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

Abstract

Proteases of the HtrA family are key factors dealing with folding stress in the periplasmatic compartment of prokaryotes. In Escherichia coli, the well-characterized HtrA family members DegS and DegP counteract the accumulation of unfolded outer-membrane proteins under stress conditions. Whereas DegS serves as a folding-stress sensor, DegP is a chaperone-protease facilitating refolding or degradation of defective outer-membrane proteins. Here, we report the 2.15-Å-resolution crystal structure of the second major chaperone-protease of the periplasm, DegQ from Legionella fallonii. DegQ assembles into large, cage-like 12-mers that form independently of unfolded substrate proteins. We provide evidence that 12-mer formation is essential for the degradation of substrate proteins but not for the chaperone activity of DegQ. In the current model for the regulation of periplasmatic chaperone-proteases, 6-meric assemblies represent important protease-resting states. However, DegQ is unable to form such 6-mers, suggesting divergent regulatory mechanisms for DegQ and DegP. To understand how the protease activity of DegQ is controlled, we probed its functional properties employing designed protein variants. Combining crystallographic, biochemical, and mutagenic data, we present a mechanistic model that suggests how protease activity of DegQ 12-mers is intrinsically regulated and how deleterious proteolysis by free DegQ 3-mers is prevented. Our study sheds light on a previously uncharacterized component of the prokaryotic stress-response system with implications for other members of the HtrA family.

Protein quality control is essential for all living cells, and complex molecular mechanisms have evolved to ensure correct folding and efficient removal of damaged or misfolded proteins (1–3). In the periplasm of many prokaryotes, proteins of the conserved HtrA family deal with folding stress (4). For pathogenic bacteria, which often encounter a hostile environment inside their host cells, HtrA proteins represent important virulence factors promoting intracellular survival (5).

In Escherichia coli, three HtrA family members, DegS, DegP, and DegQ, have been identified (6). These three proteins share a common modular domain organization comprising an N-terminal trypsin-like protease domain and one (DegS) or two (DegP, DegQ) C-terminal PDZ domains. DegS and DegP are well characterized (7, 8), and structures for both proteins have been reported (9–11). DegS is a membrane-associated, homotrimeric protease acting as a folding-stress sensor (12). Activated DegS triggers a signal-transduction pathway that ultimately induces the expression of compartment-specific chaperone and protease genes including degP (12). DegP is a bifunctional protein with tightly regulated protease and chaperone activities, facilitating the degradation or refolding of misfolded periplasmatic proteins (13). In its resting state, DegP forms compact 6-mers composed of two 3-mers arranged in a face-to-face manner (9). Every monomer of the DegP 6-mer harbors an extended loop, designated LA, which reaches into the proteolytic center of an opposing monomer. This arrangement stabilizes the 6-mer and renders all six protease sites inactive (9). In the presence of substrate proteins, 6-mers reassemble into large 12- and 24-meric complexes, which represent protease-active forms of DegP (10, 14).

In contrast to DegS and DegP, little is known about DegQ. An amino acid sequence identity of 59% with DegP (Fig. S1) and the presence of a typical signal sequence indicate DegQ as a second major chaperone-protease of the periplasm. In fact, upon overexpression, DegQ is able to rescue a degP-deficient E. coli strain at elevated temperatures (15). Interestingly, the length of loop LA, crucial for the stabilization of the 6-meric resting state of DegP, is dramatically reduced in DegQ (for E. coli: 18 residues in DegQ vs. 41 residues in DegP; see Fig. S1). Many bacterial genomes encode only two HtrA proteins: a DegS homologue and, judging from length and amino acid sequence of the LA loop, a DegQ homologue (6). Thus, DegQ alone seems to be responsible for maintaining protein homeostasis in the periplasm of many prokaryotes. According to previous studies, DegQ assembles almost exclusively into higher-order oligomers, most likely 12-mers, even in the absence of substrates (16). This raises the question if the higher-order DegQ oligomer replaces the 6-mer as a resting state or represents a protease-active form, analogous to DegP. In lack of the biochemical and structural data that allowed the development of detailed functional models for DegP and DegS, it is completely unclear how the protease activity of DegQ is regulated.

Here, we report the 2.15-Å-resolution X-ray crystal structure of the 12-meric DegQ complex from Legionella fallonii. This species is closely related to Legionella pneumophila, the causative agent of Legionnaires’ disease, a severe pneumonia with a high fatality rate (17). Together with structures of three DegQ variants that illustrate the formation of substrate complexes and the inactivation of DegQ 3-mers by domain rearrangement, we provide experimental support for a mechanistic model. The crystal structure of the DegQ 12-mer reveals an assembly mode that differs from available models for the DegP 12-mer based on cryo-EM studies (10, 14).

Results

Legionella DegQ Assembles into Large Complexes in Solution.

Mature DegQ from L. pneumophila (DegQ_Lp) and L. fallonii (DegQ_Lf) were produced, purified, and analyzed by size-exclusion chromatography (SEC) and dynamic light-scattering (DLS). SEC-elution profiles consistently showed a predominant peak indicating the presence of a large complex with an apparent molecular weight of 320 to 440 kDa (Fig. 1A, Table S1). As for large protein complexes molecular-weight determination by SEC is often inaccurate, we estimated the oligomer to consist of at least seven DegQ molecules. According to DLS analysis, the hydrodynamic radius of the particle is approximately 7 nm (Table S1). A smaller fraction of DegQ from both Legionella species formed 3-mers (156 to 164 kDa) and monomers (48 to 50 kDa) (Fig. 1A and Table S1). In contrast to a constant fraction of monomers, a pH-dependent dynamic equilibrium between the 3-mer and the larger complex was observed with the large complex prevailing at acidic to neutral conditions and the 3-mer favored at pH 9.5 (Fig. S2). Interestingly, the replacement of the active-site serine by alanine (DegQ°) in DegQ_Lp or DegQ_Lf, which inactivates the proteases, led to the sequestering of virtually all molecules into the large complex (Fig. 1A). It is possible that the formation of DegQ oligomers might be influenced by associated substrate proteins or peptides originating from the expression host that could not be degraded by the DegQ° variants. Indeed, it has been reported that the equivalent DegP°_Ec variant copurified with outer-membrane proteins (OMPs), and assembled into 12- or 24-mers (10). Although SDS-PAGE analysis of Legionella DegQ° (or DegQ) did not indicate the presence of OMPs or other substrate proteins coeluting with the large complex (or the lower molecular-weight species) (Fig. 1B), it cannot be excluded that smaller peptides escaped detection.

Fig. 1.

Legionella DegQ forms large oligomeric complexes. (A) SEC profiles of L. fallonii and L. pneumophila DegQ and DegQ° preparations. Peaks corresponding to 12-mers, 3-mers, and monomers are indicated. Elution volumes of marker proteins are shown at the top. (B) SDS-PAGE of purified DegQ_Lf and DegQ°_Lf (S) and fractions obtained after SEC. No copurified substrate molecules were identified after loading comparable amounts of protein from 12-mer, 3-mer, and monomer fractions (labeled 12, 3, and 1).

To explore the role of the PDZ domains in oligomerization, truncated DegQ_Lf variants were produced that lack both PDZ domains (DegQ_LfΔPDZ1& 2) or the C-terminal PDZ2 domain alone (DegQ_LfΔPDZ2). Both variants were highly soluble, stable, and did not show any signs of misfolding as confirmed by SEC (Fig. S3) and DLS (Table S1). The truncated proteins were not able to assemble into large complexes and formed 3-mers and monomers only (Fig. S3), implying that the protease domain alone is sufficient for 3-mer formation whereas PDZ2 is essential for the assembly of the higher-order oligomers.

Crystal Structure of the DegQ 12-mer.

To gain detailed insights into the structural organization of the observed higher-order oligomer and its functional implications, DegQ_Lf was crystallized and its three-dimensional structure determined by X-ray crystallography to a resolution of 2.15 Å (Table S2). In all four molecules of the asymmetric unit, the protease domain (residues 1–240; residues of the catalytic triad: S193, H84, and D114), the PDZ1 domain (residues 241–340), a short linker region (residues 341–352), and the PDZ2 domain (residues 353–439) were well defined by electron 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 nomenclature 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 DegQ_Lf monomers exhibit very similar conformations.

Analysis of the crystal lattice revealed the presence of a highly symmetric DegQ_Lf 12-mer (Fig. 2 A–C), which should correspond to the large complex observed in SEC. The DegQ_Lf 12-mer displays tetrahedral (332) symmetry and is composed of four homotrimers as basic building blocks, each stabilized by extensive contacts between the three protease domains. Every 3-mer interacts with the three remaining 3-mers of the 12-mer, giving rise to a spherical particle with an outer diameter of approximately 140 Å. Because of its cage-like organization, it encloses an internal cavity (Fig. 2D) with an average diameter of approximately 70 Å that lacks defined electron-density features. The active sites of the protease domain lining the inner wall of the 12-mer are accessible only from the interior of the particle (Fig. 2D). Six lateral pores (approximately 14 Å × 28 Å) connect the internal cavity with bulk solvent (Fig. 2C). Strands β1, β2, and β4 of two juxtaposed DegQ_Lf monomers partially cover each pore from the inside, restricting the size of the opening. Two LA loops connecting β1 and β2 are thus positioned in direct vicinity of every lateral pore, although the flexible loop itself could not be traced in the electron density.

Fig. 2.

Structure of the DegQ_Lf 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 DegQ_Lf 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 interactions. 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).

In the DegQ_Lf 12-mer, PDZ1 and PDZ2 are integral parts of the protein shell. The peptide-binding groove of PDZ1 is accessible from the inner cavity of the DegQ_Lf 12-mer; yet, electron-density maps show no evidence for bound peptides or substrate molecules. It is unlikely that PDZ2 is able to bind substrate molecules because its peptide-binding groove is inaccessible and lacks a positively charged amino acid residue to stabilize the C-terminal carboxyl group of substrates. Instead, PDZ2 is responsible for the structural integrity of the 12-mer by mediating interactions between adjacent 3-mers (Fig. 2 B, E, and F). Accordingly, in solution DegQ variants lacking a PDZ2 domain (DegQΔPDZ1&2 and DegQΔPDZ2) assemble into 3-mers and are incapable of forming higher-order oligomers (Fig. S3).

The 12-mer Is the Active Form of the Protease.

Wild-type DegQ_Lf was able to proteolytically degrade β-casein and unfolded BSA (via DTT treatment) but not native BSA (Fig. 3A), indicating that suitable DegQ_Lf substrates must contain partially unfolded regions. The absence of distinct cleavage intermediates suggests a processive degradation of substrate proteins into small peptides. Unlike DegP_Ec (18), DegQ_Lf was unable to process reductively unfolded lysozyme (Fig. 3A). In quantitative protease assays, DegQ_Lf was efficiently degrading resorufin-labeled β-casein, whereas, as expected, DegQ°_Lf did not show any activity (Fig. 3B).

Fig. 3.

Assembly of DegQ_Lf 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 DegQ_Lf. (B) Quantitative protease assay with DegQ_Lf proteins using resorufin-labeled casein as substrate. Residual activities of DegQ_Lf variants (c–h) are compared to wild-type DegQ_Lf (b) and a control sample without protease (a). (C) Chaperone activity of DegQ_Lf proteins. Citrate synthase (CS) was heat-inactivated for the indicated period of time in the presence of DegQ_Lf proteins or BSA (control), and residual CS activity was determined.

Deletion of the LA loop (DegQ_LfΔLA; residues 28–61 replaced by a single glycine) did not affect formation of 12-mers in solution (Fig. S3) or proteolytic activity (Fig. 3B). In contrast, DegQ_Lf variants incapable of 12-mer formation (DegQ_LfΔPDZ2, DegQ_LfΔPDZ1& 2) were completely inactive (Fig. 3B). To verify that oligomerization of DegQ_Lf and not the mere presence of a PDZ2 domain is critical for proteolytic activity, we designed a truncated protein variant lacking only the nine C-terminal residues of PDZ2 (DegQ_LfΔC9). The DegQ_Lf structure shows that these residues should be important for the stability of the 12-mer by mediating numerous interactions between PDZ domains of adjacent 3-mers. Indeed, DegQ_LfΔC9 formed exclusively 3-mers (Fig. S3) that were proteolytically inactive (Fig. 3B), further supporting that the 12-mer represents the protease-active form of DegQ_Lf.

To test if DegQ_Lf possesses chaperone-like activity as reported for DegP_Ec, we evaluated the protective effect of DegQ_Lf on heat-induced denaturation of citrate synthase. DegQ_Lf, DegQ°_Lf, and DegQ_LfΔLA, as well as the truncated variants DegQ_LfΔC9 and DegQ_LfΔPDZ2, showed comparable results (Fig. 3C), demonstrating a chaperone-like activity of DegQ_Lf independent of 12-mer formation or the presence of PDZ2.

Reorientation of PDZ1 Is Inactivating 3-meric DegQ Variants.

It is not obvious why protease activity is completely abolished in DegQ variants able to form 3-mers but not 12-mers. Assuming that the overall structure of the 3-meric building blocks is independent of incorporation into 12-mers, the exposed protease-active sites of free 3-mers should in fact promote the degradation of substrate molecules. To understand the molecular basis of the inactivation of DegQ 3-mers, we crystallized the DegQ_LfΔPDZ2 variant and determined its three-dimensional structure at 3.1-Å resolution (Table S2). As expected, the protease core of the DegQ_LfΔPDZ2 3-mer is identical to that observed in the structure of the DegQ_Lf 12-mer (rmsd for Cα atoms of protease 3-mers in DegQ_Lf and DegQ_LfΔPDZ2 is approximately 1.3 Å). Although the overall fold of the PDZ1 domain is also preserved, it is rotated by approximately 180° relative to the protease domain compared to its orientation in the 12-mer (Fig. 4A). This rotation places the peptide-binding cleft of PDZ1 and the protease-active site on opposite faces of the 3-mer. Furthermore, the linker region that connects PDZ1 and protease domain (residues 241–249) is inserted into the peptide-binding cleft of PDZ1 in an extended conformation, antiparallel to the central β-sheet of this domain, mimicking a bound substrate molecule (Fig. 4B) (19). The position of PDZ1 with respect to the protease domain is further stabilized by hydrogen bonds between E112 and H244 and between Q236 and S275. In DegQ_LfΔPDZ2, loops of the protease domain exhibit a higher flexibility than in the 12-mer. Furthermore, the catalytic triad is disrupted, as H84 is highly flexible and is positioned at a distance of 4.3 Å from the nucleophilic S193, rendering the protease inactive. Although the positioning of PDZ1 in the crystal lattice might be influenced by a Cd²⁺ ion that mediates a crystal contact to an adjacent DegQ_Lf 3-mer (CdCl₂ was used as an additive in crystallization), the structure shows that PDZ1 is not rigidly attached to the protease domain in DegQ_LfΔPDZ2. It is tempting to speculate that a very similar rotation of PDZ1 might switch off protease activity of full-length DegQ_Lf 3-mers in solution.

Fig. 4.

Structure of DegQ_LfΔPDZ2. (A) Compared to DegQ_Lf (blue), the PDZ1 domain of DegQ_LfΔPDZ2 (red) is rotated by approximately 180° relative to the protease domain. The PDZ2 domain of DegQ_Lf is not shown. (B) (Left) The PDZ1 peptide-binding groove of DegQ_LfΔPDZ2 variant (orange). The loop connecting protease and PDZ1 (residues G241–G249, shown in red) is inserted into the peptide-binding groove in a substrate-like manner. In DegQ_Lf (equivalent loop shown in green) the peptide-binding groove is accessible. (Right) For comparison, the PDZ1 domain of DegP_Ec [gray, PDB ID code 3CS0, (10)] bound to a short substrate peptide (blue).

Plasticity of the Active Site and Intrinsic Regulation of Protease Activity.

In the DegQ_Lf 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 occupancy. 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 pocket by displacing the side chain of P190 to allow for substrate binding. 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 DegQ_Lf. To get further insights into its function, we disrupted the L1 switch by replacing P190 and the preceding N189 by glycines. The 2.4-Å-resolution structure of the resulting variant, DegQ_LfL1 (Table S2), revealed that the modified L1 loop adopts a unique conformation different from the ON and OFF conformations and is unable to form a functional oxyanion hole (Fig. 5D). Accordingly, DegQ_LfL1 is proteolytically inactive (Fig. 3B). These findings corroborate the critical importance of an intact L1 switch element for the protease activity of DegQ_Lf.

Fig. 5.

Plasticity of the protease-active site of DegQ_Lf. (A) Overview on the active-site loops L1 (orange), L2 (green), L3 (blue), LD (red) of the protease domain of DegQ_Lf with flexible regions indicated by dashed lines. Residues of the peptide-binding groove of the PDZ1 domain are colored in magenta. (B–D) Comparison of active-site loops L1 and LD in DegQ_Lf, DegQ°_Lf, and DegQ_LfL1. 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.

Details on the activation mechanism of DegQ_Lf were elucidated by the 3.1-Å-resolution crystal structure of the protease-inactive DegQ°_Lf variant in complex with a peptide substrate (Table S2). Like the wild-type enzyme, this variant formed 12-mers in the crystal lattice. Additional electron density was located in the protease-active site of DegQ°_Lf, unambiguously indicating 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 acid residues of the peptide were clearly defined and included in the model. Superimposition of DegQ_Lf and DegQ°_Lf revealed distinct conformational changes in loops L1, L2, L3, and LD. In the peptide complex, the switch element L1 adopts the ON conformation (Fig. 5C) and residues I188, P190, and N192 along with N208 and I211 of L2 shape the S1 specificity pocket of the protease (Fig. 5F). This rather restricted pocket is able to accommodate small, hydrophobic residues. In contrast, the primed subsites S1′ to S3′ and the nonprimed subsites S2 to S4 lack well-defined binding pockets and seem to be less discriminatory (Fig. 5F and Fig. S4). Peptide binding is further stabilized by main-chain hydrogen-bonding interactions between the substrate (P1 and P3) and residues of L2 (T209 and I211; Fig. S5). In the peptide complex, large portions of L3 are defined by electron density, except residues 172–178 located in close proximity to the substrate binding cleft of PDZ1. A rearrangement of L3 enables a hydrogen-bonding interaction between the guanidinium group of R170 and the main-chain carbonyl of L151 residing in loop LD of an adjacent molecule (LD*; the asterisk indicates loops of neighboring DegQ molecules). LD* in turn has moved by 6–7 Å from its position in the peptide-free structure (Fig. 5 B and C) and stabilizes the ON conformation of the L1* switch element via a main-chain hydrogen bond between residues P190 and F149. Thus, the reorganization of the active-site loops suggests an interplay between PDZ1 and protease domain of adjacent monomers. It is easily conceivable that upon binding of an allosteric activator to PDZ1, a cascade of conformational rearrangements is initiated along L3 and LD* that finally stabilizes loops L1* and L2* in a proteolytically competent state to allow for efficient degradation of substrate molecules.

Discussion

Members of the HtrA-protein family have been extensively studied over more than two decades. Based on biochemical and structural analysis, functional models have been developed that shed light onto mechanism and regulation of these intriguing enzymes. In contrast to DegP_Ec and DegS_Ec, the third HtrA protein in E. coli, DegQ, has not been characterized in great detail. The notion that numerous bacteria including many pathogens encode only two HtrAs, a DegS and a DegQ homologue (6), prompted us to study DegQ from Legionella.

The predominance of stable 12-mers as the major oligomeric form of DegQ_Lf suggests an important biological function for this assembly. Using our structural data as a framework, we probed the features of DegQ_Lf 12-mers by designing protein variants that were subsequently characterized with regard to oligomerization behavior and protease as well as chaperone activity. We show that protease but not chaperone activity is dependent on 12-mer formation. Large, cage-like 12- and 24-mers have been reported to be the proteolytically active oligomeric species in E. coli DegP (10, 14). However, DegP_Ec 12- and 24-mers are formed only transiently, dependent on the presence of partially unfolded proteins, whereas DegQ_Lf 12-mers assemble independently of substrate and represent the predominant oligomeric species over a broad range of environmental conditions. In DegQ_Lf 12-mers as well as in DegP_Ec 12- and 24-mers, interactions between protease domains stabilize the 3-meric building blocks, and PDZ domains mediate contacts between neighboring 3-mers. Interestingly, the two available models for the DegP_Ec 12-mer based on cryo-EM data published by two independent groups show discrepancies regarding the assembly mode of the particle (10, 14). One model suggests that interactions between two PDZ2 domains of adjacent 3-mers stabilize the 12-mer (10). The higher-resolution reconstruction based on 8-Å-resolution cryo-EM data implicates that 12-mer formation is supported by contacts between PDZ1 and PDZ2 domains (14). Our structural and biochemical data show that intact PDZ2 domains are critical for the assembly of DegQ_Lf 12-mers. However, here PDZ2 is simultaneously interacting with three adjacent 3-mers via contacts to two PDZ2, one PDZ1, and one protease domain (Fig. 2F). Thus, a tightly interconnected, stable network is formed that is fundamentally different from the models proposed for DegP_Ec 12-mers, suggesting that the general architecture of DegQ_Lf and DegP_Ec 12-mers may be different.

The structure of DegQ_LfΔPDZ2, a variant incapable of assembly into 12-mers, suggests that the protease activity of DegQ_Lf 3-mers can be switched off by reorientation of the PDZ1 domain. Based on these results, we propose a simple working model for function and regulation of DegQ_Lf (Fig. 6). In vivo, DegQ_Lf 12-mers are the predominant oligomeric species with a smaller fraction forming 3-mers as indicated by our SEC experiments. As the protease-active sites of free 3-mers are exposed and could potentially degrade periplasmatic proteins in an uncontrolled manner, it is essential that the protease activity is switched off in this state. In the homologous DegP_Ec, free 3-mers are readily assembled into inactive 6-mers (9). Yet, 6-mers were absent in all our DegQ preparations, and the shortened LA loop would not support the formation of such a resting-state oligomer. Our data suggest that free DegQ 3-mers are inactivated by a large-scale domain movement of PDZ1 that might represent a unique safety mechanism protecting the cell from deleterious proteolytic activity. Upon (re-)integration into 12-mers, PDZ1 is reoriented to promote proteolytic activity of DegQ_Lf and allow for degradation of substrate molecules. However, in the absence of suitable substrates, the protease-active sites of DegQ_Lf 12-mers are distorted and a distinctive OFF conformation of loop L1 is strongly favored. Based on our structural data, we propose that DegQ_Lf is activated via a cascade of conformational changes in L3, LD, L2, and L1, which are most likely initiated by binding of an allosteric activator to PDZ1. The fact that, in our crystal structure, PDZ1 is free of peptides might be attributed to a release of the allosteric effector after triggering the activation cascade. As protein substrates could act as allosteric effectors promoting their own degradation, a release from the PDZ1 domain after cleavage is necessary to allow for processive substrate degradation. Similar allosteric activation cascades have been described for DegS_Ec 3-mers (20) and very recently also for DegP_Ec 24-mers (21). Our findings strongly suggest that the general mechanism for the intrinsic regulation of HtrA protease activity is conserved in DegQ, DegP, and DegS. A peptide-bound PDZ domain is assumed to be the prerequisite for protease activation in DegS and DegP, yet the detection by L3 seems to be remarkably diverse. In DegS_Ec, peptide-binding causes a reorientation of the PDZ domain, which in turn relieves inhibitory contacts between the PDZ and the protease domain (21). For DegP_Ec, it has been postulated that loop L3 senses the locked position of PDZ1 in the active 24-mer, rather than the PDZ1-bound peptide itself (21). Based on our data, we propose that the allosteric regulation of DegQ_Lf and DegP_Ec is similar, yet we cannot specify if L3 directly interacts with the PDZ1-bound peptide or with the PDZ1 domain because the tip of L3 is flexible in our crystal structure.

Fig. 6.

Model for DegQ_Lf 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 allosterically activates the protease domain via a cascade of conformational changes along protease loops L3 → LD^∗ → L1^∗/L2^∗.

The structural and biochemical data provided in this study characterize DegQ as a unique member of the HtrA family with distinct features. The high-resolution crystal structure of a 12-meric HtrA protein reveals aspects of architecture and regulation that also should be relevant for other family members, especially for the DegP 12-mer, although there are differences from models based on cryo-EM data of the latter (10, 14). Our data allowed the development of a preliminary mechanistic model, on the basis of which a number of important questions are raised. Firstly, how do substrates gain access to the protease-active sites that line the inner wall of the 12-mer? On the one hand, DegQ_Lf 12-mers could transiently disassemble into 3-mers encapsulating substrates upon reassembly; on the other hand, it is also possible that substrates are threaded through the large lateral pores present in the shell of the DegQ cage. Secondly, details of the allosteric control, especially peptide binding to the PDZ1 domain, remain to be elucidated. A crystal structure of DegQ in complex with peptides bound to both PDZ1 and protease domain would verify our proposed model of the intrinsic regulation of protease activity in DegQ. Finally, as for DegP_Ec, the molecular basis for the chaperone activity of DegQ remains obscure. Experimental results providing answers to these questions are highly desirable and will further our understanding of this fascinating protein family.

Materials and Methods

Protein Production and Purification.

Details on production and purification of Legionella DegQ proteins will be described in detail elsewhere. Briefly, N-terminally His-tagged DegQ proteins lacking the signal sequence were produced in degP-deficient E. coli KU98 harboring the pQE-31 (Qiagen) derivative pGDR and purified by nickel-affinity chromatography. Fractions containing the recombinant proteins were pooled, dialyzed against protein storage buffer [20 mM sodium acetate (pH 4.5), 200 mM sodium chloride], and concentrated to 6–20 mg/mL.

SEC.

Analytical SEC was performed using a Superdex 200 HR 10/30 column (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 (approximately 1 mg), fractions of 0.5 mL were collected and subsequently analyzed by SDS-PAGE. The SEC column was calibrated with marker proteins ranging from 670 to 29 kDa.

Protein Activity Assays.

DegQ_Lf mediated degradation of BSA (GERBU) and lysozyme (GERBU) was analyzed in presence or absence of DTT. The reaction mixture included 20 μM DegQ_Lf, 1 mg/mL BSA or lysozyme in protein storage buffer with or without 20 mM DTT. The assay was performed overnight at 42 °C, and resulting samples were analyzed by SDS-PAGE. Quantitative protease assays using resorufin-labeled casein (Roche) were performed as described previously (13). All measurements were conducted as duplicates. The chaperone activity assay was modified after Buchner et al. (22).

Crystallization, Data Collection, and Structure Determination.

DegQ_Lf constructs were crystallized using the sitting-drop vapor-diffusion technique. Details of methods used for crystallization are provided in SI Materials and Methods. X-ray diffraction data were collected at BESSY (Berlin, Germany) and MAX-lab (Lund, Sweden), integrated with MOSFLM (23), and scaled and merged with SCALA (24). Initial phases were obtained by molecular replacement with Phaser (25) using individual domains of DegP_Ec [Protein Data Bank (PDB) ID code 3CS0 (10)] as search models. Subsequent model building and refinement was performed with Coot (26) and REFMAC (27), respectively. Data collection and refinement statistics are summarized in Table S2.