The HtrA homologue DegQ is a self-compartmentizing protease that forms large 12-meric assemblies

Robert Wrasea, Hannah Scotta, Rolf Hilgenfelda,b,c,1, and Guido Hansena

aInstitute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany; bLaboratory for Structural Biology of Infection and Inflammation, Deutsches Elektronen Synchrotron, Building 22a, Notkestrasse 85, 22603 Hamburg, Germany; and cShanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China

Edited* by John Kuriyan, University of California, Berkeley, CA, and approved May 13, 2011 (received for review January 29, 2011)

Proteases of the HtrA family are key factors dealing with folding monomer of the DegP 6-mer harbors an extended loop, desig- stress in the periplasmatic compartment of prokaryotes. In Escher- nated LA, which reaches into the proteolytic center of an oppos- ichia coli, the well-characterized HtrA family members DegS and ing monomer. This arrangement stabilizes the 6-mer and renders DegP counteract the accumulation of unfolded outer-membrane all six protease sites inactive (9). In the presence of substrate pro- proteins under stress conditions. Whereas DegS serves as a fold- teins, 6-mers reassemble into large 12- and 24-meric complexes, ing-stress sensor, DegP is a chaperone-protease facilitating refold- which represent protease-active forms of DegP (10, 14). ing or degradation of defective outer-membrane proteins. Here, In contrast to DegS and DegP, little is known about DegQ. An we report the 2.15-Å-resolution crystal structure of the second amino acid sequence identity of 59% with DegP (Fig. S1) and the major chaperone-protease of the periplasm, DegQ from Legionella fallonii. DegQ assembles into large, cage-like 12-mers that form presence of a typical signal sequence indicate DegQ as a second independently of unfolded substrate proteins. We provide evi- major chaperone-protease of the periplasm. In fact, upon over- dence that 12-mer formation is essential for the degradation of expression, DegQ is able to rescue a degP-deficient E. coli strain substrate proteins but not for the chaperone activity of DegQ. at elevated temperatures (15). Interestingly, the length of loop In the current model for the regulation of periplasmatic chaper- LA, crucial for the stabilization of the 6-meric resting state of one-proteases, 6-meric assemblies represent important protease- DegP, is dramatically reduced in DegQ (for E. coli: 18 residues resting states. However, DegQ is unable to form such 6-mers, in DegQ vs. 41 residues in DegP; see Fig. S1). Many bacterial suggesting divergent regulatory mechanisms for DegQ and DegP. genomes encode only two HtrA proteins: a DegS homologue To understand how the protease activity of DegQ is controlled, we and, judging from length and amino acid sequence of the LA probed its functional properties employing designed protein var- loop, a DegQ homologue (6). Thus, DegQ alone seems to be re- iants. Combining crystallographic, biochemical, and mutagenic sponsible for maintaining protein homeostasis in the periplasm of data, we present a mechanistic model that suggests how protease many prokaryotes. According to previous studies, DegQ assem- activity of DegQ 12-mers is intrinsically regulated and how deleter- ious proteolysis by free DegQ 3-mers is prevented. Our study sheds bles almost exclusively into higher-order oligomers, most likely light on a previously uncharacterized component of the prokaryo- 12-mers, even in the absence of substrates (16). This raises the tic stress-response system with implications for other members of question if the higher-order DegQ oligomer replaces the 6-mer the HtrA family. as a resting state or represents a protease-active form, analogous to DegP. In lack of the biochemical and structural data that X-ray crystallography ∣ protein quality control ∣ oligomerization ∣ allowed the development of detailed functional models for DegP PDZ domain ∣ molecular switch and DegS, it is completely unclear how the protease activity of DegQ is regulated. rotein quality control is essential for all living cells, and com- Here, we report the 2.15-Å-resolution X-ray crystal structure Pplex molecular mechanisms have evolved to ensure correct of the 12-meric DegQ complex from Legionella fallonii. This folding and efficient removal of damaged or misfolded proteins species is closely related to , the causative (1–3). In the periplasm of many prokaryotes, proteins of the con- agent of Legionnaires’ disease, a severe with a high served HtrA family deal with folding stress (4). For pathogenic fatality rate (17). Together with structures of three DegQ variants , which often encounter a hostile environment inside that illustrate the formation of substrate complexes and the their host cells, HtrA proteins represent important virulence inactivation of DegQ 3-mers by domain rearrangement, we pro- factors promoting intracellular survival (5). In , three HtrA family members, DegS, DegP, vide experimental support for a mechanistic model. The crystal and DegQ, have been identified (6). These three proteins share a structure of the DegQ 12-mer reveals an assembly mode that common modular domain organization comprising an N-terminal differs from available models for the DegP 12-mer based on trypsin-like protease domain and one (DegS) or two (DegP, cryo-EM studies (10, 14). DegQ) C-terminal PDZ domains. DegS and DegP are well char-

acterized (7, 8), and structures for both proteins have been Author contributions: R.W., R.H., and G.H. designed research; R.W. and H.S. performed reported (9–11). DegS is a membrane-associated, homotrimeric research; R.W., H.S., and G.H. analyzed data; and R.W., R.H., and G.H. wrote the paper. protease acting as a folding-stress sensor (12). Activated DegS The authors declare no conflict of interest. triggers a signal-transduction pathway that ultimately induces *This Direct Submission article had a prearranged editor. the expression of compartment-specific chaperone and protease Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, genes including degP (12). DegP is a bifunctional protein with www.pdb.org (PDB ID codes 3PV2, 3PV3, 3PV4, and 3PV5). tightly regulated protease and chaperone activities, facilitating 1To whom correspondence should be addressed. E-mail: hilgenfeld@biochem. the degradation or refolding of misfolded periplasmatic proteins uni-luebeck.de. (13). In its resting state, DegP forms compact 6-mers composed This article contains supporting information online at www.pnas.org/lookup/suppl/ of two 3-mers arranged in a face-to-face manner (9). Every doi:10.1073/pnas.1101084108/-/DCSupplemental.

10490–10495 ∣ PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1101084108 Downloaded by guest on September 29, 2021 AB (residues 241–340), a short linker region (residues 341–352), and the PDZ2 domain (residues 353–439) were well defined by elec- tron density. Sections of loops LA (residues 31–59), LD (residues 152–157), L3 (residues 170–179), and L2 (residues 212–218) were too flexible to be traced in at least one molecule (for nomencla- ture of protease loops see Figs. S1 and S4). Low rms deviations between 0.44 and 0.81 Å after superimposition of equivalent Cα atoms indicate that all DegQLf monomers exhibit very similar conformations. Analysis of the crystal lattice revealed the presence of a highly Fig. 1. Legionella DegQ forms large oligomeric complexes. (A) SEC profiles – symmetric DegQLf 12-mer (Fig. 2 A C), which should correspond of L. fallonii and L. pneumophila DegQ and DegQ° preparations. Peaks cor- to the large complex observed in SEC. The DegQ 12-mer responding to 12-mers, 3-mers, and monomers are indicated. Elution volumes Lf of marker proteins are shown at the top. (B) SDS-PAGE of purified DegQ displays tetrahedral (332) symmetry and is composed of four Lf homotrimers as basic building blocks, each stabilized by extensive and DegQ°Lf (S) and fractions obtained after SEC. No copurified substrate molecules were identified after loading comparable amounts of protein from contacts between the three protease domains. Every 3-mer inter- 12-mer, 3-mer, and monomer fractions (labeled 12, 3, and 1). acts with the three remaining 3-mers of the 12-mer, giving rise to a spherical particle with an outer diameter of approximately Results 140 Å. Because of its cage-like organization, it encloses an inter- Legionella DegQ Assembles into Large Complexes in Solution. Mature nal cavity (Fig. 2D) with an average diameter of approximately 70 Å that lacks defined electron-density features. The active sites DegQ from L. pneumophila (DegQLp) and L. fallonii (DegQLf ) were produced, purified, and analyzed by size-exclusion chroma- of the protease domain lining the inner wall of the 12-mer are tography (SEC) and dynamic light-scattering (DLS). SEC-elution accessible only from the interior of the particle (Fig. 2D). Six lateral pores (approximately 14 Å × 28 Å) connect the internal profiles consistently showed a predominant peak indicating the β β β presence of a large complex with an apparent molecular weight cavity with bulk solvent (Fig. 2C). Strands 1, 2, and 4of two juxtaposed DegQ monomers partially cover each pore from of 320 to 440 kDa (Fig. 1A, Table S1). As for large protein com- Lf the inside, restricting the size of the opening. Two LA loops con- plexes molecular-weight determination by SEC is often inaccu- necting β1 and β2 are thus positioned in direct vicinity of every rate, we estimated the oligomer to consist of at least seven lateral pore, although the flexible loop itself could not be traced DegQ molecules. According to DLS analysis, the hydrodynamic in the electron density. radius of the particle is approximately 7 nm (Table S1). A smaller In the DegQ 12-mer, PDZ1 and PDZ2 are integral parts of fraction of DegQ from both Legionella species formed 3-mers Lf the protein shell. The peptide-binding groove of PDZ1 is acces- (156 to 164 kDa) and monomers (48 to 50 kDa) (Fig. 1A and sible from the inner cavity of the DegQLf 12-mer; yet, electron- Table S1). In contrast to a constant fraction of monomers, a density maps show no evidence for bound peptides or substrate pH-dependent dynamic equilibrium between the 3-mer and molecules. It is unlikely that PDZ2 is able to bind substrate the larger complex was observed with the large complex prevail- molecules because its peptide-binding groove is inaccessible and ing at acidic to neutral conditions and the 3-mer favored at pH 9.5 lacks a positively charged amino acid residue to stabilize the (Fig. S2). Interestingly, the replacement of the active-site serine C-terminal carboxyl group of substrates. Instead, PDZ2 is respon- by alanine (DegQ°) in DegQLp or DegQLf, which inactivates the sible for the structural integrity of the 12-mer by mediating proteases, led to the sequestering of virtually all molecules into interactions between adjacent 3-mers (Fig. 2 B, E, and F). the large complex (Fig. 1A). It is possible that the formation of Accordingly, in solution DegQ variants lacking a PDZ2 domain DegQ oligomers might be influenced by associated substrate (DegQΔPDZ1&2 and DegQΔPDZ2) assemble into 3-mers and proteins or peptides originating from the expression host that are incapable of forming higher-order oligomers (Fig. S3). could not be degraded by the DegQ° variants. Indeed, it has been reported that the equivalent DegP° variant copurified with Ec The 12-mer Is the Active Form of the Protease. Wild-type DegQLf outer-membrane proteins (OMPs), and assembled into 12- or was able to proteolytically degrade β-casein and unfolded BSA 24-mers (10). Although SDS-PAGE analysis of Legionella (via DTT treatment) but not native BSA (Fig. 3A), indicating that DegQ° (or DegQ) did not indicate the presence of OMPs or other suitable DegQLf substrates must contain partially unfolded re- substrate proteins coeluting with the large complex (or the lower gions. The absence of distinct cleavage intermediates suggests a molecular-weight species) (Fig. 1B), it cannot be excluded that processive degradation of substrate proteins into small peptides. smaller peptides escaped detection. Unlike DegPEc (18), DegQLf was unable to process reductively To explore the role of the PDZ domains in oligomerization, unfolded lysozyme (Fig. 3A). In quantitative protease assays, truncated DegQLf variants were produced that lack both PDZ DegQ was efficiently degrading resorufin-labeled β-casein, Δ 1&2 Lf domains (DegQLf PDZ ) or the C-terminal PDZ2 domain whereas, as expected, DegQ°Lf did not show any activity (Fig. 3B). Δ 2 Δ – alone (DegQLf PDZ ). Both variants were highly soluble, stable, Deletion of the LA loop (DegQLf LA; residues 28 61 re- and did not show any signs of misfolding as confirmed by SEC placed by a single glycine) did not affect formation of 12-mers (Fig. S3) and DLS (Table S1). The truncated proteins were in solution (Fig. S3) or proteolytic activity (Fig. 3B). In contrast, Δ 2 not able to assemble into large complexes and formed 3-mers DegQLf variants incapable of 12-mer formation (DegQLf PDZ , Δ 1&2 and monomers only (Fig. S3), implying that the protease domain DegQLf PDZ ) were completely inactive (Fig. 3B). To verify

alone is sufficient for 3-mer formation whereas PDZ2 is essential that oligomerization of DegQLf and not the mere presence of a BIOCHEMISTRY for the assembly of the higher-order oligomers. PDZ2 domain is critical for proteolytic activity, we designed a truncated protein variant lacking only the nine C-terminal resi- Δ 9 Crystal Structure of the DegQ 12-mer. To gain detailed insights into dues of PDZ2 (DegQLf C ). The DegQLf structure shows that the structural organization of the observed higher-order oligomer these residues should be important for the stability of the 12-mer and its functional implications, DegQLf was crystallized and its by mediating numerous interactions between PDZ domains of Δ 9 three-dimensional structure determined by X-ray crystallography adjacent 3-mers. Indeed, DegQLf C formed exclusively 3-mers to a resolution of 2.15 Å (Table S2). In all four molecules of the (Fig. S3) that were proteolytically inactive (Fig. 3B), further asymmetric unit, the protease domain (residues 1–240; residues supporting that the 12-mer represents the protease-active form of the catalytic triad: S193, H84, and D114), the PDZ1 domain of DegQLf.

Wrase et al. PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10491 Downloaded by guest on September 29, 2021 “ ” Fig. 2. Structure of the DegQLf 12-mer. (A) View along the threefold axis at the protease interface with protease domain ( Prot, blue), PDZ1 domain (orange), and PDZ2 domain (green). Individual DegQLf protomers are indicated by white contours. (B) View along the threefold axis at the PDZ2 interface. (C) View along the twofold axis at the lateral pore of the 12-mer. These pores are located in the center between the protease domains of neighboring trimers. (D) Sliced view of the 12-mer. Residues of the catalytic triad (red) lining the inner wall of the central cavity are located in close proximity to the pores. (E) Orientation as in C with one protomer displayed in cartoon representation and highlighted by a red contour. (F) Schematic representation of domain inter- actions. The highlighted protomer is shown in the same orientation as in E. Noncovalent interactions are indicated by dashed lines. The position of the pore is shown by a gray ellipse. Symmetry axes are indicated as follows: protease threefold (blue triangle), PDZ2 threefold (green triangle), pore twofold (black ellipse).

To test if DegQLf possesses chaperone-like activity as re- variant and determined its three-dimensional structure at 3.1-Å ported for DegPEc, we evaluated the protective effect of DegQLf resolution (Table S2). As expected, the protease core of the Δ 2 on heat-induced denaturation of citrate synthase. DegQLf , DegQLf PDZ 3-mer is identical to that observed in the struc- Δ α DegQ°Lf, and DegQLf LA, as well as the truncated variants ture of the DegQLf 12-mer (rmsd for C atoms of protease Δ 9 Δ 2 Δ 2 DegQLf C and DegQLf PDZ , showed comparable results 3-mers in DegQLf and DegQLf PDZ is approximately 1.3 Å). (Fig. 3C), demonstrating a chaperone-like activity of DegQLf Although the overall fold of the PDZ1 domain is also preserved, independent of 12-mer formation or the presence of PDZ2. it is rotated by approximately 180° relative to the protease domain compared to its orientation in the 12-mer (Fig. 4A). This rotation Reorientation of PDZ1 Is Inactivating 3-meric DegQ Variants. It is not places the peptide-binding cleft of PDZ1 and the protease-active obvious why protease activity is completely abolished in DegQ site on opposite faces of the 3-mer. Furthermore, the linker variants able to form 3-mers but not 12-mers. Assuming that region that connects PDZ1 and protease domain (residues the overall structure of the 3-meric building blocks is independent 241–249) is inserted into the peptide-binding cleft of PDZ1 in of incorporation into 12-mers, the exposed protease-active sites an extended conformation, antiparallel to the central β-sheet of free 3-mers should in fact promote the degradation of sub- of this domain, mimicking a bound substrate molecule (Fig. 4B) strate molecules. To understand the molecular basis of the inac- (19). The position of PDZ1 with respect to the protease domain is Δ 2 tivation of DegQ 3-mers, we crystallized the DegQLf PDZ further stabilized by hydrogen bonds between E112 and H244 ABC

Fig. 3. Assembly of DegQLf 12-mers is essential for protease, but not for chaperone activity. (A) Degradation of BSA and lysozyme. SDS-PAGE analysis of samples before (S) and after incubation at 42 °C with (+) or without (−) DTT. Unfolded BSA was processed, whereas native BSA and lysozyme (with or without

DTT) could not be degraded by DegQLf.(B) Quantitative protease assay with DegQLf proteins using resorufin-labeled casein as substrate. Residual activities of – DegQLf variants (c h) are compared to wild-type DegQLf (b) and a control sample without protease (a). (C) Chaperone activity of DegQLf proteins. Citrate synthase (CS) was heat-inactivated for the indicated period of time in the presence of DegQLf proteins or BSA (control), and residual CS activity was determined.

10492 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1101084108 Wrase et al. Downloaded by guest on September 29, 2021 A rotation of PDZ1 might switch off protease activity of full-length DegQLf 3-mers in solution.

Plasticity of the Active Site and Intrinsic Regulation of Protease Activity. In the DegQLf 12-mer, the residues of the catalytic triad assume a protease-competent conformation, but the S1 pocket and the oxyanion hole are blocked by loop L1 (Fig. 5 A and B). Interestingly, electron-density maps of this area demonstrate that L1 is flexible and adopts a second conformation with a lower oc- cupancy. In this conformation, a rotation of the entire peptide bond between P190 and G191 by approximately 180° leads to the reconstitution of the oxyanion hole and opens up the S1 pock- B et by displacing the side chain of P190 to allow for substrate bind- ing. This suggests L1 as an intrinsic switch element with defined ON and OFF conformations existing in an equilibrium. The OFF conformation seems to be favored in DegQLf . To get further in- sights into its function, we disrupted the L1 switch by replacing P190 and the preceding N189 by glycines. The 2.4-Å-resolution 1 structure of the resulting variant, DegQLf L (Table S2), revealed Δ Fig. 4. Structure of DegQLf PDZ2. (A) Compared to DegQLf (blue), the PDZ1 that the modified L1 loop adopts a unique conformation different Δ 2 domain of DegQLf PDZ (red) is rotated by approximately 180° relative to from the ON and OFF conformations and is unable to form a the protease domain. The PDZ2 domain of DegQLf is not shown. (B) (Left) functional oxyanion hole (Fig. 5D). Accordingly, DegQ L1 is Δ 2 Lf The PDZ1 peptide-binding groove of DegQLf PDZ variant (orange). The proteolytically inactive (Fig. 3B). These findings corroborate loop connecting protease and PDZ1 (residues G241–G249, shown in red) is inserted into the peptide-binding groove in a substrate-like manner. In the critical importance of an intact L1 switch element for the pro- tease activity of DegQ . DegQLf (equivalent loop shown in green) the peptide-binding groove is Lf accessible. (Right) For comparison, the PDZ1 domain of DegPEc [gray, PDB Details on the activation mechanism of DegQLf were eluci- ID code 3CS0, (10)] bound to a short substrate peptide (blue). dated by the 3.1-Å-resolution crystal structure of the protease- inactive DegQ°Lf variant in complex with a peptide substrate Δ 2 and between Q236 and S275. In DegQLf PDZ , loops of the (Table S2). Like the wild-type enzyme, this variant formed 12- protease domain exhibit a higher flexibility than in the 12-mer. mers in the crystal lattice. Additional electron density was located Furthermore, the catalytic triad is disrupted, as H84 is highly flex- in the protease-active site of DegQ°Lf, unambiguously indicating ible and is positioned at a distance of 4.3 Å from the nucleophilic a bound substrate molecule that was copurified with the protein (Fig. 5 E and F). Main-chain and Cβ atoms for six to eight amino S193, rendering the protease inactive. Although the positioning acid residues of the peptide were clearly defined and included in of PDZ1 in the crystal lattice might be influenced by a Cd2þ ion the model. Superimposition of DegQLf and DegQ°Lf revealed that mediates a crystal contact to an adjacent DegQLf 3-mer distinct conformational changes in loops L1, L2, L3, and LD. (CdCl2 was used as an additive in crystallization), the structure In the peptide complex, the switch element L1 adopts the ON shows that PDZ1 is not rigidly attached to the protease domain conformation (Fig. 5C) and residues I188, P190, and N192 along Δ 2 in DegQLf PDZ . It is tempting to speculate that a very similar with N208 and I211 of L2 shape the S1 specificity pocket of the

DegQ DegQ° DegQ L1 A B Lf C Lf D Lf

LD LD LD PDZ1 L1 L1 P190 P190 L1 Ox Ox L3 S1 Ox S193 S1 S1 P190G LD S193A S193

H84 H84 H84 LD L1 E L1 L2 F P3‘ P2‘ L2 LD L1 L1 L2 L3 P1‘ L3 S193A P1 L1 H84 PDZ1 LD PDZ1 L2 L2 BIOCHEMISTRY

P2 P3

D114 P4

Fig. 5. Plasticity of the protease-active site of DegQLf.(A) Overview on the active-site loops L1 (orange), L2 (green), L3 (blue), LD (red) of the protease domain of – DegQLf with flexible regions indicated by dashed lines. Residues of the peptide-binding groove of the PDZ1 domain are colored in magenta. (B D) Comparison 1 of active-site loops L1 and LD in DegQLf, DegQ°Lf, and DegQLfL . Positions of the oxyanion hole (Ox) and the S1 pocket are indicated. The oxyanion hole and the S1 pocket are colored in red if malformed and colored in green in the functional conformation. (E and F) Peptide bound to the active site of DegQ°Lf. Peptide residues P3 and P4 are involved in β-sheet-like contacts with L2. The side chain of P1 is pointing to the S1 pocket formed by residues of L1 and L2.

Wrase et al. PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 ∣ 10493 Downloaded by guest on September 29, 2021 protease (Fig. 5F). This rather restricted pocket is able to accom- data published by two independent groups show discrepancies modate small, hydrophobic residues. In contrast, the primed regarding the assembly mode of the particle (10, 14). One model subsites S1′ to S3′ and the nonprimed subsites S2 to S4 lack suggests that interactions between two PDZ2 domains of adja- well-defined binding pockets and seem to be less discriminatory cent 3-mers stabilize the 12-mer (10). The higher-resolution (Fig. 5F and Fig. S4). Peptide binding is further stabilized by reconstruction based on 8-Å-resolution cryo-EM data implicates main-chain hydrogen-bonding interactions between the substrate that 12-mer formation is supported by contacts between PDZ1 (P1 and P3) and residues of L2 (T209 and I211; Fig. S5). In the and PDZ2 domains (14). Our structural and biochemical data peptide complex, large portions of L3 are defined by electron show that intact PDZ2 domains are critical for the assembly of density, except residues 172–178 located in close proximity to DegQLf 12-mers. However, here PDZ2 is simultaneously inter- the substrate binding cleft of PDZ1. A rearrangement of L3 acting with three adjacent 3-mers via contacts to two PDZ2, enables a hydrogen-bonding interaction between the guanidi- one PDZ1, and one protease domain (Fig. 2F). Thus, a tightly nium group of R170 and the main-chain carbonyl of L151 resid- interconnected, stable network is formed that is fundamentally ing in loop LD of an adjacent molecule (LD*; the asterisk different from the models proposed for DegPEc 12-mers, suggest- indicates loops of neighboring DegQ molecules). LD* in turn ing that the general architecture of DegQLf and DegPEc 12-mers has moved by 6–7 Å from its position in the peptide-free structure may be different. Δ 2 (Fig. 5 B and C) and stabilizes the ON conformation of the L1* The structure of DegQLf PDZ , a variant incapable of assem- switch element via a main-chain hydrogen bond between residues bly into 12-mers, suggests that the protease activity of DegQLf P190 and F149. Thus, the reorganization of the active-site loops 3-mers can be switched off by reorientation of the PDZ1 domain. suggests an interplay between PDZ1 and protease domain of Based on these results, we propose a simple working model adjacent monomers. It is easily conceivable that upon binding for function and regulation of DegQLf (Fig. 6). In vivo, DegQLf of an allosteric activator to PDZ1, a cascade of conformational 12-mers are the predominant oligomeric species with a smaller rearrangements is initiated along L3 and LD* that finally stabi- fraction forming 3-mers as indicated by our SEC experiments. lizes loops L1* and L2* in a proteolytically competent state to As the protease-active sites of free 3-mers are exposed and could allow for efficient degradation of substrate molecules. potentially degrade periplasmatic proteins in an uncontrolled manner, it is essential that the protease activity is switched off Discussion in this state. In the homologous DegPEc, free 3-mers are readily Members of the HtrA-protein family have been extensively stu- assembled into inactive 6-mers (9). Yet, 6-mers were absent in all died over more than two decades. Based on biochemical and our DegQ preparations, and the shortened LA loop would not structural analysis, functional models have been developed that support the formation of such a resting-state oligomer. Our data shed light onto mechanism and regulation of these intriguing suggest that free DegQ 3-mers are inactivated by a large-scale enzymes. In contrast to DegPEc and DegSEc, the third HtrA pro- domain movement of PDZ1 that might represent a unique safety tein in E. coli, DegQ, has not been characterized in great detail. mechanism protecting the cell from deleterious proteolytic activ- The notion that numerous bacteria including many pathogens ity. Upon (re-)integration into 12-mers, PDZ1 is reoriented to encode only two HtrAs, a DegS and a DegQ homologue (6), promote proteolytic activity of DegQLf and allow for degradation prompted us to study DegQ from Legionella. of substrate molecules. However, in the absence of suitable sub- The predominance of stable 12-mers as the major oligomeric strates, the protease-active sites of DegQLf 12-mers are distorted form of DegQLf suggests an important biological function for this and a distinctive OFF conformation of loop L1 is strongly assembly. Using our structural data as a framework, we probed favored. Based on our structural data, we propose that DegQLf the features of DegQLf 12-mers by designing protein variants that is activated via a cascade of conformational changes in L3, LD, were subsequently characterized with regard to oligomerization L2, and L1, which are most likely initiated by binding of an behavior and protease as well as chaperone activity. We show that allosteric activator to PDZ1. The fact that, in our crystal struc- protease but not chaperone activity is dependent on 12-mer for- ture, PDZ1 is free of peptides might be attributed to a release mation. Large, cage-like 12- and 24-mers have been reported to of the allosteric effector after triggering the activation cascade. be the proteolytically active oligomeric species in E. coli DegP As protein substrates could act as allosteric effectors promoting (10, 14). However, DegPEc 12- and 24-mers are formed only tran- their own degradation, a release from the PDZ1 domain after siently, dependent on the presence of partially unfolded proteins, cleavage is necessary to allow for processive substrate degrada- whereas DegQLf 12-mers assemble independently of substrate tion. Similar allosteric activation cascades have been described and represent the predominant oligomeric species over a broad for DegSEc 3-mers (20) and very recently also for DegPEc 24-mers range of environmental conditions. In DegQLf 12-mers as well as (21). Our findings strongly suggest that the general mechanism in DegPEc 12- and 24-mers, interactions between protease do- for the intrinsic regulation of HtrA protease activity is conserved mains stabilize the 3-meric building blocks, and PDZ domains in DegQ, DegP, and DegS. A peptide-bound PDZ domain is as- mediate contacts between neighboring 3-mers. Interestingly, the sumed to be the prerequisite for protease activation in DegS and two available models for the DegPEc 12-mer based on cryo-EM DegP, yet the detection by L3 seems to be remarkably diverse. In

PDZ2

PDZ1 PDZ1 PDZ1 PDZ2 PDZ2

Prot Prot Prot

ProtProt Prot Prot Prot Prot

inactive 3-mer inactive 12-mer active 12-mer

Fig. 6. Model for DegQLf protease activation. The association of protease-inactive DegQ 3-mers into 12-mers (illustrated by a gray shading) leads to a repositioning of PDZ1 liberating its peptide-binding groove (white triangle). Substrate proteins are trapped during (re-)association of 12-mers, or threaded into preassembled 12-mers through pores in the protein shell. Subsequent binding of substrate proteins or effector peptides (green triangles) to PDZ1 allos- terically activates the protease domain via a cascade of conformational changes along protease loops L3 → LD → L1∕L2.

10494 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1101084108 Wrase et al. Downloaded by guest on September 29, 2021 10∕30 DegSEc, peptide-binding causes a reorientation of the PDZ do- SEC. Analytical SEC was performed using a Superdex 200 HR column main, which in turn relieves inhibitory contacts between the PDZ (GE Healthcare) equilibrated with two column volumes of running buffer (50 mM Hepes, 200 mM NaCl, pH 7.5). After injection of the protein sample and the protease domain (21). For DegPEc, it has been postulated that loop L3 senses the locked position of PDZ1 in the active (approximately 1 mg), fractions of 0.5 mL were collected and subsequently 24-mer, rather than the PDZ1-bound peptide itself (21). Based analyzed by SDS-PAGE. The SEC column was calibrated with marker proteins ranging from 670 to 29 kDa. on our data, we propose that the allosteric regulation of DegQLf and DegPEc is similar, yet we cannot specify if L3 directly inter- acts with the PDZ1-bound peptide or with the PDZ1 domain Protein Activity Assays. DegQLf mediated degradation of BSA (GERBU) and because the tip of L3 is flexible in our crystal structure. lysozyme (GERBU) was analyzed in presence or absence of DTT. The reaction μ 1 ∕ The structural and biochemical data provided in this study mixture included 20 M DegQLf, mg mL BSA or lysozyme in protein storage characterize DegQ as a unique member of the HtrA family buffer with or without 20 mM DTT. The assay was performed overnight at with distinct features. The high-resolution crystal structure of a 42 °C, and resulting samples were analyzed by SDS-PAGE. Quantitative 12-meric HtrA protein reveals aspects of architecture and regu- protease assays using resorufin-labeled casein (Roche) were performed as lation that also should be relevant for other family members, described previously (13). All measurements were conducted as duplicates. especially for the DegP 12-mer, although there are differences The chaperone activity assay was modified after Buchner et al. (22). from models based on cryo-EM data of the latter (10, 14). Crystallization, Data Collection, and Structure Determination. DegQ Our data allowed the development of a preliminary mechanistic Lf constructs were crystallized using the sitting-drop vapor-diffusion technique. model, on the basis of which a number of important questions are Details of methods used for crystallization are provided in SI Materials and raised. Firstly, how do substrates gain access to the protease-ac- Methods. X-ray diffraction data were collected at BESSY (Berlin, Germany) tive sites that line the inner wall of the 12-mer? On the one hand, and MAX-lab (Lund, Sweden), integrated with MOSFLM (23), and scaled and DegQ 12-mers could transiently disassemble into 3-mers encap- Lf merged with SCALA (24). Initial phases were obtained by molecular replace- sulating substrates upon reassembly; on the other hand, it is also ment with Phaser (25) using individual domains of DegPEc [Protein Data Bank possible that substrates are threaded through the large lateral (PDB) ID code 3CS0 (10)] as search models. Subsequent model building and pores present in the shell of the DegQ cage. Secondly, details refinement was performed with Coot (26) and REFMAC (27), respectively. of the allosteric control, especially peptide binding to the Data collection and refinement statistics are summarized in Table S2. PDZ1 domain, remain to be elucidated. A crystal structure of DegQ in complex with peptides bound to both PDZ1 and pro- tease domain would verify our proposed model of the intrinsic ACKNOWLEDGMENTS. We thank B. Schwarzloh and S. Schmidtke for expert regulation of protease activity in DegQ. Finally, as for DegP , technical assistance, and U. Müller (BESSY, Berlin, Germany) and T. Ursby Ec (MAX-lab, Lund, Sweden) for assistance with synchrotron data collection. the molecular basis for the chaperone activity of DegQ remains We acknowledge access to beamline BL14.1 of the BESSY II storage ring obscure. Experimental results providing answers to these ques- (Berlin, Germany) via the Joint Berlin MX-Laboratory sponsored by the tions are highly desirable and will further our understanding of Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität this fascinating protein family. Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück Centrum and the Leibniz-Institut für Molekulare Pharmakologie. Experiments at MAX-lab Materials and Methods were supported by the Integrated Infrastructure Initiative “Integrating Protein Production and Purification. Details on production and purification Activity on Synchrotron and Free Electron Laser Science” of the European of Legionella DegQ proteins will be described in detail elsewhere. Briefly, Commission (EC) (Contract R II 3-CT-2004-506008). Optimization of crystals N-terminally His-tagged DegQ proteins lacking the signal sequence were was performed within the OptiCryst project of the EC (LSH-2005-037793; produced in degP-deficient E. coli KU98 harboring the pQE-31 (Qiagen) www.opticryst.org). R.H. acknowledges support by the Deutsche Forschungs- derivative pGDR and purified by nickel-affinity chromatography. Fractions gemeinschaft Cluster of Excellence “Inflammation at Interfaces” (EXC 306) containing the recombinant proteins were pooled, dialyzed against protein and by the Fonds der Chemischen Industrie. He is also supported by a Chinese storage buffer [20 mM sodium acetate (pH 4.5), 200 mM sodium chloride], Academy of Sciences Visiting Professorship for Senior International Scientists and concentrated to 6–20 mg∕mL. (Grant 2010T1S6).

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