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• W2110528399 abstract "Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca2+ and ATP reveal that this protein can form a tetramer and that a flexible loop (the “multifunctional loop”) contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus. Synapsins are multidomain proteins that are critical for regulating neurotransmitter release in vertebrates. In the present study, two crystal structures of the C domain of rat synapsin I (rSynI-C) in complex with Ca2+ and ATP reveal that this protein can form a tetramer and that a flexible loop (the “multifunctional loop”) contacts bound ATP. Further experiments were carried out on a protein comprising the A, B, and C domains of rat synapsin I (rSynI-ABC). An ATP-stabilized tetramer of rSynI-ABC is observed during velocity sedimentation and size-exclusion chromatographic experiments. These hydrodynamic results also indicate that the A and B domains exist in an extended conformation. Calorimetric measurements of ATP binding to wild-type and mutant rSynI-ABC demonstrate that the multifunctional loop and a cross-tetramer contact are important for ATP binding. The evidence supports a view of synapsin I as an ATP-utilizing, tetrameric protein made up of monomers that have a flexible, extended N terminus. The synapsins are a family of phosphoproteins that are localized at presynaptic termini of neurons (1Südhof T.C. Czernik A.J. Kao H-T. Takei K. Johnston P.A. Horiuchi A. Kanazir S.D. Wagner M.A. Perin M.S. De Camilli P. Greengard P. Science. 1989; 245: 1474-1480Crossref PubMed Scopus (415) Google Scholar, 2Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 13371-13374Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 3Kao H.T. Porton B. Czernik A.J. Feng J. Yiu G. Haring M. Benfenati F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4667-4672Crossref PubMed Scopus (186) Google Scholar, 4Hosaka M. Hammer R.E. Südhof T.C. Neuron. 1999; 24: 377-387Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). They have a number of properties that implicate them as important cogs in the synaptic machinery. In mammals, synapsins are dynamically associated with the cytosolic surface of synaptic vesicles (4Hosaka M. Hammer R.E. Südhof T.C. Neuron. 1999; 24: 377-387Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), and they are necessary for the proper regulation of neurotransmitter release (5Llinas R. McGuinness C.S. Leonard M. Sugimori & Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3035-3039Crossref PubMed Scopus (513) Google Scholar, 6Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (615) Google Scholar, 7Ryan T.A. Li L. Chin L.S. Greengard P. Smith S.J. J. Cell Biol. 1996; 134: 1219-1227Crossref PubMed Scopus (144) Google Scholar, 8Feng J. Chi P. Blanpied T.A. Xu Y. Magarinos A.M. Ferreira A. Takahashi R.H. Kao H-T. McEwen B.X. Ryan T.A. Augustine G.J. Greengard P. J. Neurosci. 2002; 22: 4372-4380Crossref PubMed Google Scholar). These proteins are the targets of several kinases that transduce rising second-messenger concentrations into protein phosphorylation (9Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1759) Google Scholar). Synapsins also bind to several neuronal proteins (9Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1759) Google Scholar), including calmodulin, annexin VI, and cytoskeletal proteins.In vertebrates, there are five known synapsin proteins, termed Ia, Ib, IIa, IIb, and IIIa. The primary sequences of these proteins have been grouped into eight domains (1Südhof T.C. Czernik A.J. Kao H-T. Takei K. Johnston P.A. Horiuchi A. Kanazir S.D. Wagner M.A. Perin M.S. De Camilli P. Greengard P. Science. 1989; 245: 1474-1480Crossref PubMed Scopus (415) Google Scholar). At the N termini, the A domains are about 30 amino acids in length and contain a conserved phosphorylation site that regulates the ability of the synapsins to associate with synaptic vesicles (1Südhof T.C. Czernik A.J. Kao H-T. Takei K. Johnston P.A. Horiuchi A. Kanazir S.D. Wagner M.A. Perin M.S. De Camilli P. Greengard P. Science. 1989; 245: 1474-1480Crossref PubMed Scopus (415) Google Scholar, 4Hosaka M. Hammer R.E. Südhof T.C. Neuron. 1999; 24: 377-387Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The primary structures of the B domains are variable and contain a disproportional number of proline, glycine, alanine, and serine residues. The well-conserved C domains are the largest of the synapsin domains (∼300 amino acids). The crystal structure of the C domain of bovine synapsin I (bSynI-C) 11 The abbreviations used are: bSynI-C, bovine synapsin I; ATPγS, adenosine 5′-O-(thiotriphosphate); r.m.s., root mean square; VS, velocity sedimentation; ITC, isothermal titration calorimetry; GSHase, glutathione synthetase; rSynI-ABC, rat synapsin I. 11 The abbreviations used are: bSynI-C, bovine synapsin I; ATPγS, adenosine 5′-O-(thiotriphosphate); r.m.s., root mean square; VS, velocity sedimentation; ITC, isothermal titration calorimetry; GSHase, glutathione synthetase; rSynI-ABC, rat synapsin I. is known (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). The overall fold of this domain places it in a group of proteins called the “ATP-grasp superfamily” (11Galperin M.Y. Koonin E.V. Protein Sci. 1999; 6: 2639-2643Crossref Scopus (239) Google Scholar). The tertiary structure of bSynI-C is most similar to that of the ATP-grasp enzyme glutathione synthetase (GSHase). GSHase contains a flexible, catalytically essential loop, called the “multifunctional loop” (12Hibi T. Nishioka T. Kato H. Tanizawa K. Fukui T. Katsube Y. Oda J. Nat. Struct. Biol. 1996; 3: 16-18Crossref PubMed Scopus (26) Google Scholar, 13Tanaka T. Nishioka N. Oda J. Arch. Biochem. Biophys. 1997; 339: 151-156Crossref PubMed Scopus (3) Google Scholar). This loop is disordered in crystal structures of unliganded GSHase, but structures in the presence of ligands show that it can become ordered upon substrate binding (14Hara T. Kato H. Katsube Y. Oda J. Biochemistry. 1996; 35: 11967-11974Crossref PubMed Scopus (65) Google Scholar). The synapsin C domain also has a multifunctional loop, but it was disordered in crystal structures with and without bound ATPγS (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). The C-terminal domains of synapsins, D-H, are less conserved and sometimes specific to a given gene or isoform.The structure determination of bSynI-C has spurred other studies designed to characterize this domain in more detail. The crystal structure showed a tightly associated dimer of C domains (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). Subsequently, it was demonstrated that synapsins form multimers in vivo (15Hosaka M. Südhof T.C. J. Biol. Chem. 1999; 274: 16747-16753Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In vitro studies showed that ATP binds tightly to all synapsins; the half-maximal inhibitory constants of ATP competing with bound ATPγS range between 110-450 nm (2Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 13371-13374Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar, 16Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 1425-1429Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). An intriguing result of the ATP-binding investigations is the role that Ca2+ plays in this process. The ATP-binding activity of synapsin I is enhanced by Ca2+, synapsin II is indifferent to the presence of the cation, and the presence of Ca2+ is refractory to the ATP-binding activity of synapsin III (2Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 13371-13374Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 1425-1429Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). No ATPase activity has been detected for a synapsin. Despite this, it has been speculated that synapsins have enzymatic activity, based on their homology to a known class of enzymes (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar, 16Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 1425-1429Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and on the observation that the protein binds ADP with less affinity than ATP (16Hosaka M. Südhof T.C. J. Biol. Chem. 1998; 273: 1425-1429Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).In this study, we set out to characterize further the structural aspects of synapsin I. Using x-ray crystallography, we determined the crystal structures at resolutions of 2.1-2.23 Å of the C domain of rat synapsin I (rSynI-C) complexed to a Ca2+ chelate of ATP (Ca·ATP). The structures recapitulate a tetramer of synapsin C monomers that was observed before (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). Our structures also illustrate that residues from the multifunctional loop contact ATP that is bound to the C domain. Interactions across the tetramer interface appear to stabilize these contacts. Velocity sedimentation and size-exclusion chromatographic experiments on a protein comprised of the A, B, and C domains of rat synapsin I (rSynI-ABC) show that this tetramer exists in vitro and is stabilized by the presence of Ca·ATP. Isothermal titration calorimetric studies on mutant rSynI-ABC proteins demonstrate that the multifunctional loop is critical for the ATP-binding activity of the synapsin C domain, and that the tetramer participates in ATP binding.EXPERIMENTAL PROCEDURESMaterials—Unless noted otherwise, all chemicals and reagents were purchased from Sigma Chemical Corporation.Protein Purification—The plasmid pSynABC (provided by the T. C. Südhof laboratory) expresses a fusion protein comprised of glutathione S-transferase (GST) followed by a thrombin protease site and residues 2-420 of rat synapsin I. The protein was purified essentially as described (17Wang C.R. Esser L Smagula C.S. Südhof T.C. Deisenhofer J. Protein Sci. 1997; 6: 2264-2267Crossref PubMed Scopus (8) Google Scholar), with the exception that thrombin was used to cleave the protein from the glutathione-agarose beads. Further purification was performed at 4 °C. rSynI-ABC was dialyzed overnight against buffer GFA (50 mm Tris-Cl pH 8.0, 150 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol (Amresco)). The dialyzed sample was passed over a Mono Q column (Amersham Biosciences) that had been equilibrated with buffer GFA. Synapsin eluted in the flow-through. Finally, the protein solution was injected onto a Superdex 200 16/60 (Amersham Biosciences) column pre-equilibrated with buffer GFA. Fractions containing rSynI-ABC were pooled and concentrated as needed.Mutagenesis—pSynABC was used as the template for all mutageneses performed in this study. For the mutations W335F, K336R, and D290A, the Gene Editor (Promega) methodology was used, as outlined by the manufacturer. A PCR-based mutagenesis protocol (megaprimer mutagenesis) was utilized to generate the W335A and K336A mutations (18Datta A.K. Nucleic Acids Res. 1995; 23: 4530-4531Crossref PubMed Scopus (100) Google Scholar). Standard methods (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were used to restrict the PCR product and ligate it into pSynABC that was missing the corresponding wild-type region of DNA. The mutant plasmids were sequenced at The University of Texas Southwestern Medical Center at Dallas Biopolymer Facility.Chromatographic Determination of Stokes' Radius—rSynI-ABC was dialyzed against Buffer GFB (50 mm Tris-Cl pH 7.5, 150 mm NaCl, 1.5 mm CaCl2, and 1 mm dithiothreitol). In cases where ATP was included, the protein was incubated at 4 °C with 120 μm NaATP for 1 h. Proteins were applied to a Superdex 200 16/60 column that had been equilibrated with GFB ± 120 μm NaATP. A280 traces were compared against standard curves (20Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1542) Google Scholar) that had been generated using proteins of known Stokes' radii (Amersham Biosciences) in Buffer GFB both in the absence and presence of NaATP. Values for the positions of the peaks and their integrated areas were calculated with the evaluation software included with the Äkta system (Amersham Biosciences).Isothermal Titration Calorimetry—Apparent association constants for ATP binding to rSynI-ABC were determined using isothermal titration calorimetry (ITC). A solution of rSynI-ABC was dialyzed for 2 days against ITC buffer (50 mm Tris-Cl pH 7.5, 100 mm NaCl, 10 mm 2-mercaptoethanol, 500 μm CaCl2). ATP was dissolved in the same dialysis buffer used to equilibrate the protein. The concentrations of both protein and ligand were calculated using Beer's Law, c = A/ϵl, where c is the concentration, A is the absorbance (at 280 nm for the protein and 260 nm for ATP), and ϵ is the extinction coefficient. The literature ϵ for ATP was used, and the ϵ for rSynI-ABC was derived empirically (21Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3393) Google Scholar). Starting concentrations for the proteins ranged between 12 and 32 μm. The experiment consisted of a series of injections of 8 μl of the ATP solution ([ATP] = 180-600 μm) into a cell containing 1.4 ml of the protein solution equilibrated at 20 °C. In most cases, some precipitation occurred during the titration. However, several samples were centrifuged and filtered after the titration was complete, and the precipitate comprised less than 10% of the total protein. Heats associated with ATP binding were measured by a Microcal isothermal titration calorimeter (model VP-ITC). Apparent association constants derived from these data were calculated using software included with the calorimeter (Microcal ORIGIN).Analytical Ultracentrifugation—Velocity sedimentation studies were carried out at the Analytical Ultracentrifugation Core Facility at The University of Texas Southwestern Medical Center at Dallas. Purified rSynI-ABC was extensively dialyzed versus buffer GFB. rSynI-ABC samples at concentrations of 12.3 and 4.9 μm were centrifuged at 4 °C in a Beckman Model XL-I ultracentrifuge at 40,000 rpm. When ATP was included, its concentration was 120 μm and it was incubated at 4 °C with the protein for at least 1 h prior to centrifugation. The distribution of protein in the cells was monitored using absorption optics. Data were analyzed using the program DCDT+, which uses a plot of -dc/dt versus s* to derive the sedimentation coefficient, s, and the diffusion coefficient, D (22Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (237) Google Scholar). When molecular masses were estimated from these plots, the number of scans was strictly limited to obey the “rule of thumb” for Δ(ω2t) proposed by W. F. Stafford (www.bbri.org/faculty/stafford/dcdt/Rule_of_thumb.html). s0 values were derived by assembling a plot of s versus protein concentration and extrapolating to infinite dilution. The values of s0 were converted to s20,w0 by multiplying s0 by the following conversion factor shown in Equation 1, s20,w0/s0=(1-(V¯2)20,wρ20,w)(η4,b)/(1-(V¯2)4,bρ4,b)(η20,w)(Eq. 1) where (V̄2) denotes partial specific volume, ρ is solution density, and η is solution viscosity calculated under conditions of either water as the solvent at 20 °C (subscript “20,w”), or of buffer as the solvent at 4 °C (subscript “4,b”). Nonstandard values for V̄2, ρ, and η were calculated using SEDNTERP (www.rasmb.bbri.org). The minimum expected frictional coefficient, fmin, was calculated using Equation 2, fmin=6πη((3Mk(V¯2)/4πNA)1/3)(Eq. 2) where Mk is the known mass of the protein and NA is Avogadro's number. The experimental frictional coefficient, f, was calculated using the relationship in Equation 3. f=Mk(1-(V¯2))/s20,w0NA(Eq. 3) The formula used to calculate Stokes' radius (rS) is in Equation 4. rS=f/6πη20,w(Eq. 4) The sedimentation data were also analyzed with the program Sedfit (24Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). Continuous c(s) and c(M) distributions were examined, and s4,b and M values were taken to be the weighted average of the peaks in these distributions, respectively. s20,w0 values derived from these analyses were computed as outlined above.Mass Spectrometry—Electrospray mass spectrometry was carried out using standard protocols at the Biopolymers Facility of The University of Texas Southwestern Medical Center at Dallas.Crystallization and Structure Determination—One hour prior to mixing with precipitant, rSynI-ABC (7.5 mg/ml) was incubated on ice with 10 mm Ca·ATP. Crystallization was carried out using the vapor diffusion methodology by mixing a 1-μl drop of rSynI-ABC/Ca·ATP with 1 μl of reservoir solution, (150 mm Tris, pH 7.5, 15-20% (w/v) PEGMME 5000 (Fluka), and 300 mm NaCl), then hanging this drop over 1 ml of the reservoir. Crystallization was accomplished at 26 °C and usually occurred in one to 3 weeks. During the course of crystallization, the protein was partially proteolyzed such that the major product in the crystals was rSynI-C. Four crystal forms were obtained, and this report describes two of them. One occurs in space group P6522, and the other in space group P1. The crystals were stabilized by transferring them to fresh well solution, and were subsequently cryostabilized by transferring them serially to solutions containing the same buffer plus increasing amounts of ethylene glycol. Prior to cooling, the final concentration of ethylene glycol was 20% (v/v). Crystals were flash cooled in liquid propane, and the propane was then frozen in liquid nitrogen. Diffraction data were collected at beamline 19-ID at the Argonne National Laboratories Structural Biology Center at the Advanced Photon Source (APS), Cornell High Energy Synchrotron Source (CHESS) beamline F1, and National Synchrotron Light Source (NSLS) beamline X4A. An Argonne CCD detector was used at APS, and Quantum 4 CCD detectors (ADSC) were used at CHESS and NSLS. Data were integrated and scaled using the HKL2000 package (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38346) Google Scholar). Two structures of rSynI-C were determined by molecular replacement using the program EPMR (26Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D. 1999; 55: 484-491Crossref PubMed Scopus (689) Google Scholar). For the P6522 data, a single molecule of bovine synapsin I C was used as the search model. Two molecules were located in the asymmetric unit. For the P1 data, a tightly associated dimer of bSynI-C was used as the search model, and the asymmetric unit contained four such dimers. The models were adjusted using O (27Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar) and XtalView (28McRee D.E. J. Mol. Graph. 1992; 10: 44-46Crossref Google Scholar). The adjusted models were refined using the CNS (29Brünger 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. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar) protocols for simulated annealing, conjugate gradient minimization, and individual B-factor refinement. NCS restraints were used initially, but were released in the final stages of refinement with no ill effects on Rfree (30Brunger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3846) Google Scholar). The R-factors and other statistical measures of the quality of the structures are presented in Table I. In modified Ramachandran plots (31Lovell S.C. Davis I.W. Arendall III, W.B. de Bakker P.I.W. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2002; 50: 437-450Crossref Scopus (3790) Google Scholar), 99% of all residues were in the most favored regions, and none were outside allowed regions.Table ISummary of data collection and refinement statisticsData collection statisticsCrystal form (Space group)P6522P1Parametera96.070.6b96.078.4c305.8135.0α9080.6β9076.9γ12071.8BeamlineAPS ID-19CHESS F1Wavelength (Å)0.94201.008Resolution Range (Å)20-2.2320-2.1# Reflections467,389522852No. of unique reflections (F>0)40,542144,586CompletenessaNumbers in parentheses are the values for the highest-resolution shell of the data.99.8 (100.0)96.3 (92.0)Avg. 1/σ29.9 (6.1)20.9 (2.8)RsymbRsym=∑h∑iI^(h)Ii(h)/∑h∑iIi(h), where Ii(h) is the ith measurement of the intensity for Miller indices h and Î(h) represents the mean intensity value for the symmetry- (or Friedel-) equivalent reflections of Miller indices h.0.082 (0.391)0.058 (0.373)Refinement statisticsPDB accession no.1PX21PK8ParameterNo. of protein atoms4,67818,530No. of waters326665No. of Ca (II) ions28RworkcRwork includes all reflections used during refinement, whereas Rfree was calculated for a portion (5% or 10%, respectively, of the P6522 or P1 crystal forms) of the data set that was not used for refinement (30).0.2030.222RfreecRwork includes all reflections used during refinement, whereas Rfree was calculated for a portion (5% or 10%, respectively, of the P6522 or P1 crystal forms) of the data set that was not used for refinement (30).0.2400.259R.m.s. deviationsBond lengths (Å)0.0070.006Bond angles (°)1.41.3Dihedrals (°)24.624.3Impropers (°)0.490.5a Numbers in parentheses are the values for the highest-resolution shell of the data.b Rsym=∑h∑iI^(h)Ii(h)/∑h∑iIi(h), where Ii(h) is the ith measurement of the intensity for Miller indices h and Î(h) represents the mean intensity value for the symmetry- (or Friedel-) equivalent reflections of Miller indices h.c Rwork includes all reflections used during refinement, whereas Rfree was calculated for a portion (5% or 10%, respectively, of the P6522 or P1 crystal forms) of the data set that was not used for refinement (30Brunger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3846) Google Scholar). Open table in a new tab RESULTSStructural Overview of rSynI-C—We used molecular replacement to determine the structures of two crystal forms of rSynI-C complexed to a Ca2+ chelate of ATP (Fig. 1a and Table I). Although covalently intact rSynI-ABC was used in the crystallization experiments, the A and B domains were largely absent owing to proteolysis (data not shown). Crystals containing intact rSynI-ABC have been obtained; electron-density maps calculated using diffraction data from such crystals do not betray the presence of the A or B domains (data not shown). The protease sensitivity and lack of electron density of the A and B domains indicate that these domains are probably flexible and disordered under our crystallization conditions. The first crystal form of rSynI-C has the symmetry of space group P6522, with two monomers in the asymmetric unit. The second has the symmetry of space group P1, and eight monomers are found in the asymmetric unit. Thus, in our two crystal forms, we observed ten crystallographically independent monomers. The structures of rSynI-C and bSynI-C are very similar (Fig. 1b). Pairwise comparisons of the Cα atoms of the aforementioned ten rat C domains to those of two known bovine C domains (PDB accession code 1AUX) show r.m.s. deviations between the structures of 0.6 Å or less. This likeness is expected, as the primary structures of the domains differ by only four amino acids.In both of our rSynI-C structures, we observe nearly identical tetramers made up of four monomers (Fig. 1c). In the P6522 form, the asymmetric unit contains a previously described C-domain dimer (Ref. 10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar; e.g. the yellow and green monomers of Fig. 1c), and two dimers interact across a crystallographic 2-fold axis to form the tetramer. In the P1 form, four dimers exist in the asymmetric unit, and local pseudo-2-fold axes relate them, forming two of these tetramers. The dimers bury an additional 4010 Å2 of surface area (2005 Å2 per dimer) upon tetramerization. The total buried surface area of the tetramer is about 11,150 Å2. The most extensive interface between the two dimers is formed by packing the α4 helices of one dimer against the phosphate-binding loops of the other, and vice versa (Fig. 1, a and c). This oligomer was also observed in the bSynI-C structure (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar) and in other rSynI-C structures not presented in this paper (data not shown). The residues that form the tetramer interface are well conserved across all three isoforms of rat synapsin I (Fig. 2a). We therefore expect that all rat synapsin C domains can assemble in a similar tetrameric arrangement. The conservation of a key cross-tetramer contact (discussed below) across all known synapsin C domains implies that the tetramer is present in all synapsin-harboring species (Fig. 2b). A similar quaternary arrangement is observed in the tetrameric ATP-grasp enzyme GSHase (Fig. 1d).Fig. 2Multiple sequence alignments of synapsin C domains. a, conservation of the tetramer contacts in the rat synapsin C domains. A sequence alignment, performed using ClustalW (46Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55157) Google Scholar), of the C domains of the three synapsin isoforms from rat is shown. Rn stands for Rattus norvegicus, and the roman numerals denote the isoform. The numberings for the sequences are given on the right-hand side of the figure. The bottom row of the alignment shows the degree of conservation of the amino acids, as defined by ClustalW. *, all residues are identical, :, residue is conserved;., denotes a semi-conserved residue. Residues highlighted in black are those whose side chains are involved in the previously described dimer interface (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar), which forms part of the tetramer interface. Residues highlighted in gray are those whose side chains are exclusively involved in contacts between the two dimers that form the tetramer. b, multiple sequence alignment of the region near to the multifunctional loop. All C domains in the SWISS-PROT data base at the time of writing were used to obtain this alignment. Residues involved in an important cross-tetramer contact are shaded gray. In the rat synapsin I numbering, they are Asp290, Trp335, and Lys336. Rn, Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens; Cf, Canis familiaris; Xl, Xenopus laevis; Lf, Lampetra fluviatilis; Dm, Drosophila melanogaster; Ac, Aplysia californica; Ce, Caenorhabditis elegans; Lp, Lolego pealeii. Roman numerals denote the isoform of the sequence. Ac 11.1 and Lp (long isoform) were omitted from this analysis because their C domains are identical to those of other isoforms within their respective species.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structural Observations of the Multifunctional Loop and ATP binding—Our rSynI-C electron-density maps always show evidence of the multifunctional loop (residues 330-343), which was not visible in the bSynI-C structure (10Esser L. Wang C-R. Hosaka M. Smagula C.S. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). Our ten observed monomers exhibit electron density for between two and all fourteen residues of the loop (Fig. 3a). Differing local environments of the ten monomers in the crystalline lattice probably account for the differential ordering of the loop. The multifunctional loop contacts the bound ATP at portions of the nucleotide that were solvent-exposed in the bovine structure. The observed disposition of the loop makes the bound ATP essentially solvent-inaccessible (Figs. 3, b and c). Because this conformation of the loop would impede access to the AT-binding site, it is likely that it adopts" @default.
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• W2110528399 title "Tetramerization and ATP Binding by a Protein Comprising the A, B, and C Domains of Rat Synapsin I" @default.
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