Structure of the N-terminal Domain of GRP94
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m308661200
ISSN1083-351X
AutoresKaren Soldano, Arif Jivan, Christopher V. Nicchitta, D.T. Gewirth,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoGRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75–2.1 Å of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone. GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75–2.1 Å of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone. The hsp90 family of molecular chaperones are ligand-regulated proteins that participate in the conformational maturation of protein substrates involved in diverse cellular activities ranging from cell signaling to bacterial recognition (1Csermely P. Schnaider T. Soti C. Prohaszka Z. Nardai G. Pharmacol. Ther. 1998; 79: 129-168Crossref PubMed Scopus (890) Google Scholar, 2Pearl L.H. Prodromou C. Adv. Protein Chem. 2001; 59: 157-186Crossref PubMed Scopus (177) Google Scholar). They are also overexpressed in response to cell stress events, including heat shock, starvation, and oxidation. 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Given their central importance in the biology of protein folding and the cellular stress response, and their potential as targets in a variety of therapeutic strategies, the mechanism by which hsp90 chaperone activity is regulated, and the chemistry of their interactions with client substrates is under intensive investigation.GRP94 and Hsp90 exist as obligate homodimers, with each subunit consisting of an N-terminal domain, a charged region, and a C-terminal dimerization domain. Because of its relatively weak affinity (∼100 μm), the binding of ATP and ADP to Hsp90 was conclusively established only with the determination of the co-crystal structures of the N-domain in complex with bound nucleotide (15Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar). Other crystal structures of the N-domain of Hsp90 in complex with the ansamycin antibiotics geldanamycin (16Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1234) Google Scholar) and radicicol, and other inhibitors (17Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (877) Google Scholar), showed that despite their lack of structural similarity to ATP these ligands bind to the same domain and function as adenosine mimetics. The N- and C-domains are connected by a 40- to 60-residue charged region that varies in length and sequence among species and family members. Rotary shadowing electron microscopy studies have suggested that the charged region acts as a flexible tether that links the compactly folded N- and C-domains (18Maruya M. Sameshima M. Nemoto T. Yahara I. J. Mol. Biol. 1999; 285: 903-907Crossref PubMed Scopus (74) Google Scholar, 19Koyasu S. Nishida E. Kadowaki T. Matsuzaki F. Iida K. Harada F. Kasuga M. Sakai H. Yahara I. Proc. 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Sci. 2000; 25: 24-28Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar) to which Hsp90 belongs, it has been proposed that hsp90 chaperone activity is linked to an N-domain conformational change that is coupled to ATP binding and hydrolysis (27Prodromou C. Panaretou B. Chohan S. Siligardi G. O'Brien R. Ladbury J.E. Roe S.M. Piper P.W. Pearl L.H. EMBO J. 2000; 19: 4383-4392Crossref PubMed Google Scholar, 28Meyer P. Prodromou C. Hu B. Vaughan C. Roe S.M. Panaretou B. Piper P.W. Pearl L.H. Mol. Cell. 2003; 11: 647-658Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Indeed, a slow ATP hydrolysis activity has been detected in Hsp90 (29Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (613) Google Scholar, 30Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 31Grenert J.P. Johnson B.D. Toft D.O. J. Biol. Chem. 1999; 274: 17525-17533Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Mechanistic explanations of how ATP binding and hydrolysis are coupled to Hsp90 activity have been hindered, however, by the lack of any observed change in the conformation of the N-domain in response to ligands. The crystal structures of the Hsp90 N-domain, either in the apo form or in complex with ATP, ADP, and inhibitors such as geldanamycin, radicicol, and others are essentially superimposable (15Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar, 16Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1234) Google Scholar, 17Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (877) Google Scholar, 30Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 32Prodromou C. Roe S.M. Piper P.W. Pearl L.H. Nat. Struct. Biol. 1997; 4: 477-482Crossref PubMed Scopus (206) Google Scholar). The complete protein, rather than just the isolated N-domain, appears to be required for ATP hydrolysis (30Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (487) Google Scholar, 33Owen B.A. Sullivan W.P. Felts S.J. Toft D.O. J. Biol. Chem. 2002; 277: 7086-7091Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), suggesting that conformational rearrangements resulting in a close interaction with the C-domain are important for Hsp90 activity. However, the manner in which N- and C-domains associate across the flexible linker and the interfaces participating in oligomerization remain unknown. Interactions with co-chaperones may also be important catalysts for ATP hydrolysis in Hsp90s from some species as well (34Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar).Despite having over 50% sequence identity in their N-domains and complete conservation of their ligand binding cavities, Hsp90 and GRP94 differ in their interactions with regulatory ligands and may thus differ mechanistically as well. Although GRP94 responds to ansamycin inhibitors in a manner analogous to Hsp90, indicating ligand regulation (35Supino-Rosin L. Yoshimura A. Yarden Y. Elazar Z. Neumann D. J. Biol. Chem. 2000; 275: 21850-21855Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 36Vega V.L. De Maio A. Mol. Biol. Cell. 2003; 14: 764-773Crossref PubMed Scopus (47) Google Scholar, 37Nganga A. Bruneau N. Sbarra V. Lombardo D. Le Petit-Thevenin J. Biochem. J. 2000; 352: 865-874Crossref PubMed Scopus (24) Google Scholar), ATP hydrolysis above background levels has not been demonstrated for GRP94 (38Rosser M.F. Nicchitta C.V. J. Biol. Chem. 2000; 275: 22798-22805Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), nor have co-chaperones or partner proteins been identified for this paralog. Domains of Hsp90 and GRP94 also do not functionally substitute for one another (10Randow F. Seed B. Nat. Cell Biol. 2001; 3: 891-896Crossref PubMed Scopus (289) Google Scholar). In addition, although Hsp90 binds ADP/ATP with micromolar affinity (15Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar), the binding constant for these ligands to GRP94 is estimated to be several millimolar (38Rosser M.F. Nicchitta C.V. J. Biol. Chem. 2000; 275: 22798-22805Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The difference in ligand regulation between GRP94 and Hsp90 is further highlighted by their interactions with 5′-N-ethylcarboxamidoadenosine (NECA), 1The abbreviations used are: NECA5′-N-ethylcarboxamidoadenosine2ClddA2-chlorodideoxyadenosineGSTglutathione S-transferaseDTTdithiothreitolPEGpolyethylene glycolr.m.s.root mean square.1The abbreviations used are: NECA5′-N-ethylcarboxamidoadenosine2ClddA2-chlorodideoxyadenosineGSTglutathione S-transferaseDTTdithiothreitolPEGpolyethylene glycolr.m.s.root mean square. a broad-spectrum adenosine A2 receptor antagonist. Biochemical screens for NECA-binding activity identified GRP94 as the prominent cellular target of this ligand, with an equilibrium dissociation constant of 200 nm (39Hutchison K.A. Nevins B. Perini F. Fox I.H. Biochemistry. 1990; 29: 5138-5144Crossref PubMed Scopus (48) Google Scholar, 40Hutchison K.A. Fox I.H. J. Biol. Chem. 1989; 264: 19898-19903Abstract Full Text PDF PubMed Google Scholar). NECA has no detectable binding affinity for cytoplasmic Hsp90s, however (38Rosser M.F. Nicchitta C.V. J. Biol. Chem. 2000; 275: 22798-22805Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).The functional differences between GRP94 and Hsp90 underscore the fact that many aspects of hsp90 biology remain to be addressed, including the mechanism of selective regulation of the cellular paralogs, the role of ATP in GRP94 where ATP hydrolysis has proven difficult to detect, and the structural basis for tertiary interactions between hsp90 domains and quaternary associations between intact chaperones. To understand the mechanism for selective ligand responsiveness in GRP94, we have solved the structure of the N-domain of GRP94 in complex with the GRP94-specific ligand NECA, as well as with the high affinity but non-selective ligands radicicol and 2-chlorodideoxyadenosine (2ClddA). We show that an intrinsic conformational difference between cytoplasmic Hsp90 and GRP94 accounts for selectivity in ligand binding. We also show that the 5′ substituent of NECA occupies a previously unidentified second pocket adjacent to the adenosine-binding cavity. This structural difference arises in large part from a 5-amino acid insertion unique to GRP94 whose structural role until now has been unknown. These structures also identify a subdomain within the N-domain that has the potential to act as a ligandresponsive conformational switch in GRP94. Finally, to understand the structural relationship between the N- and C-domains of GRP94, the constructs we used for crystallization have included the charged region. We show here that this region forms a disordered loop whose end returns, surprisingly, to form an ordered, stable interaction with the body of the N-domain. This provides evidence for an intimate interaction between the N- and C-domains of the protein.EXPERIMENTAL PROCEDURESProtein Purification—Canine GRP94 (residues 69–337) was overexpressed in Escherichia coli as a GST fusion. BL21(DE3)pLysS cells harboring the expression plasmid were grown to an A600 of 1 at 37 °C and induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm. The cells were harvested after 3 more h of shaking, and the cell pellet was lysed in 25 mm Tris, pH 8.0, 350 mm NaCl, 5 mm DTT, 1 mm phenylmethylsulfonyl fluoride by three passes through a Microfluidics M110L homogenizer. The cell lysate was cleared by centrifugation at 25,000 × g, and the supernatant was applied to a column of glutathione-agarose (Sigma) in lysis buffer. GST-GRP94-(69–337) was eluted with a buffer containing 10 mm glutathione, 50 mm Tris, pH 8.0, 200 mm NaCl, 1 mm DTT. The fusion protein was diluted to 2.5 mg/ml and cleaved by the addition of thrombin (Hematologic Technologies) at an enzyme:protein ratio of 1:1000 (w/w) for 3.0 h at room temperature while being dialyzed against 50 mm Tris, pH 7.6, 50 mm NaCl, 1 mm DTT. The cleaved mixture was applied to a Q-Sepharose Fast Flow column (Amersham Biosciences) and eluted with a 50–600 mm NaCl gradient in 50 mm Tris, pH 7.6, 1 mm DTT. The fractions containing intact GRP94-(69–337) were combined and concentrated in a Centriprep-10 (Millipore) and further purified by gel filtration (Superdex 75, Amersham Biosciences) and high performance ion exchange (Resource Q, Amersham Biosciences) chromatography, both in 50 mm Tris, pH 7.6, buffers. Purified GRP94-(69–337) was concentrated by ultrafiltration in a Millipore Ultrafree 4–30 mg/ml, buffer exchanged into 10 mm Tris, pH 7.6, 100 mm NaCl, 1 mm DTT, and stored in small aliquots at -80 °C. GRP94-(69–337Δ40) was purified in a similar manner, except that the final Resource Q step was omitted.Ligands and Protein·Ligand Complexes—NECA and radicicol were purchased from Sigma. 2-Chlorodideoxyadenosine (2ClddA) was obtained from the Open Chemical Repository of the Drug Therapeutics Program at the NCI, National Institutes of Health (Bethesda, MD). GRP94-(69–337)·ligand complexes were prepared by adding a 3- to 5-fold molar excess of concentrated ligand (10–50 mm) in Me2SO to the 30 mg/ml protein solution and used without further purification.Crystallization and Data Collection—GRP94-(69–337)·ligand complexes were crystallized at 18 °C by the hanging drop vapor diffusion method. Two μl of protein·ligand complex was mixed with an equal volume of reservoir solution, which consisted of 100 mm Tris, pH 7.6, 200–300 mm MgCl2, and 35–45% PEG 400 for the NECA, 2ClddA, and Δ40 complexes. The reservoir for the radicicol complex was the same except that PEG 550 monomethylether was substituted for PEG 400. Diffraction quality crystals with typical dimensions of 0.6 × 0.2 × 0.1 mm grew in less than 1 week.Crystals of the NECA complex were strengthened by vapor diffusion cross-linking with glutathione following the procedure of Lusty (41Lusty C.J. J. Appl. Crystallogr. 1999; 32: 106-112Crossref Scopus (91) Google Scholar) using 5 μl of a 25% glutaraldehyde solution in a microbridge for 30 min. Following cross-linking the drop containing the crystals was placed over a fresh reservoir solution containing 100 mm Tris, pH 7.6, 220 mm MgCl2, 43% PEG 400 and gradually stabilized with this solution. Stabilized crystals were removed from the drop in nylon loops and flash frozen in liquid nitrogen. Crystals of the radicicol and Δ40 complexes were removed directly from the mother liquor and flash frozen in a stream of N2 gas cooled to -170 °C. Crystals of the 2ClddA complex were equilibrated into reservoir solution that had been supplemented with glycerol to a final concentration of 20%, removed from the drop in loops and flash frozen in liquid nitrogen.Diffraction data sets used to obtain initial phases for the NECA complex were collected at 113 K on a home source using CuKα x-rays. High resolution data were collected at 100 K on beamlines 19BM or 14-IDB at the Advanced Photon Source using charge-coupled device detectors. All data was indexed and reduced using HKL2000 (42Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar).Structure Determination and Refinement—The structures of the GRP94-(69–337)·ligand complexes were solved by molecular replacement with CNS (43Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. GrosseKunstleve 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 (16930) Google Scholar). Initial phases for the NECA complex were obtained by molecular replacement using a 2.75-Å home source data set processed in space group C2 (Native 1, 97.6% complete, I/σ = 2.2 last shell, Rmerge = 0.057), with the search model consisting of the backbone atoms only of the N-domain of yeast Hsp90 (PDB code 1AMW). The two GRP94 molecules in the asymmetric unit were located sequentially in the translation search. Electron density maps calculated from this solution revealed good density for the model through Hsp90 residue 111 (residue 181 in the GRP94 numbering). Subsequent rounds of simulated annealing refinement and manual rebuilding with O (44Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) against a 1.9-Å Advanced Photon Source 14BMC synchrotron data set (Native 2, 89.7% complete, I/σ = 2.1 last shell, Rmerge = 0.109) using NCS to restrain the two protomers in the asymmetric unit eventually revealed the course of the GRP94 polypeptide from residues 73–286 and 331–337, as well as the NECA ligand. Final refinement was carried out against a 1.75-Å C2221 data set (Native 3) using a maximum likelihood target, bulk solvent correction, and individual restrained B factor correction. The radicicol and 2ClddA complexes were solved by molecular replacement using the polypeptide from the partially refined NECA complex as the search model and were refined in a manner similar to the NECA complex. The GRP94-(69–337Δ40)-NECA complex, which has two molecules in the asymmetric unit, was solved by molecular replacement using a partially refined P21212 2ClddA complex as the search model, which also has two molecules in the asymmetric unit, and was refined as described above. Refinement statistics are given in Table I.Table ISummary of data collection and refinement statistics for GRP94·ligand complexesComplexNECARadicicol2ClddAΔ40/NECAData setNative 3NativeNativeNativeks129d2cks147c5aKn10b5aks160b2aSource/detectorAPS 19BM/CCDAPS 19BM/CCDAPS 14IDB/CCDRU200/RaxisIISpace groupC2221C2221C2221P212121a, b, c89.20, 99.18, 63.0786.75, 99.37, 63.3087.01, 98.97, 63.2565.30, 84.20, 94.09ResolutionaResolution limit was defined as the highest resolution shell where the average I/σI was 2 (Å)50—1.7550—1.8550—2.1050—2.10Last shell (Å)1.81—1.751.92—1.852.18—2.102.18—2.10Unique reflections28627235491650127334% completeness (last shell)99.7 (97.9)99.0 (95.5)98.2 (96.3)87.8 (50.2)Average I/σI (last shell)47.1 (4.45)36.3 (1.89)48.2 (2.9)40.4 (4.54)Redundancy8.27.310.96.3RmergebRmerge = ΣhklΣi|Ii(hlk) — 〈I(hlk)〉|/ΣhklΣII(hkl) (last shell)0.043 (0.293)0.055 (0.440)0.054 (0.482)0.049 (0.238)Crystallographic refinement Resolution range (Å)6.0—1.756.0—1.856.0—2.106.0—2.10 Reflections24869219261523626990 Non-solvent atoms1748178017773556 Solvent atoms231172268393 r.m.s. deviation from ideality Bond lengths (Å), angles (°)0.005, 1.230.011, 1.470.007, 1.400.006, 1.24 R valuecR = Σ|Fo — Fc|/ΣFc. 10% of the reflections were used to calculate Rfree (F > 2σF)20.7 (20.2)22.2 (21.4)20.725.0 Rfree (F > 2σF)24.4 (23.6)27.7 (26.9)27.329.9a Resolution limit was defined as the highest resolution shell where the average I/σI was 2b Rmerge = ΣhklΣi|Ii(hlk) — 〈I(hlk)〉|/ΣhklΣII(hkl)c R = Σ|Fo — Fc|/ΣFc. 10% of the reflections were used to calculate Rfree Open table in a new tab Stereochemistry was assessed using Procheck (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and clashes were identified using contact dots (46Word J.M. Lovell S.C. LaBean T.H. Taylor H.C. Zalis M.E. Presley B.K. Richardson J.S. Richardson D.C. J. Mol. Biol. 1999; 285: 1711-1733Crossref PubMed Scopus (451) Google Scholar). Graphics presented here used Ribbons (47Carson M. J. Appl. Crystallogr. 1991; 24: 958-961Crossref Scopus (783) Google Scholar) and Pymol (DeLano Scientific, San Carlos, CA). Difference distance matrices (48Richards F.M. Kundrot C.E. Proteins. 1988; 3: 71-84Crossref PubMed Scopus (362) Google Scholar) were calculated using the program DDMP, available from the Yale Center for Structural Biology (www.csb.yale.edu).Site-directed Mutagenesis—The 287–327 (“Δ40”) deletion mutant of GRP94-(69–337) was constructed with the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by sequencing.RESULTSCrystallization and Structure Determination—The structures of three complexes between GRP94-(69–337) and NECA, radicicol, and 2ClddA were solved and refined at 1.75-, 1.85-, and 2.1-Å resolution, respectively. The GRP94-(69–337) protein domain was crystallized in the presence of a 5-fold molar excess of ligand. The structure of the GRP94·NECA complex was solved by molecular replacement using the backbone atoms only from the yeast Hsp90·ADP complex (PDB code 1AMW) as the search model and refined against data to the diffraction limit. The GRP94-(69–337)·radicicol and GRP94-(69–337)·2ClddA complexes were solved by molecular replacement using the backbone atoms of the GRP94-(69–337)·NECA complex as the search model. In all three structures the charged domain residues 287–327 were disordered. A portion of an omit electron density map for the NECA complex is shown in Fig. 1. The structure of the charged domain deletion mutant GRP94(69–337Δ40) in complex with NECA was crystallized, solved, and refined in a manner similar to that of the radicicol and 2ClddA complexes.Overall Architecture—The three GRP94-(69–337)·ligand complexes fold into a conformation that closely resembles the N-domains of yeast (32Prodromou C. Roe S.M. Piper P.W. Pearl L.H. Nat. Struct. Biol. 1997; 4: 477-482Crossref PubMed Scopus (206) Google Scholar) and human (16Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1234) Google Scholar) Hsp90, with an overall r.m.s. deviation between GRP94 and the Hsp90 proteins of 2.3–2.5 Å for all backbone atoms. As will be discussed later, helices 1, 4, and 5 constitute a subdomain of GRP94 that has a different orientation relative to the remainder of the N-domain than the equivalent region in Hsp90. Excluding this region from the comparison reduces the r.m.s. deviation between GRP94 and Hsp90 to 0.90–1.1 Å for all backbone atoms, indicating a highly similar fold. The 10 residues that make up the adenine-binding cavity (Leu104, Asn107, Ala111, Asp149, Gly153, Met154, Asn162, Gly196, Phe199, and Thr245) are completely conserved between Hsp90 and GRP94 and, with the exception of Gly196, are not in the helix 1-4-5 subdomain. The r.m.s. deviation for just these 10 residues (all atoms) is 0.89 Å with yeast Hsp90 and 1.27 Å with human Hsp90.NECA Binding Is Stabilized by Interaction with Two Adjacent Cavities in GRP94 —NECA is an adenine-based nucleotide analog that binds selectively to GRP94, and not Hsp90, with a KD of 200 nm (38Rosser M.F. Nicchitta C.V. J. Biol. Chem. 2000; 275: 22798-22805Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 40Hutchison K.A. Fox I.H. J. Biol. Chem. 1989; 264: 19898-19903Abstract Full Text PDF PubMed Google Scholar). Selective ligands such as NECA may aid in the elucidation of the specific role of GRP94 in the cell and serve as a scaffold for the design of high affinity modulators of GRP94 activity. To understand the basis for NECA binding to GRP94, we determined the structure of the N-domain of GRP94 in complex with NECA. A schematic drawing of the interactions between NECA and GRP94 is shown in Fig. 2A, and a stereo representation is shown in Fig. 2B.Fig. 2Interactions between NECA and GRP94.A, schematic drawing showing the interactions. Hydrogen bonds are shown as dashed lines, and van der Waals contacts are represented by complementary double semi-circles. Red circles are water molecules. Amino acid side chains are represented by ovals, and backbone atoms are shown as squares. B, stereo view of the GRP94·NECA interaction. Selected van der Waals surfaces are depicted by dots, hydrogen bonds are depicted by dashed lines, and water molecules are shown as green spheres. Oxygen atoms are colored red, nitrogen blue, and carbon black. C, stereo view of the binding of ADP in yeast Hsp90. The coordinates were taken from PDB code 1AMW (15Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar). B and C were prepared with Ribbons (47Carson M. J. Appl. Crystallogr. 1991; 24: 958-961Crossref Scopus (783) Google Scholar).View Large Image Figure ViewerDownload (PPT)Two regions of GRP94 are observed to accommodate NECA binding (Fig. 2A). The first, as expected, is the adenine-binding cavity first identified in complexes between Hsp90 and adenosine nucleotides (15Prodromou C. Roe S.M. O'Brien R. Ladbury J.E.
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