Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF–COOH containing peptides

Journal of Structural Biology 176 (2011) 16–23

Contents lists available at ScienceDirect

Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi

Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF–COOH containing peptides Kai Fan a, Jiahai Zhang a, Xuecheng Zhang b, Xiaoming Tu a,⇑ a b

Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China School of Life Sciences, Anhui University, Hefei, Anhui 230039, PR China

a r t i c l e

i n f o

Article history: Received 15 February 2011 Received in revised form 23 June 2011 Accepted 28 June 2011 Available online 2 July 2011 Keywords: Streptococcus pneumoniae HtrA PDZ domain NMR Peptide-binding module

a b s t r a c t High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains three a-helices and five b-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX–YYF–COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays a crucial role in protein quality control in prokaryotes and eukaryotes. Bacterial HtrA proteins maintain cell functions by acting as both molecular chaperones and proteases to manage misfolded proteins. HtrA switches from chaperone to protease in a temperature-dependent manner, with the protease activity being the most apparent at elevated temperature (Spiess et al., 1999). The human homologues of HtrA are believed to be involved in cell growth, unfolded protein response, and apoptosis (Gray et al., 2000; Suzuki et al., 2001). PDZ domains are protein–protein recognition modules that play a central role in organizing diverse cell signaling assemblies. These domains were first identified from a consensus sequence of 90 amino acid residues shared by three proteins: the postsynaptic density PSD-95/SAP90, the Drosophila septate junction protein Disc-large, and the tight junction protein ZO-1. PDZ domains have a common structure consisting of six b-strands and two a-helices, which fold in an overall b-sandwich. PDZ domains mediate a wide range of specific protein–protein interactions by binding in a sequence-specific manner to the C termini of their biological partners or, in some instances, to internal sequence motifs (Harris and Lim, 2001; Hung and Sheng, 2002). It has become apparent that the highly specific nature of these protein–protein interactions is ⇑ Corresponding author. Fax: +86 551 360 0757. E-mail address: [email protected] (X. Tu). 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.06.009

important for the biological function of scaffolding proteins that contain PDZ domains (Zhang et al., 2006). From Streptococcus pneumoniae, a HtrA homologue has been identified, which contains one PDZ domain at its C terminus. S. pneumoniae (pneumococcus) is an important human pathogen, which causes serious and life-threatening infections, including pneumonia, bacteremia and meningitis (Cartwright 2002). HtrA plays a crucial role in virulence of the pneumococcus as a virulence factor and controls the bacteriocin activity of S. pneumoniae with its pleiotropic regulatory effects in several important bacterial pathways (Dawid et al., 2009). In this study, the three-dimensional solution structure of HtrA PDZ domain from S. pneumoniae was determined by NMR spectroscopy. The structure consists of a five-stand b-sandwich capped by two a-helices. In addition, the peptide-binding mode of HtrA PDZ domain was elucidated by combined experiments of surface plasmon resonance (SPR), NMR chemical shift perturbation, and sitedirected mutagenesis. 2. Materials and methods 2.1. Cloning, expression and protein purification The DNA fragment encoding residues 262–386 corresponding to the PDZ domain of HtrA was amplified by PCR and was cloned into NdeI/XhoI-cleaved plasmid pET22b(+) (Novagen). The recombinant vector was transformed into expression host BL21 (DE3). The recombinant HtrA PDZ domain was expressed and purified

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23

as described previously (Fan et al., 2010). Uniformly 15N, 13C-labeled protein was prepared with minimum medium containing 0.5 g/L 99% ammonium chloride and 2.5 g/L 99% 13C-glucose as the sole nitrogen and carbon source, respectively. NMR sample contained 0.6 mM HtrA PDZ domain, 25 mM phosphate (pH 6.8), 100 mM sodium chloride, 1.5 mM DTT in either 90% H2O/10% 2 H2O or 100% 2H2O. 2.2. NMR spectroscopy, date processing and structure calculation All NMR data were collected at 298 K on a Bruker DMX500 spectrometer. NMR data processing was carried out using NMRPipe and NMRDraw (Delaglio et al., 1995). 1H–15N HSQC, HNCACB, CACB (CO)NH, HNHA, HCC(CO)NH, H(CC)CONH, 3D 15N-edited NOESY, and 13C-edited NOESY spectra were recorded for structure analysis. The data were analyzed with Sparky 3 software (Goddard and Kneller, 1993). All softwares were run on a Linux system. 3D 15Nedited and 13C-edited NOESY spectra were used to determine the NMR distance restraints for structure calculations. Chemical shift index was carried out for Ca, Cb, C0 and Ha. The information on the u and w backbone dihedral angles was obtained by TALOS program (Shen et al., 2009). Structures were calculated using CYANA (Güntert et al., 1997). Dihedral angle restraints, hydrogen bonds, and all other NOEs were introduced in consecutive steps. A total of 200 conformers were independently calculated, and 20 lowestenergy structures were selected and analyzed by MOLMOL (Koradi et al., 1996) and PROCHECK (Laskowski et al., 1996). 2.3. Surface plasmon resonance (SPR) Interactions between HtrA PDZ domain and peptides were measured with a BIAcoreÒ 3000 instrument (BIAcore AB, Uppsala, Sweden). The peptides were coupled to a carboxymethyl-dextran CM5 sensor chip. The CM5 chip was activated using an amine coupling reagent mixture containing 0.4 M EDC [1-ethyl-3(3-dimethylaminopropyl) carbodi-imide-HCl] and 0.1 M NHS (Nhydroxysuccinimide) at a flow rate of 5 ll/min for 10 min. The peptides were diluted to a concentration of 1 mg/mL with running buffer (25 mM NaH2PO4, pH 6.8, containing 100 mM NaCl) and immobilized on the chip at a flow rate of 5 ll/min for 10 min. Ethanolamine–HCl (1.0 M, pH 8.5) was used to neutralize unbound activated sites on the chip at a flow rate of 5 lL/min for

17

10 min. The chip was washed twice with regeneration buffer (20 mM NaOH) and then with running buffer. Purified HtrA PDZ domain was diluted with running buffer. Kinetic analysis of interactions between peptide and HtrA PDZ domain was performed at six concentrations of HtrA PDZ domain (2.34, 4.67, 9.35, 18.70, 37.40 and 74.80 lM) at a flow rate of 10 lL/min for 2 min. Regeneration was performed at a flow rate of 30 lL/min for 2 min. The analysis was performed three times for each concentration. To overcome refractive index changes, data of control surface were subtracted from that of peptide-immobilized surface. Interactions between peptides and Albumin Bovine V (BSA, Sigma, 8 mg/mL) were measured as a negative control. Kinetic analyses of SPR data were performed using BIAevaluation 4.1 (Biacore Inc.). Curves were fitted with 1:1 (Langmuir) binding model. Dissociation constant (Kd) was derived from kinetic analysis. 2.4. Chemical shift perturbation To identify the residues responsible for peptide binding of HtrA PDZ domain, 0.6 mM 15N-labeled HtrA PDZ domain was titrated with unlabeled peptide to different molar ratios (1:0, 1:1, 1:3, 1:5 and 1:10 of PDZ/peptide). 1H–15N HSQC spectra were acquired for every ratio for analysis. 2.5. Site-directed mutagenesis A series of point mutations were introduced into the recombinant pET22b(+)-HtrA-PDZ vector. The plasmid pET22b(+)-HtrAPDZ was used as a template, amplified by PCR using PrimeSTARTM HS DNA polymerase (TaKaRa, Dalian, China) and two complementary (partially overlapping) primers containing the desired mutation. The 50 lL PCR reaction was carried out with 50–100 ng templates, 10 lM primer pair, 200 lM dNTPs and 2 U of DNA polymerase, and started at 98 °C for 3 min, followed by 30 cycles of 98 °C for 45 s, 60 °C for 45 s, and a final extension at 72 °C for 5 min and 30 s. After PCR reaction, the PCR product was digested with DpnI (TaKaRa, Dalian, China) overnight to remove methylated parental non-mutated plasmid, and then transformed into Escherichia coli BL21(DE3). The mutant proteins were prepared and SPR experiments were performed essentially as described above.

Fig.1. Multiple sequence alignments of different HtrA PDZ domains from different species. Alignments were performed using ClustalW2 and ESPript 2.2. Identical residues were shaded in red box, and conserved residues are shown in red letters. The secondary structure of S. pneumoniae HtrA PDZ domain determined is also included at the top of the sequence.

18

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23

residues and exhibits the same secondary structure distribution pattern as other PDZ domains (Fig. 1), which may imply their common properties not only in structure but also in ligand recognition.

Table 1 NMR structural statistics. NMR restraints in the structure calculation Intraresidue Sequential (|i  j| = 1) Medium-range (|i  j| < 5) Long-range (|i  j| P 5) Hydrogen bonds Total distance restraints Dihedral angle restraints

346 562 240 280 34 1462 126

Residual violations CYANA target functions (Å) NOE upper distance constrain violation Maximum (Å) Number >0.2 Å

0.22 ± 0.12 1±1

Dihedral angle constrain violations Maximum (°) Number >5°

2.74 ± 0.48 0±0

van der Waals violations Maximum (Å) Number >0.2 Å

0.28 ± 0.01 4±1

Average structural rmsd to the mean coordinates (Å) Secondary structure backbonea Secondary structure heavy atomsa All backbone atomsb All heavy atomsb

0.500 1.335 0.580 1.336

Ramachandran statistics, % of all residues Most favored regions Additional allowed regions Generously allowed regions Disallowed regions

75.2 17.7 7.1 0.0

1.42 ± 0.23

a

Includes residues in secondary structure. Obtained for residues A30–N126 since no long-range NOEs were identified for amino acids 1–29 and 127. b

3. Results 3.1. HtrA PDZ domain exhibits moderate sequence similarity and conserved secondary structure compared with other PDZ domains Amino acid sequence analysis showed that HtrA PDZ domain from S. pneumoniae displays moderate sequence identity and similarity in comparison with other PDZ domains (Fig. 1). HtrA PDZ domain shares less than 30% sequence identity and 49% sequence similarity with other PDZ domains whose structures have been determined. On the other hand, HtrA PDZ domain shows some features shared in PDZ domain family in that it contains the conserved

3.2. Solution structure of S. pneumoniae HtrA PDZ domain S. pneumoniae HtrA PDZ domain, containing residues 262–386 of the protein, was recombinantly expressed and purified. The solution structure of S. pneumoniae HtrA PDZ domain was calculated based on a series of NMR spectra. The NMR data used for structure calculations are summarized in Table 1. The statistical parameters in Table 1 indicate a high-quality NMR structure of HtrA PDZ domain. The minimum-energy solution structures of HtrA PDZ domain and the assembly of twenty lowest-energy structures are shown in Fig. 2. The final ensemble of 20 refined structures has been deposited in the Protein Data Bank (PDB ID code: 2l97). The solution structure of S. pneumoniae HtrA PDZ domain contains five b-strands and three a-helices. The five b-strands are as follows: strand 1 (b1, residues 34–37), strand 2 (b2, residues 59–63), strand 3 (b3, residues 79–83), strand 4 (b4, residues 106–113) and strand 5 (b5, residue 116–123). Three helices are a1 (residues 42–49), a2 (residues 69–71) and a3 (residues 91–100). The global structure of HtrA PDZ domain adopts a b-sandwich (b1– b5) fold capped by two a-helices (a1, a3). The b-sandwich contains two b-sheets: one b-sheet consists of strand 1, strand 2 and strand 3; the other consists of strand 4 and strand 5. 3.3. Structural comparison between S. pneumoniae HtrA PDZ domain and other PDZ domains S. pneumoniae HtrA PDZ domain is similar to the canonical PDZ fold such as ZO1 PDZ1 and Erbin PDZ domains, except for the helix a3 at the bottom of the b-sandwich (Fig. 3; Appleton et al., 2006). The topology comparison of HtrA PDZ domain with other PDZ domains indicates a slightly difference, but with the secondary structure elements distribution essentially similar (Fig. 3). As the case in other PDZ domains, a deep binding groove for substrates is formed by the N-terminal loop, b1 and a3 of HtrA PDZ domain. The structure of S. pneumoniae HtrA PDZ domain was submitted to the structure recognition program DALI (Holm and Sander, 1995) to search for its similar structures. Not surprisingly, the result showed that the structures most similar to S. pneumoniae HtrA PDZ domain are those of the PDZ domains of human and E. coli HtrA family members. The Ca RMSD between S. pneumoniae HtrA

Fig.2. Structure of S. pneumoniae HtrA PDZ domain determined by NMR spectroscopy. (A) The ensemble of 20 lowest-energy conformers calculated for S. pneumoniae HtrA PDZ domain (residues 25–127). (B) Ribbon structure of a representative conformer of S. pneumoniae HtrA PDZ domain with the secondary structure elements highlighted (residues 25–127). Both figures were generated using MOLMOL.

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23

19

Fig.3. Structural comparison (upper) between (A) S. pneumoniae HtrA PDZ domain (residues 25–127), (B) Erbin PDZ domain and (C) Human HtrA1 PDZ domain. The corresponding SSEs in spatial structures are indicated with diagrams (lower).

PDZ domain and E. coli DegS (a homologue of HtrA) PDZ domain, human HtrA2 PDZ domain and human HtrA1 PDZ domain are 2.9 Å, 3.1 Å and 3.2 Å, with corresponding Z-scores of 9.8, 9.4 and 8.3, respectively. Although sequence comparison of these HtrA proteins reveals a low degree of sequence conservation, with 20% identity among the PDZ domains (Fig. 1), the similarity of three dimensional structures of these PDZ domains might indicate an evolutionary conservation of HtrA family proteins. 3.4. Interactions between S. pneumoniae HtrA PDZ domain and ligand peptides The PDZ domains of the HtrA family appear to have evolved to mediate specific ligand recognition. The optimal ligand-binding profiles for human and E. coli HtrA PDZ domains have been studied

(Zhang et al., 2007; Runyon et al., 2007; Walsh et al., 2003). The PDZ domain of E. coli Degs specifically recognizes the C-terminal short sequence motifs (–YYF–COOH) of misfolded OMPs (outermembrane porins). Here we investigated the interactions of the S. pneumoniae PDZ domain with four synthetic peptides (KRVYYF, GNVYYF, PIGHVYYF, and GHVYYF) corresponding to the motifs – YYF–COOH by surface plasmon resonance (SPR). The results indicated that S. pneumoniae HtrA PDZ domain bound specifically to the peptides (data not shown). The dissociation constant (Kd) for the peptide KRVYYF was estimated to be 11 lM (Fig. 4). Besides, the SPR experiments indicated that the S. pneumoniae HtrA PDZ domain also binds to the peptides VKIMVI and GQYYFV (data not shown), which are the favored recognition sequences for mammalian HtrA1 and HtrA2 PDZ domains respectively (Murwantoko et al., 2004).

Fig.4. Interaction between S. pneumoniae HtrA PDZ domain and the peptide KRVYYF observed by SPR.

20

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23

3.5. S. pneumoniae HtrA PDZ domain bound to the peptides via the groove formed by b1 and a3 To determine the residues of S. pneumoniae HtrA PDZ domain involved in peptide recognition, chemical shift perturbation experiments were performed using the representative peptide KRVYYF. 1 H–15N HSQC experiments were recorded for 15N-labeled HtrA PDZ domain before and after the addition of increasing amounts of unlabeled peptide to different molar ratios of PDZ/peptide. The peptide KRVYYF induced a number of significant chemical shift perturbations in the resonances of backbone amide protons of HtrA PDZ domain in the 1H–15N HSQC spectrum (Fig. 5A). These chemical shift changes increased gradually throughout the titration with

increasing amount of peptide. The residues, R28, L31 and I33 (belong to N-terminal loop), Q34, M35, V36 and N37 (belong to b1), S39, V41 and G58 (belong to the loop between b1 and b2), V59, I60, V61, R62 and S63 (belong to b2), Q65 (belong to the loop between b2 and b3), A70 (belong to a2), I80 (belongs to b3), S91, T92, D93, L94, Q95, S96, A97, L98 and N100 (belong to a3), showed obviously reduced peak intensity or substantial shift of resonance (Fig. 5A and B). Interestingly, the most perturbed residues cluster in a space composed of b1, b2 and a3, contributing to the hydrophobic pocket of S. pneumoniae HtrA PDZ domain (Fig. 6). This indicates that S. pneumoniae HtrA PDZ domain binds to the peptide in a canonical fashion as in the cases of other PDZ domains, wherein the peptides occupies the cleft between b1 and a3. Perturbation

Fig.5. The interactions between S. pneumoniae HtrA PDZ domain and the peptide KRVYYF studied by NMR. (A) The 1H–15N HSQC spectra of S. pneumoniae HtrA PDZ domain titrated with unlabeled peptide at various molar ratios. (B) The representative residues of S. pneumoniae HtrA PDZ domain with chemical shift or peak intensity significantly changed in the perturbation. Panel S91 only shows the shifted peak after titrated with ratio 1:5, while the original peak of S91 could not be seen in the area, as the dS91NH = 9.88 ppm. Three colors represent different molar ratios of PDZ/peptide: Green, 1:0; blue, 1:1; and red, 1:5.(For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.)

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23

21

Fig.6. (A) Ribbon representation and (B) molecular surface of S. pneumoniae HtrA PDZ domain displaying residues (in green color) involved in its interaction with the peptide KRVYYF. (C) Electrostatic surface diagram of S. pneumoniae HtrA PDZ domain.

Fig.7. The interactions between S. pneumoniae HtrA PDZ mutants and the peptide KRVYYF investigated by SPR: (A) S91A mutant; (B) S63A mutant.

of Q65 and A70, both located in the loop between b2 and b3, implied that this loop might be involved in ligand interaction as well.

and T92 are supposed to be critical for the ligand binding of HtrA PDZ domain. 4. Discussion

3.6. Site-directed mutagenesis indicated the critical residues responsible for HtrA PDZ domain binding to the peptide Since residues L31, I33, V36, R62, S63, A70, S91 and T92 of HtrA PDZ domain showed the most significant perturbation (Fig. 5B), point mutations at these sites were introduced individually. Their effects on the peptide binding ability were then evaluated by SPR (Fig. 7, Table 2 and Supplement Fig. 1). The mutation of most residues made little difference to the peptide binding affinity of HtrA PDZ domain. However, the mutant S91A abolished the peptidebinding completely and the mutants S63A and T92A decreased the affinity constant obviously. Therefore, the residues S91, S63

As a major virulence factor of S. pneumoniae (pneumococcus), an important human pathogen, HtrA is involved in the ability of the pneumococcus to grow at high temperatures, to resist oxidative stress, and to undergo genetic transformation. The S. pneumoniae HtrA contains a trypsin-like protease domain and one PDZ domain as other HtrAs do. PDZ domains are protein modules that mediate specific protein–protein interactions and bind preferentially the C-terminal 3–4 residues of target proteins. A unique feature of HtrAs is that the relative orientation of the PDZ and protease domains is fixed by an extended C-terminus that promotes additional interaction between the two domains. The PDZ

22

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23 Table 2 Binding constants of S. pneumoniae HtrA PDZ domain and KRVYYF–COOH determined by SPR. Mutation

Location

Kd (M)

Wild type L31A I33A V36A R62A S63A A70I S91A T92A

N terminal loop N terminal loop b1 b2 b2 a2 a3 a3

1.1e5 8.6e6 1.7e5 4.9e6 6.4e6 1.6e4 5.3e6 No binding 5.1e4

domain represents a binding platform for an allosteric activator, thereby coupling binding of an unfolded protein with activation of proteolytic activity. The interplay between PDZ and protease domain enables the HtrA proteases to reversibly switch between active and inactive states and thus adjust their enzymatic activity to fulfill diverse needs of the cells. It has been shown for HtrAs that PDZ domains are involved in sensing protein folding stress, as well as in regulation of catalytic activity and, in some cases, in assembly of various oligomeric states (Wilken et al., 2004; Murwantoko et al., 2004; Gupta et al., 2004; Iwanczyk et al., 2007; Hasselblatt et al., 2007). In this study, we solved the solution structure of S. pneumoniae HtrA PDZ domain, which possesses five b-strands and three a-helices. The global structure of HtrA PDZ domain adopts a five-strand b-sandwich (b1–b5) fold capped by two a-helices (a1, a3). Structural comparison indicated that S. pneumoniae HtrA PDZ domain shares the canonical tertiary structure with other HtrAs PDZ domains. Especially, there is the same binding groove formed by b1 and a3, indicating that S. pneumoniae HtrA PDZ domain might interact with its ligands in a similar mode with that of other PDZ domains. The optimal ligand-binding profiles for human HtrAs, mammalian HtrAs and DegS have been demonstrated (Zhang et al., 2007; Runyon et al., 2007; Walsh et al., 2003; Murwantoko et al., 2004). The PDZ domains of HtrA family can recognize C-terminal and internal stretches of hydrophobic sequence. Here, we performed the SPR experiments to investigate the interactions of the S. pneumoniae PDZ domain with synthetic peptides: KRVYYF, GNVYYF, PIGHVYYF, GHVYYF (corresponding to the –YYF–COOH motifs recognized by DegS PDZ domains), VKIMVI and GQYYFV (recognized by mammalian HtrA PDZ domains). The results indicated that all of these C-terminal hydrophobic peptides bind to the S. pneumoniae PDZ domain, supporting that all the HtrA PDZ domains might have the similar interaction mode. On the other hand, in contrast to Erbin and ZO1 PDZ domains, the more promiscuous specificity of the S. pneumoniae HtrA PDZ domain may be required for its recognition of misfolded proteins for proteins quality control as protease/chaperone. The interaction interface on S. pneumoniae HtrA PDZ domain for binding to the peptide KRVYYF was further characterized by chemical shift perturbation. It was shown that most perturbed residues come from b1, b2 and a3, which contribute to the formation of a hydrophobic pocket, similar to the case of other HtrA PDZ domains. Sequence analysis indicated that the peptide binding cleft is composed of a number of conserved or similar residues, revealing that S. pneumoniae HtrA PDZ domain binds to ligand peptides in a similar mode with that of other HtrA PDZ domains. The residues located at the binding clefts interact with ligands mainly by hydrophobic rather than electrostatic or hydrogen-bonding force. Furthermore, we designed a series of point mutations at the sites with the most significant perturbation to determine the crucial residues for peptide recognition. SPR experiments revealed

that the individual substitutions of Ala for residues L31, I33, V36, and R62 and the substitution of Ile for residue A70 might not affect the hydrophobic pocket accommodating the peptide, as the affinity constants showed no significant changes. Notably, the mutant S91A abolished its binding to peptide completely and the mutants S63A and T92A decreased the binding affinity constants obviously. The comparison with the counterpart residues of the structures of human HtrA PDZ domains in complex with peptides suggested that S63, S91 and T92 might interact with the residues V(3), Y(2), Y(2) of peptide K(5)R(4)V(3)Y(2)Y(1)F(0), respectively. Since the major difference of the side chains between Ala and Ser or Thr is the lack of hydroxyl in Ala residue, it is suggested that, beside the predominantly hydrophobic interactions, the hydrogen bonds formed by the hydroxyls of S63, S91 and T92 might be also crucial for the ligand binding of S. pneumoniae HtrA PDZ domain.

5. Conclusion The HtrA family of serine proteases has been shown to play an important role in the proteins quality control, which is mediated by unique domain architecture: the combination of a protease domain and a C-terminal PDZ domain. Here, we determined the solution structure of the S. pneumoniae HtrA PDZ domain and identified its interactions with some ligand peptides. Our results demonstrate that S. pneumoniae HtrA PDZ domain recognizes its binding partners in a canonical manner, and belongs to the class 2 PDZ domain, as it prefers C-terminal hydrophobic polypeptide motifs, especially at position 2, 0 (Songyang et al., 1997; Harris and Lim, 2001). Unlike the class 1 PDZ domains, the HtrA PDZ domain is considered to be a promiscuous peptide-binding module, which is consistent with the major biological function of HtrA: recognition of misfolded proteins for proteins quality control as protease/chaperone. A common activation mechanism has been proposed for HtrA proteins: ligands bind to the PDZ domain and induce conformational changes, which are then transmitted to the protease domain. The conserved structure and similar ligand recognition of S. pneumoniae HtrA PDZ domain in comparison to other HtrA PDZ domains might imply a similar activation mechanism for S. pneumoniae HtrA. Acknowledgments We thank F. Delaglio and A. Bax for providing NMRPipe and NMRDraw, T.D. Goddard and D. Kneller for Sparky, P. Güntert and K. Wüthrich for CYANA, R. Koradi and K. Wuthrich for MOLMOL. This work was supported by the Knowledge Innovation Program of the Chinese Academy of Science [grant number KSCX2-EW-Q-4], the National High-Tech R&D Program [grant number 2006AA02A315] and the National Basic Research Program of China (973 Program) [grant numbers 2007CB914503 and 2009CB918804].

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jsb.2011.06.009. References Appleton, B.A., Zhang, Y., Wu, P., Yin, J.P., Hunziker, W., et al., 2006. Comparative structural analysis of the Erbin-PDZ domain and the first PDZ domain of ZO-1: insights into determinants of PDZ domain specificity. J. Biol. Chem. 281, 22312– 22320. Cartwright, K., 2002. Pneumococcal disease in Western Europe: burden of disease, antibiotic resistance and management. Eur. J. Pediatr. 161, 188–195.

K. Fan et al. / Journal of Structural Biology 176 (2011) 16–23 Dawid, S., Sebert, M.E., Weiser, J.N., 2009. Bacteriocin activity of Streptococcus pneumoniae is controlled by the serine protease HtrA via posttranscriptional regulation. J. Bacteriol. 191, 1509–1518. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., et al., 1995. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293. Fan, K., Zhang, J., Shang, Q., Tu, X., 2010. 1H, 13C and 15N resonance assignment of the PDZ domain of HtrA from Streptococcus pneumoniae. Biomol. NMR Assign. 4, 79– 82. Goddard, T.D., Kneller, D.G., 1993. SPARKY 3, University of California, San Francisco. Gray, C.W., Ward, R.V., Karran, E., Turconi, S., Rowles, A., et al., 2000. Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response. Eur. J. Biochem. 267, 5699–5710. Güntert, P., Mumenthaler, C., Wüthrich, K., 1997. Torsion angle dynamics for NMR structure calculation with new program DYANA. J. Mol. Biol. 273, 283–329. Gupta, S., Gupta, S., Singh, R., Datta, P., Zhang, Z., et al., 2004. The C-terminal tail of presenilin regulates Omi/HtrA2 protease activity. J. Biol. Chem. 279, 45844– 45854. Harris, B.Z., Lim, W.A., 2001. Mechanism and role of PDZ domains in signaling complex assembly. J. Cell Sci. 114, 3219–3231. Hasselblatt, H., Kurzbauer, R., Wilken, C., Krojer, T., Sawa, J., et al., 2007. Regulation of the sigmaE stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress. Genes Dev. 21, 2659–2670. Holm, L., Sander, C., 1995. Trends Biochem. Sci. 20, 478–480. Hung, A.Y., Sheng, M., 2002. PDZ domains: structural modules for protein complex assembly. J. Biol. Chem. 277, 5699–5702. Iwanczyk, J., Damjanovic, D., Kooistra, J., Leong, V., Jomaa, A., et al., 2007. Role of the PDZ domains in Escherichia coli DegP protein. J. Bacteriol. 189, 3176–3186. Koradi, R., Billeter, M., Wuthrich, K., 1996. J. Mol. Graph. 14, 51–55.

23

Laskowski, R.A., Rullmann, J.A., MacArthur, M.W., Kaptein, R., Thornton, J.M., 1996. J. Biomol. NMR 8, 477–486. Murwantoko, Yano, M., Ueta, Y., Murasaki, A., Kanda, H., et al., 2004. Binding of proteins to the PDZ domain regulates proteolytic activity of HtrA1 serine protease. Biochem. J. 381, 895–904. Runyon, S.T., Zhang, Y., Appleton, B.A., Sazinsky, S.L., Wu, P., et al., 2007. Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3. Protein Sci. 16, 2454–2471. Shen, Y., Delaglio, F., Cornilescu, G., Bax, A., 2009. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223. Songyang, Z., Fanning, A.S., Fu, C., Xu, J., Marfatia, S.M., et al., 1997. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275, 73–77. Spiess, C., Beil, A., Ehrmann, M., 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339–347. Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., et al., 2001. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell 8, 613–621. Walsh, N.P., Alba, B.M., Bose, B., Gross, C.A., Sauer, R.T., 2003. OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain. Cell 113, 61–71. Wilken, C., Kitzing, K., Kurzbauer, R., Ehrmann, M., Clausen, T., 2004. Crystal structure of the DegS stress sensor: how a PDZ domain recognizes misfolded protein and activates a protease domain. Cell 117, 483–494. Zhang, Y., Appleton, B.A., Wu, P., Wiesmann, C., Sidhu, S.S., 2007. Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain. Protein Sci. 16, 1738–1750. Zhang, Y., Yeh, S., Appleton, B.A., Held, H.A., Kausalya, P.J., et al., 2006. Convergent and divergent ligand specificity among PDZ domains of the LAP and zonula occludens (ZO) families. J. Biol. Chem. 281, 22299–22311.