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• W2038009022 abstract "ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation. ROCK or Rho-associated kinase, a serine/threonine kinase, is an effector of Rho-dependent signaling and is involved in actin-cytoskeleton assembly and cell motility and contraction. The ROCK protein consists of several domains: an N-terminal region, a kinase catalytic domain, a coiled-coil domain containing a RhoA binding site, and a pleckstrin homology domain. The C-terminal region of ROCK binds to and inhibits the kinase catalytic domains, and this inhibition is reversed by binding RhoA, a small GTPase. Here we present the structure of the N-terminal region and the kinase domain. In our structure, two N-terminal regions interact to form a dimerization domain linking two kinase domains together. This spatial arrangement presents the kinase active sites and regulatory sequences on a common face affording the possibility of both kinases simultaneously interacting with a dimeric inhibitory domain or with a dimeric substrate. The kinase domain adopts a catalytically competent conformation; however, no phosphorylation of active site residues is observed in the structure. We also determined the structures of ROCK bound to four different ATP-competitive small molecule inhibitors (Y-27632, fasudil, hydroxyfasudil, and H-1152P). Each of these compounds binds with reduced affinity to cAMP-dependent kinase (PKA), a highly homologous kinase. Subtle differences exist between the ROCK- and PKA-bound conformations of the inhibitors that suggest that interactions with a single amino acid of the active site (Ala215 in ROCK and Thr183 in PKA) determine the relative selectivity of these compounds. Hydroxyfasudil, a metabolite of fasudil, may be selective for ROCK over PKA through a reversed binding orientation. Protein phosphorylation is one of the major posttranslational modifications required for regulation of cellular activities, and kinases that catalyze these reactions are one of the largest families of enzymes in eukaryotic cells. Disruption of a kinase activity, either through mutation or deregulation, can have profound consequences on the functioning of a cell, leading to a wide variety of disease states. Further, because kinases play a regulatory role in pathways involved in signal transduction and cell cycle control, modulation of kinase activity presents an opportunity for therapeutic intervention. One such kinase is ROCK (Rho-associated, coiled-coil containing protein kinase; reviewed in Ref. 1Riento K. Ridley A.J. Nat. Rev. Mol. Cell. Biol. 2003; 4: 446-456Crossref PubMed Scopus (1564) Google Scholar), a Ser/Thr kinase with potential roles in several therapeutic indications including hypertension (2Mukai Y. Shimokawa H. Matoba T. Kandabashi T. Satoh S. Hiroki J. Kaibuchi K. Takeshita A. FASEB J. 2001; 15: 1062-1064PubMed Google Scholar), atherosclerosis (3Shimokawa H. J. Cardiovasc. Pharmacol. 2002; 39: 319-327Crossref PubMed Scopus (251) Google Scholar), and immunosuppression (4Ohki S. Iizuka K. Ishikawa S. Kano M. Dobashi K. Yoshii A. Shimizu Y. Mori M. Morishita Y. J. Heart Lung Transplant. 2001; 20: 956-963Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Because desired therapeutic outcomes would likely result from a reduction of ROCK activity, much research has focused on designing small molecule inhibitors of ROCK. There are two isoforms of ROCK, known as ROCK I and II, or Rho-kinase β and α, respectively (5Leung T. Chen X.Q. Manser E. Lim L. Mol. Cell. Biol. 1996; 16: 5313-5327Crossref PubMed Google Scholar). These two kinases regulate the activity of muscle myosin regulatory light chain (RLC) 2The abbreviations used are: RLCregulatory light chainPKAcAMP-dependent kinaseMES4-morpholineethanesulfonic acid.2The abbreviations used are: RLCregulatory light chainPKAcAMP-dependent kinaseMES4-morpholineethanesulfonic acid. proteins by direct phosphorylation, (6Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar, 7Kawano Y. Fukata Y. Oshiro N. Amano M. Nakamura T. Ito M. Matsumura F. Inagaki M. Kaibuchi K. J. Cell Biol. 1999; 147: 1023-1038Crossref PubMed Scopus (474) Google Scholar) and by phosphorylation and inhibition of the myosin binding subunit of myosin phosphatase. This leads to increased levels of phosphorylated myosin light chain and subsequent muscle contraction (8Kureishi Y. Kobayashi S. Amano M. Kimura K. Kanaide H. Nakano T. Kaibuchi K. Ito M. J. Biol. Chem. 1997; 272: 12257-12260Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). The Rho/ROCK pathway is also involved in nonmuscle myosin regulation and has been implicated in stress fiber and focal adhesion formation (9Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar), neurite retraction (10Hirose M. Ishizaki T. Watanabe N. Uehata M. Kranenburg O. Moolenaar W.H. Matsumura F. Maekawa M. Bito H. Narumiya S. J. Cell Biol. 1998; 141: 1625-1636Crossref PubMed Scopus (410) Google Scholar), and tumor cell invasion (11Yoshioka K. Matsumura F. Akedo H. Itoh K. J. Biol. Chem. 1998; 273: 5146-5154Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Given the importance of ROCK in regulating such key processes involved in cytoskeleton rearrangement and cell adhesion, considerable effort has been expended on delineating the details of its mechanism of action. regulatory light chain cAMP-dependent kinase 4-morpholineethanesulfonic acid. regulatory light chain cAMP-dependent kinase 4-morpholineethanesulfonic acid. ROCK is composed of a catalytic kinase domain (residues 73–405), a coiled-coil region (residues 425–1100), and a C-terminal pleckstrin homology domain (residues 1103–1230). A similar domain organization is shared with three closely related kinases: myotonic dystrophy kinase (DMPK) (12Bush E.W. Helmke S.M. Birnbaum R.A. Perryman M.B. Biochemistry. 2000; 39: 8480-8490Crossref PubMed Scopus (39) Google Scholar), myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) (13Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (67) Google Scholar), and citron kinase (CRIK) (14Di Cunto F. Calautti E. Hsiao J. Ong L. Topley G. Turco E. Dotto G.P. J. Biol. Chem. 1998; 273: 29706-29711Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The work described herein represents the first structure of a DMPK family member. Full-length ROCK, in the absence of effector molecules, exists as an autoinhibited structure. The C-terminal region of ROCK (coiled-coil and pleckstrin homology domains) has been shown to partially inhibit the kinase catalytic activity by binding directly to the kinase domain (15Amano M. Chihara K. Nakamura N. Kaneko T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1999; 274: 32418-32424Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). When GTP-bound RhoA binds to the Rho-binding region of the coiled-coil domain, the interactions between the catalytic kinase domain and the C-terminal region are disrupted, relieving the inhibition (16Ishizaki T. Maekawa M. Fujisawa K. Okawa K. Iwamatsu A. Fujita A. Watanabe N. Saito Y. Kakizuka A. Morii N. Narumiya S. EMBO J. 1996; 15: 1885-1893Crossref PubMed Scopus (791) Google Scholar). Cleavage of the C-terminal inhibitory domain from the catalytic domain by caspase-3 during apoptosis also activates the enzyme (17Coleman M.L. Sahai E.A. Yeo M. Bosch M. Dewar A. Olson M.F. Nat. Cell Biol. 2001; 3: 339-345Crossref PubMed Scopus (971) Google Scholar, 18Sebbagh M. Renvoize C. Hamelin J. Riche N. Bertoglio J. Breard J. Nat. Cell Biol. 2001; 3: 346-352Crossref PubMed Scopus (701) Google Scholar). We have previously characterized the oligomerization state of full-length ROCK and four truncated constructs using light scattering and analytical ultracentrifugation methods (19Doran J.D. Liu X. Taslimi P. Saadat A. Fox T. Biochem. J. 2004; 384: 255-262Crossref PubMed Scopus (56) Google Scholar). Protein constructs containing residues 6–415 exist as dimers, whereas smaller constructs are predominantly monomeric. ROCK (6–415) proved to be highly homogenous in solution and yielded diffraction quality crystals suitable for x-ray structure determination. This construct lacks the C-terminal inhibitory domains and is similar to the caspase-3-activated form of ROCK. An analogous form of truncated CRIK has also been observed in vivo (14Di Cunto F. Calautti E. Hsiao J. Ong L. Topley G. Turco E. Dotto G.P. J. Biol. Chem. 1998; 273: 29706-29711Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Here we report the x-ray crystal structure of ROCK bound to ATP-competitive inhibitors. The structure reveals two kinase domains linked by an N-terminal dimerization domain comprised of five α-helices from each monomer. In this arrangement, the active sites of the two kinases share a common face, possibly facilitating interactions with dimeric substrates or inhibitory domains. Each kinase domain appears to be have a catalytically competent conformation in the absence of phosphorylation. In full-length ROCK, interactions with the C-terminal domains may change this arrangement of the kinase domains or alter the structure of a regulatory element such as the activation loop. Crystal structures of four ROCK protein-ligand complexes were determined containing the inhibitors Y-27632, fasudil (HA-1077), hydroxyfasudil (HA-1100), and a dimethylated analog of fasudil (H-1152P). Y-27632 is a pyridine compound that is chemically distinct from the other three isoquinoline based ligands. In a prior study, Breitenlechner et al. (20Breitenlechner C. Gassel M. Hidaka H. Kinzel V. Huber R. Engh R.A. Bossemeyer D. Structure (Camb.). 2003; 11: 1595-1607Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) determined the structures of Y-27632, fasudil, and H-1152P bound to PKA. To allow comparisons among all four ligands in both ROCK and PKA, we also determined the structure of PKA bound to hydroxyfasudil. All four compounds are competitive inhibitors at the ATP binding site. Several previous studies have established that Y-27632 is a selective inhibitor of ROCK, and it is used to assess the effects of ROCK inhibition in a variety of cellular and animal models (4Ohki S. Iizuka K. Ishikawa S. Kano M. Dobashi K. Yoshii A. Shimizu Y. Mori M. Morishita Y. J. Heart Lung Transplant. 2001; 20: 956-963Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 21Ikeda F. Terajima H. Shimahara Y. Kondo T. Yamaoka Y. J. Surg. Res. 2003; 109: 155-160Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22Sinnett-Smith J. Lunn J.A. Leopoldt D. Rozengurt E. Exp. Cell Res. 2001; 266: 292-302Crossref PubMed Scopus (65) Google Scholar, 23Narumiya S. Ishizaki T. Uehata M. Methods Enzymol. 2000; 325: 273-284Crossref PubMed Google Scholar). Fasudil, a vasodilator used for the treatment of cerebral vasospasm, is an approved drug whose mechanism of action is believed to stem from ROCK inhibition (24Masaoka H. Takasato Y. Nojiri T. Hayakawa T. Akimoto H. Yatsushige H. Toumori H. Miyazaki Y. Honma M. Acta Neurochir. 2001; 77: 209-211Crossref Scopus (14) Google Scholar, 25Shibuya M. Asano T. Sasaki Y. Acta Neurochir. 2001; 77: 201-204Crossref Scopus (33) Google Scholar, 26Nagata K. Kondoh Y. Satoh Y. Watahiki Y. Yokoyama E. Yuya H. Hirata Y. Shishido F. Hatazawa J. Kanno I. Sone T. Clin. Neuropharmacol. 1993; 16: 501-510Crossref PubMed Scopus (23) Google Scholar). Hydroxyfasudil, first identified as a metabolite of fasudil (27Shibuya M. Suzuki Y. Sugita K. Saito I. Sasaki T. Takakura K. Nagata I. Kikuchi H. Takemae T. Hidaka H. Nakashima M. J. Neurosurg. 1992; 76: 571-577Crossref PubMed Scopus (370) Google Scholar), is a more selective inhibitor of ROCK compared with its parent. The dimethylated analog of fasudil, H-1152P, is the most potent inhibitor of the four ligands toward ROCK by 2 orders of magnitude (28Sasaki Y. Suzuki M. Hidaka H. Pharmacol. Ther. 2002; 93: 225-232Crossref PubMed Scopus (229) Google Scholar). Comparison of these four ROCK ligand structures both among themselves and against their corresponding counterparts in PKA suggests that a very specific active site contact is important for selective ROCK inhibition. Cloning and Expression—Rock I (Swiss-Prot code Q13464) was isolated from a human leukocyte cDNA library. The cDNA encoding the ROCK protein comprising residues 6–553 and 6–415 were cloned into a baculoviral transfer vector, pBEV10, and expressed in insect cells as described (29Chambers S.P. Austen D.A. Fulghum J.R. Kim W.M. Protein Expression Purif. 2004; 36: 40-47Crossref PubMed Scopus (63) Google Scholar). The proteins were engineered to include a hexahistidine tag (His6) at the N terminus to facilitate purification. Protein Purification—The protein used for crystallization was first produced by proteolytic cleavage of a larger construct and later through construction of a DNA expression vector containing sequences corresponding to the desired proteolytic product. The ROCK Ser6–Leu553 construct was metal affinity-purified as described (19Doran J.D. Liu X. Taslimi P. Saadat A. Fox T. Biochem. J. 2004; 384: 255-262Crossref PubMed Scopus (56) Google Scholar). After thrombin cleavage of the His tag, the sample was treated with clostripain (Washington Chemicals) at a ratio of 1:100 (w/w) and 10 mm CaCl2 for 3 h at room temperature followed by overnight incubation at 4 °C. This yielded a C-terminal truncation whose limits were determined to be 6–415 by N-terminal sequencing (Applied Biosystems Procise Sequencing System) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Applied Biosystems Voyager-DE STR Biospectrometry work station). The protein was then diluted 10-fold with 20 mm HEPES, pH 7.4, loaded onto a MonoQ HR 5/5 ion exchange column (Amersham Biosciences), and eluted with a 100–400 mm NaCl gradient over 40 column volumes. The sample was then loaded onto a HiLoad 16/60 Superdex 200 column (Amersham Biosciences) equilibrated with 20 mm HEPES, pH 7.4, 200 mm NaCl, and 2 mm β-mercaptoethanol. The main peak was pooled and appeared to be 99% pure as judged by SDS-PAGE analysis. The ROCK 6–415 construct was purified using an identical procedure except that the clostripain proteolysis step was no longer required. Ligand Inhibition Measurements—The inhibition constant, Ki, for inhibiting PKA and ROCK were obtained from dose-response titration curves. The residual enzyme activity remaining was determined by the spectrophotometric coupled enzyme assay as previously described (30Fox T. Coll J.T. Xie X. Ford P.J. Germann U.A. Porter M.D. Pazhanisamy S. Fleming M.A. Galullo V. Su M.S. Wilson K.P. Protein Sci. 1998; 7: 2249-2255Crossref PubMed Scopus (129) Google Scholar). In this assay, every mole of ADP generated in the kinase reaction is coupled to the generation of NAD from NADH using pyruvate kinase and lactate dehydrogenase. The final concentration of the assay components were as follows: 0.1 m HEPES, pH 7.6, 10 mm MgCl2, 1 mm dithiothreitol, 2.5 mm phosphoenolpyruvate, 200 μm NADH, 2.5% Me2SO, 50 μg/ml pyruvate kinase, and 10 μg/ml lactate dehydrogenase. The ATP and peptide concentrations were 10 and 15 μm (LRRASLG) for PKA and 15 μm and 45 μm (KKRNRTLSV) for ROCK I assays, respectively. Assay plates were incubated at 30 °C for 10 min, and the absorbance change at 340 nm was monitored (Table 1).TABLE 1Inhibition constants determined for ROCK and PKA Open table in a new tab Crystallization and X-ray Analysis—ROCK crystals were grown by the vapor diffusion method at 22 °C. Equal volumes of protein stock solution (20 mg/ml protein, 20 mm HEPES, pH 7.8, 100 mm NaCl, 2 mm 2-mercaptoethanol) and well solution (3–8% polyethylene glycol 3350, 100 mm MES, pH 5.5, 50 mm CaCl2, 10 mm dithiothreitol) were mixed and suspended over 1 ml of well solution. Over 4 days, the crystals reached a final size of ∼200 μm. The crystals were harvested and flashfrozen in a solution composed of the well solution with 25–30% (v/v) glycerol. The Y-27632 (BIOMOL International L.P.) complex with ROCK was made by adding the ligand (2 mm) to the clostripain treated 6–553 construct prior to crystallization. Subsequent complexes with fasudil (Toronto Research Chemicals), hydroxyfasudil (prepared as in Ref. 31Hidaka, H., and Sone, T., Patent 4,678,783, pp. 1-21, Asahi Kasei Kogyo Kabushiki Kaisha, JapanGoogle Scholar), and H-1152P (Calbiochem) were made by soaking unliganded crystals (ROCK 6–415) with 500 μm compound and 5% Me2SO (final concentration) for 48 h at room temperature. The Y-27632/ROCK diffraction data were recorded at the COMCAT (32-ID) Beamline at the Advanced Photon Source (Argonne National Laboratories and Emerald Biosciences). All other diffraction data were recorded at Beamline 5.0.2 at the Advanced Light Source (Lawrence Berkeley Laboratories). Intensities were integrated and scaled using the programs DENZO and SCALEPACK (32Otwinoski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38526) Google Scholar) and CrystalClear (33CrystalClear: An Integrated Program for the Collection and Processing of Area Detector Data. Rigaku/MSC, The Woodlands, TX1997–2002Google Scholar). The structure was determined by molecular replacement using homology models based upon PKA (Protein Data Bank code 1ATP). The molecular replacement solution was determined using AMORE (34Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar, 35Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1367-1372Crossref PubMed Scopus (658) Google Scholar). The crystals belong to the space group P3121. The asymmetric unit consists of two monomers that are well ordered. The asymmetric unit also contains the N-terminal region of a third molecule that interacts with a symmetry-related molecule across a 2-fold axis. The electron density for this region is quite poor, and the remainder of the molecule (kinase domain) is not visible at all. The protein model was built using QUANTA and refined with CNX (Accelrys) (36Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar, 37Rice L.M. Brunger A.T. Proteins. 1994; 19: 277-290Crossref PubMed Scopus (382) Google Scholar) (Table 2).TABLE 2Data collection and refinement statisticsROCKPKA hydroxyfasudilY27632FasudilHydroxyfasudilH-1152PData collection X-ray sourceAPSALSALSALSRigaku Raxis Space groupP3121P3121P3121P3121P212121 Unit cell parameters (Å)a = b = 180.9, c = 91.5a = b = 181.0, c = 89.4a = b = 181.8, c = 91.7a = b = 183.6, c = 91.7a = 72.8, b = 75.8, c = 80.0 Resolution (Å)20-2.620-3.220-2.9520-3.320-2.2 Redundancy6.74.47.36.14.9 Completeness (%)aValues for the highest resolution shell are shown in parentheses.90.8 (53.6)92.2 (90.2)93.3 (76.2)99.4 (99.4)96.6 (84.5) RmergeaValues for the highest resolution shell are shown in parentheses.0.086 (0.40)0.12 (0.38)0.062 (0.34)0.12 (0.37)0.075 (0.34) 〈I/o 〉aValues for the highest resolution shell are shown in parentheses.10.6 (2.3)6.3 (2.0)17.2 (4.5)11.7 (5.0)13.6 (3.4)Refinement Reflections used47,18423,47133,68426,47022,170 Test reflections22831176166513441525 R factor0.2310.2210.2500.2340.223 Free R factor0.2630.2680.2910.2800.287 RMS deviationsBond lengths (Å)0.0070.0090.0100.0100.009Bond angles (°)1.31.31.41.41.6Dihedral angles (°)22.622.222.722.422.3Rmerge = ΣhklΣi|I(hkl)i — 〈I(hkl) 〉|/ΣhklΣi 〈 |I(hkl)i 〉 over i observations of reflection hkl.R factor = Σ∥Fobs| — |Fcalc∥/Σ|Fobs| where Fobs and Fcalc are the observed and calculated structure factors, respectively. Free R factor is calculated from a randomly chosen subset of reflections not used for refinement. The same reflection subset was used for all four ROCK data sets.a Values for the highest resolution shell are shown in parentheses. Open table in a new tab Rmerge = ΣhklΣi|I(hkl)i — 〈I(hkl) 〉|/ΣhklΣi 〈 |I(hkl)i 〉 over i observations of reflection hkl. R factor = Σ∥Fobs| — |Fcalc∥/Σ|Fobs| where Fobs and Fcalc are the observed and calculated structure factors, respectively. Free R factor is calculated from a randomly chosen subset of reflections not used for refinement. The same reflection subset was used for all four ROCK data sets. Two Kinase Domains Interact via a Dimerization Domain—The protein used in this study consists of the N-terminal region comprising approximately one-third of the full-length sequence of ROCK. The crystal structure reveals two protein molecules in a head-to-head arrangement, related by a pseudo-2-fold rotation noncrystallographic symmetry (Fig. 1A). Each monomer consists of an N-terminal helical domain (residues 5–72), a bilobed kinase domain (residues 73–356), and a kinase tail (residues 357–405). Helices from the two N-terminal domains interact with each other to form a single structure (Fig. 1, B and D). This region will be referred to hereafter as the dimerization domain. The kinase tail of each molecule lies across its own kinase domain and interacts with the dimerization domain. The kinase domain of ROCK has a global fold typical of serine/threonine kinases, consisting of two lobes linked by a hinge region. The smaller, N-terminal lobe (residues 73–153) contains a twisted five-stranded anti-parallel β-sheet and a single α-helix. The C-terminal lobe (residues 159–356) is largely α-helical. The ATP-binding pocket of the active site is formed by a groove at the interface between these two domains and is enclosed by the hinge region (residues 154–158), the glycine-rich loop (residues 81–91), and the activation loop (residues 216–231). The ROCK kinase domain structure was compared with PKA, the most similar kinase with respect to sequence identity for which the three-dimensional structure is known. When secondary structural elements are aligned, a root mean square difference of 1.3 Å for C-α atom positions was observed between ROCK and PKA (Protein Data Bank code 1ATP) (using 220 residues) (38Zheng J. Knighton D.R. ten Eyck L.F. Karlsson R. Xuong N. Taylor S.S. Sowadski J.M. Biochemistry. 1993; 32: 2154-2161Crossref PubMed Scopus (513) Google Scholar). The N-terminal helices and the kinase tail of each monomer in the asymmetric unit interact over an extensive interface to form a dimer. The dimerization domain is made from two sets of five helices, one set from each protein monomer, and part of the kinase tail from each monomer (residues 383–405). The kinase tail lies roughly parallel to helix 2 and packs between its own kinase domain and helix 2 and also helix 4 of the noncrystallographic symmetry-related monomer (Fig. 1). Most of the contact surface area is comprised of the helices of one chain interacting with the helices of the other monomer. The two kinase tails do not interact with each other except via contacts between Phe387 on each chain. The extensive interface between the two chains is largely hydrophobic and buries ∼4120 Å2 solvent-accessible surface area/dimer. For comparison, the buried surface area in complexes between a protein antigen and an antibody are typically less than 2000 Å2 (39Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1761) Google Scholar). These dimer interactions orient the two monomers such that the active sites and regulatory elements of the kinase domains appear on a single face of the dimer (Fig. 1B). Active Conformation Independent of Phosphorylation—ROCK is activated by disrupting the interactions between the kinase domain and regulatory elements in the C terminus, either through RhoA binding or cleavage of the C terminus by caspase-3. Because the crystallization construct lacks these inhibitory C-terminal domains, we would expect to observe a catalytically competent conformation. In particular, the residues of the active site should be aligned as in other catalytically active kinase structures, and the activation loop should not occlude the peptide substrate binding groove. The catalytic residues of the kinase domain were compared with the PKA structure to determine whether the ROCK structure represents a catalytically active conformation (40Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1355) Google Scholar). The positions and side chain rotamers of the catalytic residues resemble those observed in the phosphorylated PKA-ATP-peptide complex (Protein Data Bank code 1ATP). In PKA, Asn171 forms a hydrogen bond to and thus orients Asp166, which in turn forms a hydrogen bond with the Ser or Thr hydroxyl group of the substrate at the site of phosphorylation. The corresponding residues in ROCK, Asn203 and Asp198, have the same position and side chain conformations. Likewise, the residues of PKA that interact with the ATP phosphates and Mg2+ atoms (Lys72, Asn171, and Asp184) are conserved both in sequence and position in ROCK (Lys105, Asn203, and Asp216) (Fig. 2). An ion pair between the active site lysine and a glutamate on the C-helix is a conserved feature of catalytically active kinase conformations. As in the active conformation of PKA, the salt bridge in ROCK between Glu124 and Lys105 is intact. Among protein kinases, the conformation of the activation loop varies widely (reviewed in Ref. 41Nolen B. Taylor S. Ghosh G. Mol Cell. 2004; 15: 661-675Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar). In inactive kinase conformations, the activation loop often blocks the active site, interfering with peptide and ATP substrate binding. In active kinases, this loop lies parallel to helices E and F, and the active site is accessible. Although the sequence and conformation of the activation loop vary among active kinase conformations, they all share the same global conformation. In the ROCK structure, even though no phosphorylation is observed in the structure, the position of the activation loop resembles an active conformation. The activation loops of the two ROCK molecules in the asymmetric unit are very similar (0.3 Å root mean square difference over main chain atoms), diverging only at the region between the activation loop and the EF helix (Asp232–Pro238), where each of the two monomers makes different crystal lattice contacts. In this region, one ROCK monomer (chain A) most closely resembles the corresponding region in PKA, whereas these residues in the other monomer (chain B) move away from the active site by about 5 Å. These two different conformations indicate that these loop regions are likely to be flexible. In many kinases, a catalytically competent conformation is stabilized by interactions with a phosphorylated residue on the activation loop. In the ROCK structure no phosphorylation is observed in the electron density map. Furthermore, the residues that typically interact with the phosphate group are not conserved in ROCK. In PKA for example, the active conformation of the loop is stabilized by interactions between a phosphothreonine (Thr197) and three basic residues (His87, Arg165, and Lys189) and one polar residue (Thr195). In ROCK, the corresponding residue (Thr233) appears unmodified and lies about 4 Å outside the active site relative to PKA. Also three of the four corresponding residues that are in a position to interact with the phosphothreonine in ROCK are hydrophobic (Phe120, Met221, and Cys231) (Fig. 3). In the absence of interactions with a phosphorylated residue, we would expect other interactions to stabilize the loop conformation. Indeed, the interactions between the activation loop and the αEF/αF loop are more extensive in ROCK than in PKA. Both the αEF/αF loop and the activation loop are each four residues longer than the corresponding sequences in PKA (Fig. 2). These insertions almost double the buried surface between the two" @default.
• W2038009022 created "2016-06-24" @default.
• W2038009022 creator A5021471617 @default.
• W2038009022 creator A5037467232 @default.
• W2038009022 creator A5046873578 @default.
• W2038009022 creator A5051943188 @default.
• W2038009022 creator A5067239399 @default.
• W2038009022 creator A5085298568 @default.
• W2038009022 creator A5086182257 @default.
• W2038009022 date "2006-01-01" @default.
• W2038009022 modified "2023-10-17" @default.
• W2038009022 title "The Structure of Dimeric ROCK I Reveals the Mechanism for Ligand Selectivity" @default.
• W2038009022 cites W1539796472 @default.
• W2038009022 cites W1588069091 @default.
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