Crystal Structure of the Protease Domain of a Heat-shock Protein HtrA from Thermotoga maritima
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m208148200
ISSN1083-351X
AutoresDong Young Kim, Dong Ryoung Kim, Sung Chul Ha, N.K. Lokanath, Chang Jun Lee, Hye‐Yeon Hwang, Kyeong Kyu Kim,
Tópico(s)Protein Structure and Dynamics
ResumoHtrA (high temperature requirement A), a periplasmic heat-shock protein, functions as a molecular chaperone at low temperatures, and its proteolytic activity is turned on at elevated temperatures. To investigate the mechanism of functional switch to protease, we determined the crystal structure of the NH2-terminal protease domain (PD) of HtrA fromThermotoga maritima, which was shown to retain both proteolytic and chaperone-like activities. Three subunits of HtrA PD compose a trimer, and multimerization architecture is similar to that found in the crystal structures of intact HtrA hexamer fromEscherichia coli and human HtrA2 trimer. HtrA PD shares the same fold with chymotrypsin-like serine proteases, but it contains an additional lid that blocks access the of substrates to the active site. A corresponding lid found in E. coli HtrA is a long loop that also blocks the active site of another subunit. These results suggest that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change, which includes the opening of the helical lid to expose the active site and subsequent rearrangement of a catalytic triad and an oxyanion hole. HtrA (high temperature requirement A), a periplasmic heat-shock protein, functions as a molecular chaperone at low temperatures, and its proteolytic activity is turned on at elevated temperatures. To investigate the mechanism of functional switch to protease, we determined the crystal structure of the NH2-terminal protease domain (PD) of HtrA fromThermotoga maritima, which was shown to retain both proteolytic and chaperone-like activities. Three subunits of HtrA PD compose a trimer, and multimerization architecture is similar to that found in the crystal structures of intact HtrA hexamer fromEscherichia coli and human HtrA2 trimer. HtrA PD shares the same fold with chymotrypsin-like serine proteases, but it contains an additional lid that blocks access the of substrates to the active site. A corresponding lid found in E. coli HtrA is a long loop that also blocks the active site of another subunit. These results suggest that the activation of the proteolytic function of HtrA at elevated temperatures might occur by a conformational change, which includes the opening of the helical lid to expose the active site and subsequent rearrangement of a catalytic triad and an oxyanion hole. Protein quality control, which is essential for cell viability, is tightly controlled by proteases and molecular chaperones (1Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Google Scholar). Especially under stress conditions such as high temperature, heat-shock proteins, which are mostly proteases or molecular chaperones, are induced to protect cells from toxic denatured proteins (2Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Google Scholar). Molecular chaperones bind to the hydrophobic patches on denatured proteins to prevent further aggregation and help them to fold back into their native states (3Büchner J. FASEB J. 1996; 10: 10-19Google Scholar). The regulatory subunits of heat-shock proteases (4Gottesman S. Maurizi M.R. Wickner S. Cell. 1997; 91: 435-438Google Scholar) recognize the hydrophobic surface on unfolded proteins and eliminate them mostly in an ATP-dependent manner (5Goldberg A.L. Eur. J. Biochem. 1992; 203: 9-23Google Scholar). High temperature requirement A (HtrA, 1The abbreviations used are: HtrA, high temperature requirement A; CS, citrate synthase; NCS, noncrystallographic symmetry; PD, protease domain; r.m.s.d., root mean square deviations; Tm, Thermotoga maritima. also called DegP or protease Do) is a heat-shock protease localized in the periplasmic space of bacteria (6Pallen M.J. Wren B.W. Mol. Microbiol. 1997; 26: 209-221Google Scholar). It shows an ATP-independent proteolytic activity and plays an important role in the degradation of misfolded proteins accumulated by heat shock or other stresses (7Lipinska B. Fayet O. Baird L. Georgopoulos C. J. Bacteriol. 1989; 171: 1574-1584Google Scholar). Therefore, its activity seems to be essential for bacterial thermotolerance and for cell survival at high temperatures (7Lipinska B. Fayet O. Baird L. Georgopoulos C. J. Bacteriol. 1989; 171: 1574-1584Google Scholar). HtrA is also involved in pathogenesis of Gram-negative and Gram-positive bacteria by degrading damaged proteins that are produced by reactive oxygen species released from the host defense system (8Jones C.H. Bolken T.C. Jones K.F. Zeller G.O. Hruby D.E. Infect. Immun. 2001; 69: 5538-5545Google Scholar). Therefore, HtrA is considered as a target for development of broad-spectrum antibiotics (8Jones C.H. Bolken T.C. Jones K.F. Zeller G.O. Hruby D.E. Infect. Immun. 2001; 69: 5538-5545Google Scholar). In addition to proteolytic activity, HtrA is known to have a molecular chaperone activity (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar, 10Misra R. Castillo-Keller K. Deng M. J. Bacteriol. 2000; 182: 4882-4888Google Scholar). The chaperone function is dominant at low temperatures, whereas the proteolytic activity is turned on at elevated temperatures (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar). This temperature-dependent functional switch is necessary for controlling protein stability as well as eliminating denatured proteins to maintain cellular viability (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar). HtrA is a highly conserved protein found in species ranging from bacteria to humans. Two known human homologues of bacterial HtrA (HtrA1 and HtrA2) are also expected to be involved in mammalian stress response pathways (11Gray C.W. Ward R.V. Karran E. Turconi S. Rowles A. Viglienghi D. Southan C. Barton A. Fantom K.G. West A. Savopoulos J. Hassan N.J. Clinkenbeard H. Hanning C. Amegadzie B. Davis J.B. Dingwall C. Livi G.P. Creasy C.L. Eur. J. Biochem. 2000; 267: 5699-5710Google Scholar, 12Faccio L. Fusco C. Chen A. Martinotti S. Bonventre J.V. Zervos A.S. J. Biol. Chem. 2000; 275: 2581-2588Google Scholar). However, because HtrA2 showed proteolytic activity even at room temperature, temperature-dependent activation of proteolytic activity seems to be absent from mammalian HtrAs (13Savopoulos J.W. Carter P.S. Turconi S. Pettman G.R. Karran E.H. Gray C.W. Ward R.V. Jenkins O. Creasy C.L. Protein Expr. Purif. 2000; 19: 227-234Google Scholar). HtrA is a serine protease with a catalytic triad in its active site. Recent crystal structure analyses revealed that Escherichia coli HtrA forms a hexameric complex composed of two trimers (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar) and human HtrA2 forms a homotrimer (15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). Each subunit is composed of one protease domain at the amino terminus and one or two PDZ (named after three proteins, PSD-95, Discs-large, and ZO-1) domains at the carboxyl terminus. The protease domain of E. coli HtrA fully retains the molecular chaperone activity, although the proteolytic activity is absent (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar). The PDZ domains, also found in the Clp/Hsp100 family of heat-shock proteins, are known to play a role in substrate recognition (16Levchenko I. Smith C.K. Walsh N.P. Sauer R.T. Baker T.A. Cell. 1997; 91: 939-947Google Scholar). In the crystal structures of E. coli HtrA and human HtrA2, PDZ domains are proposed to mediate the initial binding of substrates (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar) or to be involved in modulation of protease activity (15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). However, PDZ domains do not participate in multimerization in both E. coli and human HtrAs (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar, 15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). Unlike other proteases of the Clp/Hsp100 family, HtrA does not have a regulatory component or an ATP binding domain because it is an ATP-independent heat-shock protease. So far two crystal structures of HtrAs have been reported (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar, 15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar), and they seem to differ in structural architecture, multimerization, and activation mechanism. For a better understanding of the dual role of HtrA and the activation mechanism of the proteolytic function, we have solved the crystal structure of the protease domain (PD, residues 24–262, Fig. 1) of Thermotoga maritima HtrA (Tm HtrA), which displays both molecular chaperone and proteolytic activities. The crystal structure indicates that the rearrangement of the active site of bacterial HtrA is necessary for the proteolytic activity and that oligomerization architecture of HtrA might vary depending on the presence of the lid covering the active site. The protease domain of HtrA from T. maritima (PD, residues 24–262, Fig. 1) was cloned, purified, and crystallized as described elsewhere (18Kim D.Y. Kim K.K. Acta Crystallogr. Sect. D. 2002; 58: 170-172Google Scholar). The putative signal sequence (residues 1–23) was deleted in the construct. For the translational start, a methionine residue was added in front of Asp24. Intact HtrA was also prepared in the same way as HtrA PD (18Kim D.Y. Kim K.K. Acta Crystallogr. Sect. D. 2002; 58: 170-172Google Scholar). HtrA PD was crystallized in the cubic space group P213, with the unit cell parameters a =b = c = 120.55 Å by the hanging drop vapor diffusion method at 22 °C from a reservoir solution containing 100 mm phosphate-citrate (pH 4.4), 110 mm Li2SO4, and 5% (v/v) PEG 1000 (18Kim D.Y. Kim K.K. Acta Crystallogr. Sect. D. 2002; 58: 170-172Google Scholar). There are two molecules in an asymmetric unit. The chaperone-like activities of HtrA and HtrA PD were measured as described previously (19Büchner J. Grallert H. Jakob U. Methods Enzymol. 1998; 290: 323-338Google Scholar) with some modifications using pig heart citrate synthase (CS) as the substrate (Sigma). CS (final 65 μm monomer) was denatured in a solution containing 100 mm Tris-HCl (pH 8.0), 100 mm NaCl, 6 m guanidinium hydrochloride, and 40 mm dithiothreitol for at least 2 h at room temperature. Chemically denatured CS was rapidly diluted 250-fold into a refolding buffer containing 10 mm Tris-HCl (pH 8.0) and HtrA or HtrA PD to reach the indicated molar ratios (Fig. 2). Light scatterings from aggregated proteins were monitored by measuring the absorbance at 320 nm with Spectra MAX Plus (Molecular Device) at 25 °C. Initial velocities of aggregate formation were calculated by measuring the absorbance change for the initial 15 s where the rate of aggregate formation is constant. Thirty micrograms of the reduced form of α-lactalbumin (Sigma) and 28 μg of HtrA PD or 48 μg of HtrA (at 1:2 molar ratio of protease to substrate) were incubated in 60 μl of reaction buffer containing 5 mmTris (pH 7.5) and 1.5 mm dithiothreitol at 25, 45, 65, and 85 °C. The reduced form of α-lactalbumin was prepared by incubating 3 mg/ml protein in 5 mm Tris (pH 7.5) containing 10 mm dithiothreitol at 4 °C for 2 days. HtrA and HtrA PD were preincubated at each indicated temperature prior to the addition of the substrate protein. The reaction was performed in 100-μl tubes to minimize the evaporation effect. After incubation for 30 min at each temperature, 17 μl of 5× SDS-PAGE sample buffer was added to stop the reaction. Then, the samples were analyzed by 17% SDS-PAGE. Multiwavelength anomalous diffraction data were collected from a frozen crystal of HtrA PD at the Pohang Accelerator Laboratory beamline 6B with a MacScience 2030b area detector. Data collected at three wavelengths (edge, peak, and remote) were processed and integrated by DENZO and scaled by SCALEPACK using the HKL program suite (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar). Native data of HtrA PD were also collected at the Pohang Accelerator Laboratory and processed using the HKL program suite (Table I). Two selenium sites (one methionine in one subunit) of PD were found and used for phase calculation in the program SOLVE (21Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Google Scholar). A relatively low figure of merit (0.25) is explained by the presence of only one selenium atom per 238 residues in the subunit.Table IData collection, phasing, and refinement statisticsCrystalPDPD-SeMetData collection and phasingWavelength (Å)0.97170.97910.97930.9715Resolution (Å)20.0–2.8 (2.9–2.8)30.0–3.0 (3.11–3.0)30.0–3.0 (3.11–3.30)30.0–3.0 (3.11–3.00)R merge(%)aRmerge = Σ‖I − ‖/ΣI.6.6 (27.0)8.0 (32.5)6.3 (28.8)5.8 (29.7)Completeness (%)95.1 (97.2)87.4 (90.6)87.4 (90.3)87.4 (91.1)No. of unique reflections13,843 (1376)10,379 (1047)10,401 (1053)10,423 (1066)Redundancy2.433.203.263.28I/ς15.714.515.415.4Overall figure of meritBefore density modification0.25After density modification0.54RefinementResolution (Å)20.0–2.8R factor/R free(%)bRfactor = Σ‖F obs −F calc‖/ΣF obs.22.2/28.4No. reflections (no cutoff)13,621No. solvent atoms58No. protein atoms3,464Mean B factors (Å2)54.8r.m.s.d. in bonds (Å)0.008r.m.s.d. in angles (°)1.53a Rmerge = Σ‖I − ‖/ΣI.b Rfactor = Σ‖F obs −F calc‖/ΣF obs. Open table in a new tab Solvent flattening and 2-fold noncrystallographic symmetry (NCS) averaged by RESOLVE (22Terwilliger T.C. Acta Crystallogr. Sect. D. 2000; 55: 965-972Google Scholar) resulted in a high quality electron density map sufficient for model building. Amino acids were assigned using the program O (23Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar). Several cycles of rigid body refinement, positional refinement, and simulated annealing were performed at 3.0-Å resolution with CNS (24Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilgen M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Google Scholar). The refinements were continued at 2.8-Å resolution using the data collected from the native HtrA PD. Successive refinement with temperature factors and addition of solvents resulted in anR-value of 22.2% and an R free value of 28.4%, with a bulk solvent correction and overall anisotropic thermal factor refinement. R free was calculated with 10% of the reflections. NCS restraints were enforced during the refinement except flexible regions (LA and L2), in which two subunits in an asymmetric unit showed different conformations. The final model includes residues 24–48 and 51–251 and 58 water molecules (Table I). Structural evaluation of the refined model using PROCHECK (25Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Google Scholar) reveals that the structure has good geometric parameters (Table I), and no residue falls in the disallowed region of the Ramachandran plot. Statistical analysis of B-factor distribution was performed by t test and the Wilcoxon rank sum test.p < 0.001 was considered to be significant. The figures in the article were drawn using the programs MOLSCRIPT (26Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Google Scholar) and GRASP (27Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Google Scholar). The final coordinates and structure factors have been deposited in the Protein Data Bank (PDB; accession number 1L1J). Tm HtrA and Tm HtrA PD were overexpressed in E. coli and purified. The chaperone-like activities of both Tm HtrA and Tm HtrA PD were measured by their abilities to suppress the aggregation of CS, which has been widely used for molecular chaperone assays (19Büchner J. Grallert H. Jakob U. Methods Enzymol. 1998; 290: 323-338Google Scholar). The aggregation of CS was monitored by light scattering at 320 nm after chemically denatured CS was diluted in the refolding buffer. Tm HtrA or Tm HtrA PD suppressed CS aggregation, decreasing the initial velocity of aggregate formation (Fig. 2, A andB). By addition of 2- and 4-fold molar excesses of Tm HtrA to CS, the initial velocities of aggregation were decreased to 40.6 and 13.7%, respectively, compared with the initial velocity in the absence of HtrA or HtrA PD. It was decreased to 68.3% when a 4-fold molar excess of Tm HtrA PD was added, whereas 2-fold addition of Tm HtrA PD essentially did not change it (data not shown). An 8-fold excess of either Tm HtrA or Tm HtrA PD was enough to decrease the aggregation rate to about zero. Under the same assay conditions, the initial velocity of the reaction is not decreased when bovine serum albumin was used for the control protein (Fig. 2, A and B). These results indicate that Tm HtrA PD as well as the intact Tm HtrA have the chaperone-like activity to inhibit the aggregation of CS. However, because the enzyme activity of CS was not recovered (data not shown), it seems that Tm HtrA does not assist the refolding of CS, although the aggregation of denaturated CS was completely suppressed by incubation with Tm HtrA. It can be inferred that Tm HtrA exhibited only chaperone-like activity against CS, as is observed for other heat-shock proteins such as α-crystallin (28Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Google Scholar). The molar ratios of chaperones to substrates required for suppression of CS aggregation are different between intact HtrA and HtrA PD (Fig.2), which is also observed for E. coli HtrA and E. coli HtrA PD. In the case of E. coli HtrA, a higher concentration of the protease domain was required for refolding of MalS protein than intact HtrA (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar). Such a requirement for higher molar ratios of HtrA PD might be explained by the absence of PDZ domains, which are known to be involved in substrate recognition (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar, 15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar, 16Levchenko I. Smith C.K. Walsh N.P. Sauer R.T. Baker T.A. Cell. 1997; 91: 939-947Google Scholar). α-Lactalbumin, which is commonly used for the assays of heat-shock proteases such as 20 S proteosome (29Wenzel T. Baumeister W. Nat. Struct. Biol. 1995; 2: 199-204Google Scholar) and E. coli HtrA (30Kim K.I. Park S.C. Kang S.H. Cheong G.W. Chung C.H. J. Mol. Biol. 1999; 294: 1363-1374Google Scholar), was employed as a substrate to investigate the proteolytic activity of Tm HtrA. Clearly, Tm HtrA displayed the proteolytic activity at elevated temperatures, and maximal activity was observed at 85 °C (Fig. 2 C). As reported for E. coli HtrA (9Spiess C. Beil A. Ehrmann M. Cell. 1999; 97: 339-347Google Scholar, 31Skorko-Glonek J. Krzewski K. Lipinska B. Bertoli E. Tanfani F. J. Biol. Chem. 1995; 270: 11140-11146Google Scholar), the proteolytic activity of Tm HtrA increased with temperature, and Tm HtrA is autodegraded at high temperatures (Fig.2 C). Compared with Tm HtrA, Tm HtrA PD showed a relatively weak proteolytic activity, although its activity also increased with temperature. The degradation products of Tm HtrA PD were long enough to be visible on 17% SDS-PAGE, whereas intact Tm HtrA completely degraded the substrate into short peptides that are not shown in the gel (Fig. 2 C). Such a weak proteolytic activity of Tm HtrA can also be explained by the absence of a PDZ domain. In both E. coli HtrA and human HtrA2, PDZ domains play key roles in substrate binding and formation of the chamber near the active site (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar, 15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). Tm HtrA PD generates longer degradation products, because substrates are freely released from Tm HtrA PD after cleavage. In contrast, within the chamber of intact Tm HtrA, substrates are cleaved into small peptides simultaneously at adjacent active sites. The crystal structure of Tm HtrA PD (residues 24–262) has been solved by multiwavelength anomalous diffraction at 3.0 Å resolution and refined to 2.8-Å resolution (Table I). The experimental electron density map calculated with multiwavelength anomalous diffraction phases and improved by solvent flattening and NCS averaging was of sufficient quality to locate most main chains and some side chains. Tm HtrA PD is composed of two β-barrel domains connected by a long loop between β6 and β7 (Figs. 3 and4 A). Residues in the catalytic triad, Asp127-His97-Ser206, are located in the cleft of two β-barrels. The structural comparison by DALI server (32Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Google Scholar) reveals that its topology is similar to proteases in the chymotrypsin family (33Perona J.J. Craik C.S. Protein Sci. 1995; 4: 337-360Google Scholar). Among the members of the chymotrypsin family, α-lytic protease (PDB accession number 1qq4; Ref. 34Derman A.I. Agard D.A. Nat. Struct. Biol. 2000; 7: 394-397Google Scholar) can be superimposed on Tm HtrA PD with the lowest r.m.s.d. of 2.5 Å for 161 Cα atoms of 198 Cα atoms of α-lytic protease (Figs. 4 Band 5). However, its fold is different from those of proteolytic cores of ATP-dependent proteases such as ClpP (35Wang J. Hartling J.A. Flanagan J.M. Cell. 1997; 91: 447-456Google Scholar) and HslV (36Bochtler M. Ditzel L. Groll M. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6070-6074Google Scholar).Figure 4Ribbon diagrams of Tm HtrA PD (A), α-lytic protease (B), E. coli HtrA PD (C), and human HtrA2 PD (D).α-Lytic protease, E. coli HtrA PD, and human HtrA2 PD were positioned in the same orientation with Tm HtrA PD. Two β-barrel domains are green and blue. The regions (LA) connecting β1 and β2 in Tm HtrA PD, E. coli HtrA PD, human HtrA2 PD, and α-lytic protease aremagenta and labeled. In E. coli HtrA PD the loopLA* containing residues 38–79 is pink. LA* is the loop belonging to the subunit in the opposite trimer; E. coli HtrA PD forms a dimer by an interaction at the interface between the loop LA and LA*. Residues 48 and 51 are connected by a dashed line because residues 49 and 50 are not modeled in Tm HtrA PD. The residues in the catalytic triads (Asp, His, and Ser) are drawn as orange stick models. These residues in Tm HtrA PD and E. coli HtrA are blocked by LA or LA*, whereas they are open to the solvent in α-lytic proteases and human HtrA2. Disulfide bonds of α-lytic protease are drawn as yellow lines. These bonds are not found in HtrAs, however. Each secondary structure and loop of Tm HtrA PD is labeled.View Large Image Figure ViewerDownload (PPT)Figure 5Comparison of three HtrA PDs and α-lytic protease. A stereo Cα trace of Tm HtrA PD (green), E. coli HtrA PD (blue), human HtrA2 PD (red), and α-lytic protease (magenta). Three residues of the catalytic triad of Tm HtrA PD are drawn as black stick models. Most structures in the core region containing the central β-barrel and α-helices are well conserved in the four proteins, whereas loop regions display differences. The loop LA of each HtrA, where a large conformational movement is observed, is labeled and emphasized by athicker line.View Large Image Figure ViewerDownload (PPT) It is notable, however, that several structural differences exist between Tm HtrA PD and α-lytic protease. The most significant of them is the length of LA (loop A connecting β1 and β2, according to the nomenclature in Ref. 33Perona J.J. Craik C.S. Protein Sci. 1995; 4: 337-360Google Scholar) in two proteins (Figs. 4 and5). LA of Tm HtrA (residues 47–76), including an amphipathic helical lid (α2, residues 55–66) and α3, is located on top of the catalytic residue Ser206(Figs. 4 A and 6 A). Interestingly, the residues corresponding to the helical lid of Tm HtrA PD are found only in bacterial HtrAs, not in human homologues (Fig. 1), and this lid is expected to have the important functional or structural roles in bacterial HtrAs. There are differences in several other loops connecting β-strands (Fig. 5). Among them, structural changes near the loop containing the putative oxyanion hole and the catalytic residue Ser206 (residues 202–206) seem to be significant for explaining the functional differences of Tm HtrA and α-lytic protease (Figs. 5 and 6). Three conserved disulfide bonds found in almost all members of the chymotrypsin family of serine proteases are absent from all known HtrAs (Fig. 4). Overall structure of Tm HtrA PD turns out to be similar with the protease domains of E. coli HtrA and human HtrA2 with an r.m.s.d. of 2.2 Å for 179 Cα atoms of 215 Cα atoms ofE. coli HtrA and 1.9 Å for 181 Cα atoms of 196 Cα atoms of human HtrA2, respectively (Figs. 4 and 5). The large r.m.s.d. among HtrAs are mostly caused by the structural difference near the active site and loop LA (Fig. 5). Interestingly, Tm HtrA shows more structural resemblance with human HtrA than E. coliHtrA. Trimeric interactions found in E. coli HtrA hexamer or human HtrA2 trimer seem to be conserved in Tm HtrA PD (Fig.7), in which three subunits of Tm HtrA PD related by 3-fold crystallographic rotation symmetry are tightly packed by hydrophobic interactions. A hydrophobic patch composed of the residues near α1, β7, β8, and β11 is involved in the hydrophobic packing in a trimer (Figs. 4 A and7 A). Those hydrophobic residues are quite well conserved in most other HtrAs (Fig. 1), implying that hydrophobic packing in a trimer is a general feature of HtrAs. By trimerization, the 6700 Å2 surface area of Tm HtrA PD is buried, which is comparable with the 6044 Å2 in human HtrA2 (15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). Taken together, it appears that the Tm HtrA forms a trimer by hydrophobic interaction mediated by the protease domain, as observed in E. coli HtrA and human HtrA2 (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar, 15Li W. Srinivasula S.M. Chai J. Li P. Wu J.-W. Zhang Z. Alnemri E.S. Shi Y. Nature Struct. Biol. 2002; 9: 436-441Google Scholar). Analytical ultracentrifugation and gel filtration experiments also support the existence of Tm HtrA PD as a trimer in solution (data not shown). The main differences among HtrAs might be the size and conformation ofLA (Figs. 1, 4, and 5). E. coli HtrA has a long loop reaching the active site of the opposite subunit (Figs.4 C, 6 C, and 7 B). In addition, β1 and β2 in E. coli HtrA is long enough to make a β-sheet with two other β-strands from the trimer in the other side, leading to a hexamer structure (Fig. 7 B) (14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar). In contrast, LAin Tm HtrA PD is mainly composed of a helix (α2) covering the active site of the same subunit in the current structure (Figs. 4 A,6 A, and 7 A) and is shorter than its counterpart in E. coli HtrA (Figs. 1 and 7). Interestingly, human HtrA2 has a very short LA, which is not involved in the dimerization of trimers or the covering of the active site (Figs.4 D, 6 D, and 7 C). Two molecules of HtrA PD in an asymmetric unit related by 2-fold NCS are associated by the minimal hydrophobic interactions among a few residues (Tyr25, Pro28, Val32, and Ala35; figure not shown) in the NH2-terminal helix (α1). Therefore, the presence of two molecules in the asymmetric unit seems to have no biological relevance. The two molecules in the asymmetric unit show identical conformations except the regions near LA and L2 (loop 2 connecting β11 and β12), suggesting those regions are relatively flexible. Most molecular chaperones have hydrophobic substrate binding sites on their surfaces to recognize and bind to the exposed hydrophobic patches of substrates (3Büchner J. FASEB J. 1996; 10: 10-19Google Scholar). However, in Tm HtrA PD trimer, most of the hydrophobic surface near the active site is buried and no noticeable hydrophobic region is exposed (Fig. 7 D). Therefore, certain conformational changes might occur to expose the hydrophobic substrate binding site when Tm HtrA or Tm HtrA PD shows the chaperone-like activity. Most hydrophobic residues in the helical lid of LA form wide contacts by hydrophobic interactions with Leu80 in β2, Pro163 and Leu164 in LD (loop D connecting β7 and β8), Pro203 and Gly204 in L1 (loop 1 connecting β9 and β10), and Ala223 and Ile224 in L2 (Fig. 6 A). BecauseLA* of E. coli HtrA makes intimate contact withL1 and L2 (the asterisk denotes the loops in the neighboring subunit, see Figs. 4 C,6 C, and 7 B; Ref. 14Krojer T. Garrido-Franco M. Huber R. Ehrmann M. Clausen T. Nature. 2002; 416: 455-459Google Scholar), the hydrophobic interactions between the loops near the active site and lid seem to be common in bacterial HtrAs. However, in other HtrAs residues interacting with LA appear to vary depending on the size of the lid (Fig. 1). Possible substrate binding sites of Tm HtrA (S3, S2, S1, S1′, S2′, and S3′ defined in Ref. 33Perona J.J. Craik C.S. Protein Sci. 1995; 4: 337-360Google Scholar) a
Referência(s)