Crystal Structure of the Unliganded Alkaline Protease from Pseudomonas Aeruginosa IF03O8O and Its Conformational Changes on Ligand Binding 1
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J. Biochem. 118, 474-479 (1995) Crystal Structure of the Unliganded Alkaline Protease from Pseudomonas aeruginosa IF03O8O and Its Conformational Changes on Ligand Binding 1 Hideyuki Miyatake,* Yasuo Hata,*,2 Tomomi Fujii,* Kensaku Hamada,•õ Kazuyuki Morihara,•ö and Yukiteru Katsube * Institute for Chemical Research , Kyoto University, Uji, Kyoto 611; •õFaculty of Science, Shimane University, Matsue, Shimane 690; •öInstitute for Applied Life Science, University of East Asia, 2-1 Ichinomiya-Gakuen-cho, Shimonoseki, Yamaguchi 751; and Institute for Protein Research, Osaka University, Suita, Osaka 565 Received for publication, May 31, 1995 The crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IF03080 has been determined at 2.0 A resolution by the X-ray method. The enzyme consists of N-terminal catalytic and C-terminal ƒÀ-helix domains. On structural comparison between the present unliganded enzyme and structurally-known liganded enzyme, some structural changes were observed around the active site. In the unliganded enzyme, Y216 serves as the fifth ligand for the active site zinc ion. On ligand binding, Y216 may move to form a hydrogen-bond with the carbonyl oxygen of the P1 residue of a ligand peptide. D191 in the flexible loop, Y190 to D196, over the active site cleft forms hydrogen-bonds with the backbone atoms of the P1 and P2 residues of the ligand to close the entrance of the cleft. The water molecule which is the fourth ligand for the zinc ion is replaced by the carbonyl oxygen of the P1 residue. These structural changes around the active site may reflect the substrate-binding mode during the enzymatic reaction. Key words: alkaline protease, ƒÀ-helix, crystal structure, metalloprotease, Pseudomonas aeruginosa. Pseudomonas aeruginosa is well known to secrete two alkaline protease. Neutral metalloproteases such as ther metalloproteases: a 50 kDa alkaline protease and a 33 kDa molysin and P. aeruginosa elastase, which are more com elastase, which belong to the serralysin and thermolysin mon than alkaline metalloproteases, are typical "N(NH2) families, respectively. These enzymes were originally type" enzymes which hydrolyze the peptide bonds of isolated from P. aeruginosa IF03080 and IF03455, and certain amino acid residues on the amino group side through characterized as zinc-dependent metalloproteases by specific recognition. On the contrary, a P. aeruginosa Morihara et al. (1-3). In particular, the alkaline protease alkaline protease is a "C (COOH) -type" endoprotease which from P. aeruginosa has been paid much attention in the digests its substrate on the carboxyl side of certain amino medical field for the development of anti-infection agents acid residues (13), and has a hydrophobic binding site (S2') against the bacterium (4). The enzyme is composed of a in addition to the major recognition site (S1) for Arg or polypeptide chain and one catalytic zinc atom, and requires probably Lys (14). Thus, the alkaline protease is an Arg (or several calcium ions for stabilization of its folding (2). The Lys)-specific C-type metalloprotease, although its sub amino acid sequences of 470 residues for P. aeruginosa strate specificity is relatively broad (12, 15). The patho alkaline proteases have been deduced from the DNA logical aspects of the alkaline protease have been extensive sequences of its strains, IF03455 (5) and PA01 (6). The ly investigated already. The enzyme exhibits the potential crystal structure of the alkaline protease from P. aeru anti-coagulant capacity to hydrolyze natural substrates of ginosa was determined using a complex of the enzyme with plasmin, such as fibrin and fibrinogen, with similar specific a mixture of tetrapeptide products (7). The structure was activities to plasmin (13). From these properties of the used to determine the crystal structure of the 50 kDa alkaline protease from P. aeruginosa , it is inferred that the metalloprotease from Serratia marcescens (8), a member enzyme may play a key role in infection of host cells by the of the serralysin family (9-11). From biochemical studies bacterium through the inactivation of various physiological on the catalysis by a P. aeruginosa alkaline protease, it is activators such as some complement components , im well known that the potential catalytic capability of the munoglobulins A and G, and many protease inhibitors (16). enzyme is optimum in the alkaline pH range of 8 to 10 (1, In order to elucidate the characteristic mechanism of its 12). The alkaline optimum pH is characteristic of the catalysis, and to determine the reasons for its alkaline optimum pH and the C(COOH)-type recognition on sub 1 This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 06235204) from the Ministry of strate cleavage, we have determined the crystal structure Education, Science and Culture of Japan. of the alkaline protease from P. aeruginosa IF03080 by the 2 To whom correspondence should be addressed . Phone: +81-774-32 X-ray diffraction method . Here we report the three-dimen 3111 (Ext. 2159), Fax: +81-774-33-1247 sional structure of the unliganded alkaline protease from P. 474 J. Biochem. Unliganded Alkaline Protease Structure 475 aeruginosa determined at 2,0 A resolution, and the struc technique (19). The solvent-flattened map was good enough tural differences between the unliganded and liganded to follow the entire main-chain folding and most of the forms of the enzyme. side-chain orientations of the enzyme molecule. An initial A lyophilized sample of the alkaline protease from P. model of the enzyme was built with the program package, aeruginosa IFO3080, which was a gift from Nagase Bio TURBO-FRODO (20), on a computer graphics system, chemical, was subjected to crystallization without further IRIS Indigo Elan. The initial crystallographic R value was purification. Crystallization trials were conducted using the 43.5% for 16,182 independent reflections [I>3ƒÐ(I)] hanging drop vapor diffusion method. Small crystals were within the resolution of 10.0 to 2.8A. The model was obtained at 25•‹C using a precipitant solution of 6% (w/v) refined by means of simulated annealing techniques using polyethylene glycol 6000 (50mM acetate buffer including 1 the molecular dynamics program, X-PLOR (21, 22), in mM NaN3, pH 5.6). For the crystallization, the droplets stalled on a CRAY Y-MP2E/264 supercomputer. After were prepared by adding 2.5,ƒÊl of the precipitant solution several cycles of refinement, manual rebuilding of the to an equal volume of a 2% protein solution (pH 7.0) model was carried out using 2Fo -Fc and Fo -Fc maps. The including 5mM CaCl2 and 1mM NaN3. The macroseeding resolution was gradually increased to 2.0 A during iteration technique was effective for growing the small crystals to a of refinement and rebuilding. The total numbers of 335 size (1.0 x 0.6 x 0.3mm) large enough for X-ray experi water molecules and 8 calcium ions were included for the ments. The prismatic crystals have the orthorhombic space further refinement below 2.5 A resolution. The final model group, P2,212,, with cell dimensions of a=77.16, b= has an R factor of 19.8% for 33,698 independent reflections 176.69, and c=51.12 A. They contain four enzyme mole [I > 2ƒÐ (I), 80.0% complete] within the resolution of 8.0 to cules in the unit cell, and exhibit a Vm value of 3.56 A3/Da, 2.0 A. The root-mean-square (r.m.s) deviations from ideal which corresponds to the solvent content, 66%, in the bond distances and angles were 0.009 A and 1.58•‹, respec crystals. Three kinds of isomorphous heavy-atom deriva tively. The model exhibits a reasonable conformation with tive crystals were prepared by soaking the native crystals excellent geometry. The model was inspected with the in heavy-atom solutions of 15mM CH3HgC1, for 3 days, 2.5 program, PROCHECK (23). All residues were observed in mM HgCl2, for 1 day, and 1mM UO2(CH3COO)2, for 1 day, the allowed region of the Ramachandran plot, and 87.6% of respectively. the total residues lay in the most favored region. The All diffraction intensity data were collected using the coordinates of the final model will be deposited in the synchrotron radiation X-ray source (wavelength = 1.00 Brookhaven Protein Data Bank. A) at the Photon Factory of the National Laboratory for Stereoscopic drawings and a folding diagram of the High Energy Physics, Tsukuba, Japan. X-ray diffraction alkaline protease are shown in Figs. 1 and 2, respectively. patterns were recorded on Fuji-imaging plates with a The enzyme molecule has an elongated ellipsoidal shape screenless Weissenberg camera at the BL6A2 station. Two with approximate dimensions of 90 X 42 x 35 A. Its struc native crystals were mounted with the crystallographic b* ture comprises two structurally distinct domains: N and and c* axes parallel to the spindle axis, respectively, and C-terminal domains. the derivative crystals with the c* axis parallel to the Apart from the a -helix comprising the first 17 residues, spindle. The intensity data were processed with the pro which is associated with the C-terminal domain, the N gram, WEIS (17), and calculations of phases and electron terminal domain, residues 18-250, is a catalytic domain density maps were carried out with the program, PHASES with one zinc ion in the active center and shares a basically (18). The statistics for data collection and phase calculation common folding topology with other zinc metalloendopro are summarized in Table I. teases, as suggested by Blundell (24). The catalytic domain A 2.8 A resolution electron density map of the alkaline has an open-sandwich topology, as shown in Fig. 2, in which protease calculated by the isomorphous replacement two ƒ¿-helices (ƒ¿5 and ƒ¿6) lie against a twisted mixed method was modified by means of the solvent-flattening ƒÀ-sheet of five strands (ƒÀ3-ƒÀ7).