbity) Favored (%)/outlier (%)

Values in parentheses are for highest resolution shell. asu, asymmetric unit; rmsd, root mean square deviation. a Rmerge = ShSijIi(h)  j/ShSiI(h), where Ii(h) is the ith measurement. b Rwork is the crystallographic R-factor (Rcryst) for the working set used for the refinement. Rcryst = ShjjFobs(h)j  jFcalc(h)jj/Shj Fobs(h)j, where Fobs(h) and Fcalc(h) are the observed and calculated structure factors. c Rfree is Rcryst calculated for the test set consisting of 5% of reflections excluded from the refinement.

The entire AaPDZ tandem fragment adopts an overall ‘‘clamshaped’’ structure (Figure 2), where the putative ligand-binding grooves of the two PDZ domains are aligned in an antiparallel manner on the same side of the molecule. With this arrangement, the two grooves are combined and constitute a single internal ‘‘pocket-like’’ structure with a rather hydrophobic interior (Figure 2C). Consistent with the topology of the EcPDZ domains,

both of the two PDZ domains (AaPDZ-N and -C) showed a circular permutation compared with the topology of the canonical PDZ fold (Figures S1A and S1B available online). The canonical PDZ domain is composed of six b strands (bA–bF) and two a helices (aA and aB) in which the N terminus is located before bA and the C terminus is after bF. The RseP-type PDZ domains possess the N terminus before bC and the C terminus after bB, whereas bA and bF are connected with a loop (Figures S1C and S1D). The three-residue inter-PDZ linker (L206 to K208) connects bB of AaPDZ-N and bC of AaPDZ-C, thereby enabling the formation of the pocket-like structure by tethering the two putative ligand-binding grooves to one another. Inspection of the individual structures suggested that the canonical mode of accommodation for a long peptide ligand is unfavorable in the ligand-binding grooves of AaPDZ-N and -C, as was reported for the EcPDZ-N and -C domains (Inaba et al., 2008; Li et al., 2009). Generally, a PDZ domain captures its ligand peptide as an antiparallel extension of the b sheet off of the bB strand (Harris and Lim, 2001). Both of AaPDZ-N and -C possess a proline residue in bB (P205 and P289, respectively) that should disturb formation of the sheet extension (Figures 3A and 3B). In addition, a tryptophan residue in aB (W157 and W242 for AaPDZ-N and -C, respectively) makes the groove narrow and shallow by placing its bulky side chain within the groove. These proline and tryptophan residues are completely conserved in EcPDZ-N and -C (Figures 3C and 3D). A loop upstream of bB, termed the carboxylate-binding loop, serves as an acceptor for the carboxylate group of the ligand in the canonical PDZ structure. In contrast, AaPDZ-N contains an acidic residue, E200, in the vicinity of the carboxylate-binding loop (Figure 3A), which remains solvent exposed and renders the surface of the putative ligand-binding groove negatively charged (Figure 2C) and, consequently, unfavorable for interaction with a carboxylate group. In contrast, EcPDZ-N appears to use a different strategy to prevent carboxylate binding. The putative ligand-binding groove in EcPDZ-N is capped by a helical element immediately upstream of the carboxylate-binding loop (Figure 3C). This capping helix is formed by the residues 208–213, and the side chain of V210 sticks into the hydrophobic groove between aB and bB. These observations implicate that the PDZ domains of the RseP-type S2P homologs might be involved in some noncanonical functions other than the recognition of the C terminus of the ligand peptide. SAXS Analysis of the PDZ Tandem Fragments of E. coli RseP and the Homolog The inter-PDZ angles deviate significantly between the two crystal forms of the AaPDZ tandem (Figures 2B and 2F; Figure S2), although the individual AaPDZ-N and -C domains are almost identical in structure (Figure S3). The PDZ pocket constituted by the two ligand-binding grooves assumes a ‘‘semi-open’’ conformation in the Fab-complex form, in which the two grooves draw close to one another in a face-to-face configuration (Figure 2F; Figure S2A). In contrast, the pocket assumes a more ‘‘open’’ conformation in the Fab-free crystal (Figures 2B; Figure S2B). Superposition of the PDZ-N part between the two forms showed the PDZ-C part in two different positions related by an approximately 60
swing-motion around the inter-PDZ linker (Figures S2C and S2D). Closer inspection suggests that

328 Structure 22, 326–336, February 4, 2014 ª2014 Elsevier Ltd All rights reserved

Structure RseP Discriminates Substrates by Size Exclusion

Figure 2. Crystal Structures of AaPDZ Tandem in Fab-Free and Fab-Complex Forms (A and B) Ribbon representation of the crystal structure of the Fab-free form shown in side view (A) and top view (B). All of the residues included in the construct (from E115 to E292) were assigned in this model. PDZ-N and -C are shown in green and cyan, respectively. Additional regions in the N and C termini and the inter-PDZ linker are colored in light pink. Residues P117-E122 in the N terminus assume a helical structure. The carboxylatebinding loops of the two PDZ domains are highlighted in red and the directions of the putative ligand-binding in the canonical binding mode are indicated with magenta arrows. The PDZ pocket is indicated with a red dotted line. (C and D) Electrostatic surface of AaPDZ tandem in the Fab-free form in side view (C) and top view (D). The molecular surface is colored according to the electrostatic surface potential from +10 kT/e (blue) to 10 kT/e (red). The two ligand-binding grooves constituting the PDZ pocket are enclosed with red dotted lines. (E and F) Ribbon representation of crystal structure in the Fab-complex form in side view (E) and top view (F). One of the two AaPDZ tandem fragments in the asymmetric unit of the crystal (chain B) is extracted and shown in two different views. Compared with the Fab-free form, the two ligand-binding grooves are closer to one another, and the PDZ pocket (red dotted line) assumes a ‘‘semi-open’’ conformation.

the arrangement of the two PDZ domains is affected by the crystal-packing interactions in both crystal forms. In the Fab-complex, the complementarity-determining regions (CDRs) grasp a protruding loop of AaPDZ-C, K276 to Y283, as their epitope (Figure S3C). The third CDR of the heavy chain is inserted deeply into the PDZ pocket and seems to restrict the closing motion of the two PDZ domains (Figure S2A). In the Fab-free crystal, the inter-PDZ angle is locked by an intimate interaction with a neighbor molecule related by two-fold crystallographic symmetry (Figure S2B). We therefore tried to analyze the structure in solution by SAXS (Figure S2E). The calculated radius of gyration (Rg) indicated that the solution structure is closer to the Fabcomplex form (semi-open state) rather than the Fab-free form (open state), although the pair-distance distribution function [P(r)] indicated that not only the inter-PDZ angle but also the inter-PDZ distance fluctuated to some extent (Figure S2F). Our previous SAXS analysis indicated that the EcPDZ tandem forms a ‘‘bent-dumbbell-like’’ structure that excluded

the possibility that the two PDZ domains are arranged in either extended or tightly packed configurations (Inaba et al., 2008). This previous observation coincides well with the structural features of the AaPDZ tandem. Nevertheless, the ellipsoidal structures of the two separate EcPDZ domains could be fit with multiple possible orientations into the dummy-residue model from our previous SAXS study due to the lack of characteristic features. With the AaPDZ tandem structure from this study as a reference, we reanalyzed our previous SAXS data to attempt building a structural model of the entire EcPDZ tandem fragment. We first superposed the EcPDZ-N and -C domains on their counterparts in the AaPDZ tandem in the Fab-complex (semi-open) form. Next, we adjusted the distance between the two domains without changing their relative orientation so that the entire fragment fit into the envelope (Figures 4A and 4B). The theoretical scattering curve calculated from the resulting model fits with the experimental curve, with a c2 value of 0.855. The Rg value calculated for the model was 22.4 A˚ and is in excellent agreement with that calculated from the experimental scattering curve, 22.1 ± 0.8 A˚ (Figure 4C). Taken together, it is highly possible that the formation of the ‘‘clamshaped’’ structure with the combined ‘‘pocket-like’’ grooves is a common feature among the PDZ tandems of the RsePtype S2P homologs.

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Structure RseP Discriminates Substrates by Size Exclusion

Figure 3. Ribbon Representation of the RseP-Type PDZ Domains For RseP-type PDZ domains AaPDZ-N (A), AaPDZ-C (B), EcPDZ-N (C), and EcPDZ-C (D), the residues lining the putative ligand-binding groove are highlighted by representing their side chains as the stick-models. The main chain of the carboxylate-binding loop is indicated in red. (A and C) In AaPDZ-N, the putative binding site for the C terminus of the ligand is solvent exposed (the magenta dotted circle). The ligand binding in the canonical mode (the magenta arrow) is, however, not favorable due to the presence of W157 in aB and P205 in bB. These proline and tryptophan residues are conserved among all of the four PDZdomains (as colored in orange). In addition, the acidic residue E200 upstream of the carboxylatebinding loop (as indicated in orange) renders the groove negatively charged. In EcPDZ-N, the carboxylate-binding loop is blocked with the capping helix (orange) and the side chain of V210 sticks into the hydrophobic pocket of the putative ligand-binding groove (magenta dotted arc). (B and D) The structures of AaPDZ-C and EcPDZ-C are quite similar, and the residues lining the groove are highly conserved between the two PDZ-C domains.

Arrangement of the PDZ Tandem in the Full-Length RseP on the Membrane Thereafter, we tried to analyze the spatial arrangement of the PDZ tandem in the context of the full-length RseP confined in the lipid bilayer. Although there are no PDZ domains in MjS2P, it is the only S2P family protein for which there is a full-length 3D structure available (Protein Data Bank [PDB] ID code: 3B4R) (Feng et al., 2007). Using this structure, we predicted the boundaries of the TM domains and the periplasmic region containing the PDZ tandem. Despite the difference in domain organization, TM2–TM4 of MjS2P showed significant sequence similarities to TM1–TM3 of both EcRseP and AaRseP (Figure S4). Sequence alignment indicated that PDZ-N from EcRseP is linked to TM2 with a linker comprised of eight residues (I121 to R128). It is therefore suggested that the PDZ pocket lies just above the helix-bundle of TM1–TM3 that houses the active center and the substrate-binding site within the membrane (Figure 5A). Assuming that the PDZ pocket is pointed toward the membrane surface, the interior of the pocket is separated from the periplasmic space and connected to the outside through a narrow gap between the PDZ domains. We then experimentally examined steric accessibility in the PDZ tandem on the membrane via modification with the bulky membrane-impermeable reagent methoxypolyethylene

glycol 5000 maleimide (mal-PEG-5k; MW 5,000). We introduced cysteine residues into solvent-exposed positions on the PDZ tandem of a cysteine-less EcRseP derivative, where C-terminally His6-Myc-tagged EcRseP proteins (hereafter simply referred to as EcRseP) were expressed in E. coli, and we compared the extent of chemical modification in the spheroplast (Figure 5B). Consistent with our prediction, no detectable mal-PEG-5k modification was observed for cysteine residues introduced inside the pocket (V261C and I222C), whereas those outside the pocket (Q252C and A136C) were modified rapidly. S211C in the capping helix, located at the entrance of the pocket, was modified relatively slowly. However, all of these mutants were modified with mal-PEG-5k under detergent-solubilized conditions. Furthermore, V261C and S211C were reactive to the smaller membrane-impermeable alkylating reagent 4-acetamido-40 -maleimidylstilbene-2,20 -disulfonic acid (MW 536.44), even in the absence of detergent (Figure S5). These results indicate that the PDZ pocket is inaccessible to the bulky mal-PEG-5k when EcRseP resides on the membrane. Interestingly, two residues outside the pocket, D163C and L167C, were not modified by mal-PEG-5k in the spheroplast, but they become modifiable when the membrane was solubilized with the detergent. It is possible that these residues are located proximal to the membrane surface and are inaccessible to the reagent due to steric hindrance from the membrane. In contrast, I304C in the carboxylate-binding loop, the side chain of which is expected to be buried inside the molecule, showed no strong modification by mal-PEG-5k even with the addition of the detergent. This supports the key assumption that the EcPDZ-N mutant fragment remains folded under the detergent-solubilizing conditions. In this

330 Structure 22, 326–336, February 4, 2014 ª2014 Elsevier Ltd All rights reserved

Structure RseP Discriminates Substrates by Size Exclusion

Figure 4. Modeling of the EcPDZ Tandem Structure (A and B) Superposition of the crystal structures of EcPDZ-N and -C domains with the envelope of the dummy-residue model in top view (A) and side view (B). The ribbon representation of the two domains is colored as in Figures 3A and 3B. The relative distance of the EcPDZ-N and -C domains was adjusted on the assumption that the EcPDZ tandem adopts a semi-open conformation similar to the crystal structure of the AaPDZ tandem in the Fab-complex form. The PDZ pocket constituted by the two putative ligand-binding grooves is indicated with a red dotted line. (C) Fit of the theoretical scattering profile for the predicted model with the experimental SAXS data. The Rg and c2 values are shown in the inset table.

experiment, we also confirmed that all of the mutants are able to cleave RseA only after the first cleavage by DegS (Figure S6). Based on the above-mentioned sequence analysis and chemical modifications, it is likely that the PDZ tandem constitutes a steric obstacle to substrate accommodation and that the substrates must pass through the gap between the two PDZ domains to approach the active center. In that case, only substrates containing a compact periplasmic domain would be able to fit into the PDZ pocket such that their TM segments could be accommodated into the substrate-binding site sequestrated within the membrane. To test this hypothesis, we examined the DegS dependency of intramembrane cleavage with EcRseP(DPDZ-NC), an EcRseP derivative devoid of the entire PDZ tandem (residues 127–309) that was constructed in our previous study (Hizukuri and Akiyama, 2012). We introduced N-terminally hemagglutinin (HA)-tagged RseA, HA-RseA, and the EcRseP proteins into an rseA/rseP/degS-triple deletion strain. As expected, immunoblotting revealed that EcRseP(DPDZ-NC) cleaved the full-length HARseA independently of prior cleavage by DegS (Figure 5C). This result is similar to the previously reported deregulation of sequential cleavage for the L151P mutant (Inaba et al., 2008). Destabilization of the PDZ Tandem Induced by Mutations Affecting the Capping Helix and Its Effect on the Sequential Cleavage Many deregulation-causing mutations have been identified around the putative ligand-binding groove of EcPDZ-N, shown

in Figure 6A (Inaba et al., 2008). Most of those mutations are expected to weaken the interactions that hold the capping helix against the ligand-binding groove. One example is L151P, a mutation that has been reported to abolish the DegS dependency. This mutation might induce a conformational change of the capping helix by changing the structure of the putative ligand-binding groove where the capping helix sits above L151 at the bottom of the hydrophobic pocket of the groove. In contrast, another deregulation-causing mutation, L213P, affects the boundary between the capping helix and the carboxylatebinding loop. This mutation would still be expected to cause a substantial conformational change around the capping helix. To test this hypothesis, we constructed an EcRseP mutant lacking the capping helix, EcRseP(DCH), and we examined its behavior in the cleavage of RseA. We first assessed its cleavage activity by using a model substrate that mimics the DegScleavage product and allows for monitoring of cleavage by immunoblotting, HA-MBP-RseA140, in which the N-terminal cytoplasmic domain of RseA is substituted with an HA-tag and a maltose-binding protein (MBP) sequence, and the C-terminal periplasmic region was truncated at residue 140 (Akiyama et al., 2004). The helix deletion slightly reduced the cleavage activity, but the mutant was still capable of cleaving HA-MBPRseA140, similarly to the deregulated mutants L151P and L213P (Figure 6B). More remarkably, HA-RseA in the presence of RseB and absence of DegS was degraded by EcRseP(DCH) (Figure 6C), indicating that the mutant was capable of cleaving the full-length RseA without the prior cleavage by DegS. These results exhibit that the complete removal of the capping helix exerts similar effects on the cleavage of RseA to those caused by the point mutations in the putative ligand-binding region of the PDZ-N domain. For EcPDZ-N and mutants expressed as soluble fragments, analysis using circular dichroism spectroscopy, analytical sizeexclusion chromatography, and SAXS indicated that the structure of EcPDZ-N domain is largely unfolded by the mutations

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Figure 5. Structural Model of EcRseP and Effect of Removal of PDZ Tandem (A) Model of the full-length EcRseP. The structural model of the EcPDZ tandem is represented as in Figure 4. The PDZ-pocket (as indicated with red in the envelope) is presumed to lie just above the active center constituted by the helix-bundle of the TM domains. The residues replaced with cysteine are highlighted with stick-models in magenta. (B) The mal-PEG-5k modifiability of introduced Cys residues. C-terminally His6-Myc-tagged EcRseP(Cys-less) or its single cysteine derivatives in E. coli spheroplasts were treated with malPEG-5k in the presence or absence of Triton X-100 for the indicated times. Trichloroacetic acid (TCA)-precipitated proteins were analyzed by SDS-PAGE and immunoblotting using anti-Myc monoclonal antibodies. Positions of molecular size markers (in kilodaltons) are indicated on the left. The closed arrowhead indicates the malPEG-5k-labeled form of EcRseP derivatives. The asterisk indicates EcRseP derivatives modified with a minor mal-PEG-5k component with a smaller mass, as reported previously (Koide et al., 2007). The mutations were classified into five categories depending on their location and are indicated with differently colored squares. (C) DegS dependency of RseA cleavage by EcRseP derivatives. HA-RseA was expressed in rseA/rseP/degS-triple deletion cells together with EcRseP or its derivatives; lane 3 is vector control using pTWV228. TCA-precipitated proteins were analyzed by SDS-PAGE and immunoblotting using anti-HA polyclonal and anti-Myc monoclonal antibodies. The asterisk indicates C-terminally cleaved products of HA-RseA that were produced by the action of an unidentified protease, as reported previously (Kanehara et al., 2003). EcRseP(DPDZ-NC) and EcRseP(DPDZ-NC/E23Q) were overproduced from pUC-based high copyplasmids (as indicated with ‘‘o.p.’’) due to their lower stability in E. coli.

(Figure S7). However, the full-length mutant proteins stably accumulated in E. coli as reported previously (Inaba et al., 2008), although we would expect degradation after membrane insertion for a largely unfolded region. One possibility is that the regions flanking the PDZ domains, such as the TM regions and the rest of the periplasmic region, would prevent complete unfolding of the entire mutant protein. In fact, this scenario would be in line with our previous report demonstrating that the deregulation-causing mutations resulted in specific cleavage of EcRseP by trypsin (Inaba et al., 2008). These mutants remained stable throughout the process of expression and analysis until the addition of exogenous trypsin. The primary cleavage site was predicted to be near the boundary between PDZ-N and -C, indicating that the mutations were presumed to affect the tertiary structure of the PDZ tandem such that the interPDZ linker is exposed for cleavage (Figure 7A). Here we examined that both of the EcRseP(DCH) and L213P mutants also

showed trypsin sensitivity, producing major tryptic fragment with almost the same size as that produced for L151P mutants, whereas the wild-type was stable and almost no tryptic fragments were observed (Figure 7B). Furthermore, the cleavage profiles that include a minor tryptic fragment are almost fully consistent between the EcRseP(DCH) and L213P mutants, suggesting that a similar structural change was induced by these two different mutations. All present information considered, it is probable that these mutations largely destabilize the tertiary structure of the PDZ tandem while the structural integrity of the rest of the protein, including the active center, is maintained to an extent such that the mutants are still capable of cleaving the model substrates, as shown in Figure 6B. A Model for Substrate Discrimination through the PDZ Tandem in Regulated Intramembrane Proteolysis of RseA by RseP The sum of our experiments prompt us to propose here a hypothetical model for the mechanism of regulation, namely, that the

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Structure RseP Discriminates Substrates by Size Exclusion

Figure 6. Mutational Analysis Targeted to EcPDZ-N (A) Point mutations in EcPDZ-N that have been identified as causing the deregulation of the sequential cleavage are labeled, and the positions of the mutated residues are highlighted in magenta on the ribbon representation of the EcPDZ-N domain. The capping helix is indicated in orange. The labels for L151 and L213, the residues mutated in the cleavage assay in (B) and (C), are outlined with boxes and the residues are shown as stick-models. (B) In vivo cleavage of the RseA-derived model substrate. HA-MBP-RseA140 was expressed in rseA/rseP-double deletion cells together with EcRseP or its derivatives. Lane 2 is vector control using pTWV228. TCA-precipitated proteins were analyzed by SDS-PAGE and immunoblotting using anti-HA monoclonal and anti-Myc monoclonal antibodies. ‘‘Uncleaved’’ and ‘‘Cleaved’’ indicate EcRseP-uncleaved and cleaved forms of HA-MBP-RseA140, respectively. (C) DegS-independent cleavage of the full-length RseA. HA-RseA was expressed in rseA/rseP/ degS-triple deletion cells together with EcRseP or its derivatives and analyzed as described in (B). Asterisk indicates the C-terminally cleaved product of HA-RseA (see legend of Figure 5).

PDZ tandem with a pocket-like structure serves as a size-exclusion filter for RseA entry to the RseP active site (Figure 8). This model explains the mechanism by which the second intramembrane cleavage is suppressed by the glutamine-rich region of RseA, RseB, and the PDZ tandem of RseP in E. coli. Close localization of all of these components to the periplasmic face of the cell membrane is crucial for this sterics-based model. Under normal growth conditions, the TM segment of RseA cannot approach the active center because the periplasmic domain of RseA binds with RseB via the sequence close to the glutamine-rich region. Upon exposure to extracytoplasmic stress, malfolded outer membrane proteins (OMPs) trigger DegS activation and simultaneously release the RseB inhibition of RseP (Chaba et al., 2011; Kulp and Kuehn, 2011). The activated DegS cleaves off the large periplasmic portion of RseA including the glutamine-rich region; consequently, RseA can no longer bind with RseB. The sum drastic size reduction in the periplasmic domain enables truncated RseA to pass through the PDZ filter, and its TM segment is then permitted to approach the active center for the second cleavage to occur. As shown above, complete removal of the PDZ domains deregulates the process (Figure S9A), and point mutations expected to perturb the tertiary structure of the PDZ tandem are well explained by our model of regulation (Figure 7A). Mutations that would be predicted to disrupt the ‘‘clam-shaped’’ structure of the PDZ tandem deregulate the RIP process and leave the PDZ tandem susceptible to trypsin proteolysis. In this model, we suppose that the PDZ tandem discriminates between the intact and the DegS-cleaved RseA based on the size of the periplasmic region rather than on a specific interaction with the C-terminal sequence of RseA. Although it cannot be formally ruled out that PDZ-C interacts with the C-terminal residue V148 in the DegS-cleavage product of RseA, our recent results strongly indicate that the exposure of the hydrophobic

C terminus is not essential for recognition at least in the in vivo cleavage reaction (Hizukuri and Akiyama, 2012). As discussed above, the structural features of the RseP-type PDZ domains strongly suggest that both of the PDZ-N and -C domains are intrinsically unsuitable to interact with the C terminus of a ligand peptide. The circular permutation of the RseP-type PDZ domains seems to play a pivotal role in the formation of the size-exclusion filter. Although the antiparallel arrangement of the ligand-binding grooves is often identified in the other PDZ tandems such as PSD-95 and syntenin (PDB ID codes: 3GSL and 1W9Q, respectively), it would be difficult for such PDZ tandems with the canonical topology to assume a closed pocket-like conformation because the two PDZ domains in those tandems are connected at the opposite side of the ligand-binding grooves (compare Figure S8 with Figures 2A–2D). Furthermore, both of the N and C termini of the canonical PDZ tandem are also present distal to the grooves. With such connections, the ligand-binding grooves could not be positioned toward the active site sequestrated within the membrane. Our size-exclusion model can indeed explain most of the previous observations in the genetic and biochemical analyses on the sE activation pathway. For instance, even the full-length RseA has some susceptibility to cleavage by RseP in the DrseB strain (Grigorova et al., 2004). The RseA periplasmic domain might behave like an intrinsically disordered region, only adopting a rigid conformation when bound to RseB (Walsh et al., 2003). It is also known that point mutations to the RseA periplasmic domain around the glutamine-rich region or the truncation of this region, both of which are expected to abolish the interactions between RseA and RseB, result in the DegS-independent cleavage of RseA (Kanehara et al., 2003). In all cases without RseB, the RseA periplasmic domain should be flexible enough to fit into the PDZ pocket even in the DegS-uncleaved form (Figure S9B).

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Figure 7. Trypsin Sensitivity of Wild-Type and Mutated EcRseP (A) Model for the generation of the major tryptic fragment. The deregulation-causing mutations are presumed to induce not only a local conformational change around the capping helix (as indicated by an orange column with ‘‘CH’’ on the left or by an orange line on the right) but also a destabilization of the tertiary structure of the entire PDZ tandem (as indicated with magenta dotted arrows) that consequently increases the sensitivity of the inter-PDZ region to trypsin. The C-terminal half of the EcRseP protein containing the Myc-tag was detected as tryptic fragment (TF) by the immunoblotting. (B) Analysis of the tryptic cleavage pattern. Cells expressing EcRseP or its derivatives were converted to spheroplasts by sucrose-lysozyme treatment and further treated with trypsin for the indicated times, as described previously (Inaba et al., 2008). TCA-precipitated proteins were analyzed by SDS-PAGE and immunoblotting using anti-Myc monoclonal antibodies. Full and TF indicate the full-length EcRseP and a major tryptic fragment, respectively, generated by cleavage around the inter-PDZ region. Single and double asterisks indicate minor tryptic fragments.

The DegS-independent cleavage of RseA has been identified in physiological processes. The Salmonella enterica serovar Typhimurium possesses an RseP homolog implicated in the sE stress response. A recent study has revealed that Salmonella sE is activated by acid stress as well as extracytoplasmic stress (Muller et al., 2009). Under acid stress conditions, the full-length RseA is degraded by RseP in a DegS-independent manner where the RseP PDZ domain is indispensable for the acid induction of sE activation. In Salmonella, acid stress might induce the structural alteration of the PDZ tandem so that the intact RseA could be accommodated into the PDZ pocket even in the presence of RseB (Figure S9C). Although an acid-induced sE activation pathway has not been identified in E. coli at this point, there might be another DegS-independent stress response pathway. Another possibility is that accommodation of the substrate periplasmic region into the PDZ pocket might change the catalytic center from an inactive, or autoinhibited, state to an activated state. Such a mechanism could operate in place of or in cooperation with our proposed size-exclusion model. A conformational change in the PDZ tandem, such as the opening motion of the two PDZ domains, might be transmitted to the TM region and induce an active conformation of the catalytic center. In fact, it is known that the deregulation effect of the PDZ-N mutants was significantly enhanced by the additional mutation A115V within TM2 (Inaba et al., 2008). This observation suggests that the PDZ tandem and the TM region are functionally correlated with one another, but they can also be incorporated into our model. If the A115V mutation changes the relative orientation of the TM helices and places tension on the tethers to the PDZ tandem, the ‘‘clam-shaped’’ closed conformations of the tandem would be disfavored. The net result would be a similar scenario to the structural perturbations caused by single point mutations and the capping helix deletion that affect the structural integrity of the PDZ tandem.

For PDZ-containing S2P homologs in general, clearance of bulky periplasmic/luminal moieties seems to be a common regulatory mechanism for substrate acceptance in the catalysis of intramembrane proteolysis. Our recent study has shown that RseP is also involved in the degradation of remnant signal peptides of secreted proteins (Saito et al., 2011). Cleavage of the signal peptide from b-lactamase by RseP requires prior detachment of the peptide from the bulky mature form of b-lactamase that also fits with our size-exclusion model. Furthermore, it was reported for RIP of ATF6 by human S2P that the size of the luminal domain of ATF6 affects the cleavage efficiency (Shen and Prywes, 2004). Substrates possessing short luminal domains are susceptible to intramembrane cleavage by S2P, independently of prior cleavage by S1Ps. Interestingly, the S1P dependency of the S2P cleavage was restored by the addition of artificial domains, such as the luminal domain of IRE1a, onto the short luminal domains of ATF6. Human S2P is predicted to be comprised of six TM domains and to contain a single PDZ domain between TM4 and TM5 (Kinch et al., 2006). According to the TM prediction, the N terminus of the PDZ domain is also connected to TM4 almost directly, indicating that a single PDZ domain also lies above the active center. The entire luminal domain including the PDZ domain is composed of 200 amino acid residues, the size of which is comparable to that of the RseP periplasmic region. The single PDZ domain and the rest of the luminal domain might constitute a certain architecture serving as a size-exclusion filter to suppress the entry of ATF6 containing a bulky luminal domain in human S2P. Conclusions In the present study, we have determined the crystal structure of the PDZ tandem fragment of a bacterial RseP homolog at an atomic level. In addition, we analyzed the structure

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Figure 8. Model for the Negative Regulation of the Intramembrane Proteolysis of RseA through the PDZ Tandem (A) Suppression of the intramembrane proteolysis under unstressed conditions. Before the first cleavage by DegS, RseA forms a complex with RseB in the periplasmic region, and its entry into the active center is blocked through the sizefiltering function of the PDZ tandem. The combined putative ligand-binding grooves within the PDZ tandem are enclosed within the dotted line. The capping helix (CH) found in the E. coli PDZ-N domain is indicated with the orange column. (B) Entry of RseA into the RseP active center under stress conditions. After the first cleavage by DegS, the reduced-size periplasmic region of RseA becomes capable of entering into the PDZ pocket. As a consequence, the TM segment of RseA is susceptible to cleavage by RseP. The upper panels represent side views parallel to the membrane plane, and the lower panels are top views perpendicular to the membrane plane from the periplasmic side.

of the PDZ tandem of E. coli RseP using SAXS and chemical modification of engineered cysteine residues. Based on the structural properties of the PDZ tandem in consideration of the mutational data, we have made the proposal that the PDZ tandem serves as a size-exclusion filter for substrate entry during two-step sequential cleavage. In this model, we propose that RseP discriminates the substrates from nonsubstrates by the size of their periplasmic regions, rather than by the recognition of a specific sequence/motif. We presume that this regulation mechanism should reflect the in vivo cleavage process where the relative arrangement of RseP and the substrates is restricted by the cell membrane. Further understanding of the mechanism by which RseP senses the prior periplasmic cleavage of the substrates and catalyzes the intramembrane cleavage demands the determination of a complete structure for RseP; this structure will unambiguously reveal the arrangement of the two PDZ domains in the context of the full-length protein confined in the lipid bilayer.

Biochemical Analysis of EcRseP In vivo cleavage efficiency against the model substrates was analyzed using the E. coli rseA/rsePdouble deletion strain KK211 (Kanehara et al., 2002). DegS dependency of the intramembrane cleavage was examined by expressing EcRseP or its mutants together with HA-RseA in the E. coli rseA/ rseP/degS-triple deletion strain AD1840 (Kanehara et al., 2002). The trypsinsensitivity test for EcRseP was performed as described previously (Inaba et al., 2008). The mal-PEG-5k modification of the single-cysteine EcRseP mutants was also performed essentially as described previously (Koide et al., 2007). A full description of experimental procedures, including the generation of anti-AaPDZ tandem IgG and the characterization of the EcPDZ-N mutant fragments, is given in the Supplemental Information. ACCESSION NUMBERS The atomic coordinates and structure factors of the Fab-free and Fabcomplex forms have been deposited in the Protein Data Bank as accession numbers 3WKL and 3WKM, respectively. The cDNA sequences for the heavy and light chains of anti-AaPDZ tandem IgG Fab are deposited in the DNA Data Bank of Japan with accession numbers AB701676 and AB701677, respectively. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, nine figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.str.2013.12.003.

EXPERIMENTAL PROCEDURES Structural Analysis of AaPDZ Tandem The AaPDZ tandem fragment was produced in E. coli as a glutathione S-transferase-fusion protein, whereas the Fab fragment was prepared through papain treatment of anti-AaPDZ tandem immunoglobulin G (IgG). The AaPDZ tandem fragment was subjected to crystallization in solitary and in complex with Fab. The X-ray diffraction data were collected at Photon Factory BL-17A (Tsukuba, Japan) and SPring-8 BL-44XU (Hyogo, Japan). The structure was solved by molecular replacement using the CCP4 program suite (Collaborative Computational Project Number 4, 1994). Data collection and refinement statistics are summarized in Table 1. The SAXS data were measured at SPring-8 BL45XU or using the BioSAXS-1000 system (Rigaku) at our laboratory and subsequently analyzed using the ATSAS package (http://www.embl-hamburg.de/ biosaxs/software.html).

ACKNOWLEDGMENTS We are grateful to the staff of beamlines BL-17A at Photon Factory and BL44XU and BL-45XU at SPring-8 for providing data collection facilities and support. We thank Mai Toriyama and Makiko Neyazaki for technical support in the protein preparation and Samuel Thompson for editing the manuscript. We thank Harald Huber (University of Regensburg, Germany) for providing the genomic DNA from A. aeolicus. We also thank Shuji Akiyama (Institute for Molecular Science, Japan) for providing the SAXS data of the EcPDZ tandem. This work was supported in part by research grants from the Japan Society for the Promotion of Science (JSPS, to T.N., Y.A., and Y.H.); the Ministry of Education, Culture, Sports, Science and Technology (to Y.A.); the Astellas

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Foundation for Research on Metabolic Disorders (to T.N.); the Sumitomo Foundation (to T.N.); and the Institute for Fermentation, Osaka (to Y.A.). Y.H. was supported by JSPS Research Fellowships for Young Scientists. Received: May 24, 2013 Revised: December 4, 2013 Accepted: December 4, 2013 Published: January 2, 2014

Kinch, L.N., Ginalski, K., and Grishin, N.V. (2006). Site-2 protease regulated intramembrane proteolysis: sequence homologs suggest an ancient signaling cascade. Protein Sci. 15, 84–93. Koide, K., Maegawa, S., Ito, K., and Akiyama, Y. (2007). Environment of the active site region of RseP, an Escherichia coli regulated intramembrane proteolysis protease, assessed by site-directed cysteine alkylation. J. Biol. Chem. 282, 4553–4560.

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