The Evolutionarily Conserved Trimeric Structure of CutA1 Proteins Suggests a Role in Signal Transduction
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m304398200
ISSN1083-351X
AutoresFabio Arnesano, Lucia Banci, Manuela Benvenuti, Ivano Bertini, V. Calderone, Stefano Mangani, Maria Silvia Viezzoli,
Tópico(s)Cellular Mechanics and Interactions
ResumoCutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit β-sheet formation. Each subunit consists of a ferredoxin-like (β1α1β2β3α2β4) fold with an additional strand (β5), a C-terminal helix (α3), and an unusual extended β-hairpin involving strands β2 and β3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits. CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit β-sheet formation. Each subunit consists of a ferredoxin-like (β1α1β2β3α2β4) fold with an additional strand (β5), a C-terminal helix (α3), and an unusual extended β-hairpin involving strands β2 and β3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits. CutA1 is a widespread protein of about 12 kDa found in bacteria, plants, and animals, including humans. The protein was originally identified in a gene locus of Escherichia coli called cutA involved in divalent metal tolerance (1Fong S.T. Camakaris J. Lee B.T. Mol. Microbiol. 1995; 15: 1127-1137Crossref PubMed Scopus (98) Google Scholar). The cutA locus consists of two operons, one containing a single gene encoding a cytoplasmic protein, CutA1, and the other composed of two genes encoding a 50-kDa (CutA2) and a 24-kDa (CutA3) inner membrane proteins. Molecular genetics studies on the E. coli cutA locus showed that some mutations lead to copper sensitivity due to its increased uptake (1Fong S.T. Camakaris J. Lee B.T. Mol. Microbiol. 1995; 15: 1127-1137Crossref PubMed Scopus (98) Google Scholar). However, the specific function of CutA1 in E. coli is still unknown. On the other hand recent studies from two independent groups highlighted a possible role of mammalian CutA1 in the anchoring of the enzyme acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; EXAFS, extended x-ray absorption fine structure; r.m.s.d., root mean square deviation; XAS, x-ray absorption spectroscopy. in neuronal cell membranes (2Perrier A.L. Cousin X. Boschetti N. Haas R. Chatel J.M. Bon S. Roberts W.L. Pickett S.R. Massoulie J. Rosenberry T.L. Krejci E. J. Biol. Chem. 2000; 275: 34260-34265Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 3Navaratnam D.S. Fernando F.S. Priddle J.D. Giles K. Clegg S.M. Pappin D.J. Craig I. Smith A.D. J. Neurochem. 2000; 74: 2146-2153Crossref PubMed Scopus (29) Google Scholar). CutA1 does not directly interact with AChE (2Perrier A.L. Cousin X. Boschetti N. Haas R. Chatel J.M. Bon S. Roberts W.L. Pickett S.R. Massoulie J. Rosenberry T.L. Krejci E. J. Biol. Chem. 2000; 275: 34260-34265Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), but the CutA1 gene is widely expressed in different regions of the brain with an expression pattern that parallels that of AChE (3Navaratnam D.S. Fernando F.S. Priddle J.D. Giles K. Clegg S.M. Pappin D.J. Craig I. Smith A.D. J. Neurochem. 2000; 74: 2146-2153Crossref PubMed Scopus (29) Google Scholar). In addition CutA1 copurified with AChE from human caudate nucleus (3Navaratnam D.S. Fernando F.S. Priddle J.D. Giles K. Clegg S.M. Pappin D.J. Craig I. Smith A.D. J. Neurochem. 2000; 74: 2146-2153Crossref PubMed Scopus (29) Google Scholar). CutA1, thus, might provide an intriguing link between copper tolerance in bacteria and a complex process in the brain of the most evolved organisms. The function of CutA1 in plants is still unknown. Copper is a transition metal essential to all organisms since it is involved in many redox reactions and in several biological processes (4Linder M.C. Biochemistry of Copper. Plenum Press, New York1991: 1-13Crossref Google Scholar). Although essential for cellular metabolism, copper is highly toxic when it exceeds cellular needs and accumulates in the cell. Proteins which bind copper are involved in several human neurological pathologies, such as the Menkes and Wilson diseases, the Alzheimer pathology, and the Creutzfeld-Jacob syndrome (5Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (714) Google Scholar). For this reason, all organisms must have homeostatic mechanisms that allow the intake of the necessary amount of copper, thus preventing its accumulation beyond the level of toxicity (6Rosenzweig A.C. O'Halloran T.V. Curr. Opin. Chem. Biol. 2000; 4: 140-147Crossref PubMed Scopus (175) Google Scholar, 7Pena M.M.O. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (628) Google Scholar); these mechanisms are carried out by proteins that specifically bind copper in the cell. Intriguingly, although CutA1 from several organisms was annotated as a "divalent cation tolerant protein" in GenBank™, it was also suggested to be possibly involved in at least two unrelated processes in bacteria and mammals, thus fulfilling different functions (1Fong S.T. Camakaris J. Lee B.T. Mol. Microbiol. 1995; 15: 1127-1137Crossref PubMed Scopus (98) Google Scholar, 2Perrier A.L. Cousin X. Boschetti N. Haas R. Chatel J.M. Bon S. Roberts W.L. Pickett S.R. Massoulie J. Rosenberry T.L. Krejci E. J. Biol. Chem. 2000; 275: 34260-34265Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In an attempt to understand this peculiar behavior and unravel its function, we have determined the crystal structure of one representative protein from bacteria (E. coli CutA1) and one from mammals (Rat CutA1) and characterized their copper binding properties. The structural arrangement of CutA1 from both organisms shows a striking similarity to the trimeric assembly of signal transduction proteins, called PII (8Cheah E. Carr P.D. Suffolk P.M. Vasudevan S.G. Dixon N.E. Ollis D.L. Structure (Lond.). 1994; 2: 981-990Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), which are involved in the nitrogen regulatory response in bacterial cells and eukaryotic chloroplasts (9Ninfa A.J. Atkinson M.R. Trends Microbiol. 2000; 8: 172-179Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 10Arcondeguy T. Jack R. Merrick M. Microbiol. Mol. Biol. Rev. 2001; 65: 80-105Crossref PubMed Scopus (353) Google Scholar). In addition, the E. coli protein is able to bind a Cu(II) ion in a site structurally equivalent to the ATP binding site in PII proteins (11Xu Y. Cheah E. Carr P.D. van Heeswijk W.C. Westerhoff H.V. Vasudevan S.G. Ollis D.L. J. Mol. Biol. 1998; 202: 149-165Crossref Scopus (135) Google Scholar, 12Xu Y. Carr P.D. Huber T. Vasudevan S.G. Ollis D.L. Eur. J. Biochem. 2001; 268: 2028-2037Crossref PubMed Scopus (50) Google Scholar). The conserved quaternary structure between bacterial and mammalian CutA1 proteins represents an important breakthrough for the comprehension of their function. Genome Analysis—CutA1 sequences were searched in GenBank™ (www.ncbi.nlm.nih.gov/Entrez) using BLAST (www.ncbi.nlm.nih.gov/BLAST). Conservation was calculated from multiple alignment of CutA1 sequences using MULTALIN (pbil.ibcp.fr). STRING (www.bork.embl-heidelberg.de/STRING) was used to identify possible functional associations between CutA1 and neighboring genes. Cloning, Expression and Purification of CutA1—Genomic DNA from E. coli (strain LE392) was extracted and purified using the DNeasy tissue kit (Qiagen). The plasmid encoding the CutA1 gene from Rattus norvegicus was a kind gift of Prof. Eric Krejci. E. coli strain DH5α was used as the cloning host, and the strain BL21(DE3) Gold was used for protein expression (Stratagene). The expression vector was constructed by amplifying the CutA1 gene from E. coli genomic DNA and from rat cDNA through PCR. The primers were designed to introduce NcoI and BamHI sites at either end of the gene to facilitate cloning into the expression vector pET16-b (Novagen). The correct DNA sequences of the obtained plasmids were confirmed by DNA sequencing analysis. For protein production E. coli BL21(DE3) Gold competent cells were transformed with the constructed plasmids and selected with 100 μg/ml ampicillin. E. coli cultures were grown aerobically in 2× yeast-tryptone (YT) medium supplemented with 200 μg/ml ampicillin to mid-exponential phase and induced with 0.8 mm isopropyl-β-d-thiogalactopyranoside for 16 h at 37 °C. Cells were harvested, resuspended, and disrupted through sonication (8 cycles, 30 s each). Both proteins were purified through anionic exchange chromatography using a DEAE-Sepharose CL6B (Amersham Biosciences) column equilibrated with 50 mm Tris-HCl, pH 8, and then eluted with two linear gradients (0-0.3 m NaCl in 50 mm Tris, pH 8 (total volume 200 ml), and 0.3-1.0 m NaCl in 50 mm Tris, pH 8 (total volume 900 ml)). Fractions containing the CutA1 proteins and detected through SDS-PAGE were pooled together and concentrated. Both the proteins were obtained in high yield (∼150 mg/liter of culture). Protein Characterization—Electrospray mass spectra were taken with an Applied Biosystems electrospray ionization-time of flight Mariner mass spectrometer. The actual molecular mass was determined by gel filtration chromatography on a Superdex 75 column (Amersham Biosciences) equilibrated with 10 mm phosphate buffer, 100 mm NaCl, pH 7.0. To obtain a calibration line mixtures of various size standards were loaded. Transferrin was used in each mixture to determine the void volume. 1H,15N and 1H,13C heteronuclear single quantum coherence NMR experiments were recorded on a 13C,15N-enriched apoCutA1 sample on Ultra Shield 700 Bruker spectrometer using a triple resonance (TXI) 5-mm probe equipped with pulsed field gradients along the xyz axes. Binding of copper(II) to CutA1 was followed through electronic spectroscopy using a Varian Cary 50 spectrophotometer. Copper content was evaluated through atomic absorption measurements with a PerkinElmer Life Sciences 2380 instrument. X-band EPR spectra were acquired with a magnetic field modulation frequency and amplitude of 100 kHz and 12.00 G, respectively, at 10 K on a BRUKER EMX (9.5 GHz) EPR spectrometer equipped with an ESR 900 helium flow cryostat (Oxford Instruments). Crystallization of CutA1 from E. coli and Rat—Crystals of CutA1 were grown in a few days using the vapor diffusion technique at 20 °C from about 12 mg/ml E. coli and 10 mg/ml rat CutA1 (∼1 mm protein solutions). For E. coli CutA1, the solution contained 0.1 m Hepes, pH 7.5, 2.0 m (NH4)2SO4, 2% PEG 400 (polyethylene glycol), and 3 mm 4-(hydroxymercuri)benzoic acid, and for rat CutA1, the solution contained 0.1 m sodium acetate, pH 4.6, 30% 2-methyl-2,4-pentanediol (MPD), 5 mm CuSO4, 20 mm CaCl2. The crystals of CutA1 both from E. coli and from rat were flash-frozen under a cold nitrogen stream at 100 K without the addition of cryoprotectants. Data Collection and Structure Determination—A 2.1-Å resolution MAD data set at the mercury edge was collected at 100 K on CutA1 from E. coli using synchrotron radiation (European Molecular Biology Laboratory PX beamline BW7B at the DORIS storage ring, DESY, Hamburg, Germany). Furthermore, a monochromatic data set at 0.93 Å was collected at the same beamline on a crystal that diffracted up to 1.7 Å resolution. A monochromatic data set at 0.93 Å was collected at 100 K on rat CutA1 using synchrotron radiation (European Synchrotron Radiation Facility ID14-1 beamline, Grenoble, France). The crystals diffracted up to 2.1 Å resolution. All the data sets were processed using MOSFLM 6.2 (13Leslie A.G.W. Moras D. Podjarny A.D. Thierry J.-C. Crystallographic Computing V. Oxford University Press, Oxford1991: 50-61Google Scholar, 14Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar) and scaled with SCALA (14Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar, 15Evans P.R. Joint CCP4 and ESF-EACBM Newsletter. 1997; 33: 22-24Google Scholar). The phasing on CutA1 from E. coli was performed with the program SOLVE (16Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3225) Google Scholar). The density modification and a partial chain tracing were performed with the program RESOLVE (16Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3225) Google Scholar). The subsequent exhaustive chain tracing was carried out on the high resolution monochromatic data set with the program ARP/WARP 6.0 (17Perrakis A. Morris R.J.H. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2571) Google Scholar). The structure of rat CutA1 was solved using the molecular replacement method and the software MOLREP (14Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar, 18Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4224) Google Scholar); a monomer of CutA1 from E. coli was used as the model in the rotation and translation function. The rat CutA1 structure shows some degree of disorder in the loop encompassing residues 82-90. In subunit A the electron density for the side chains of residues Tyr-86 and Glu-87 is very weak, and His-84 appears to be present in at least two conformers. Similar disorder of this loop is present in subunits E and F, which prevents the building of side chains of Tyr-86 and Glu-87 in E and of Glu-87 in F. Both structures were then refined, and water molecules were added using REFMAC5 (14Collaborative Computational Project 4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar, 19Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (14023) Google Scholar) and CNS (20Brunger 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 (17023) Google Scholar). Table I shows the data collection and refinement statistics for the two proteins.Table IData collection, MAD phasing and refinement statistics for CutA1 from E. coli and ratData collection statisticsE. coli CutA1Rat CutA1, remote (0.934 Å)Peak (1.005231 Å)Inflex (1.00870 Å)Remote (0.932 Å)High resolution remote (0.932 Å)Space groupP212121Cell dimensions (Å)a = 55.44a = 55.99a = 70.39b = 89.17b = 89.56b = 88.27c = 121.63c = 122.30c = 125.85Resolution (Å)40.0-2.040.0-2.040.0-2.040.0-1.740-2.15Total reflections275,560 (44,905)276,182 (45,035)205,853643,844 (90,452)300,781 (32,115)Unique reflections37,431 (5,921)37,408 (5,922)28,53265,739 (9,337)46,060 (6,646)Completeness91.8 (91.8)91.8 (91.8)92.4 (91.6)97.2 (96.3)100 (100)Anomalous completeness91.6 (90.6)91.5 (90.5)92.4 (91.3)97.1 (96.0)R sym (%)9.8 (32.7)10.0 (38.1)9.5 (23.2)9.1 (51.9)7.3 (47.0)R anom (%)9.5 (15.8)8.9 (16.1)7.9 (19.0)8.0 (26.4)Multiplicity7.3 (7.0)7.3 (7.3)7.2 (6.7)9.7 (9.6)6.5 (4.8)I/σI4.3 (2.2)4.3 (1.9)4.9 (1.7)5.7 (1.1)5.3 (1.7)Refinement statisticsResolution (Å)20-1.720-2.15R cryst (%)20.318.9R free (%)25.526.0Protein atoms4,8315171Ligands113Water molecules343404B protein (Å2)20.438.7B solvent (Å2)24.551.6r.m.s.d. bond length (Å)0.020.03r.m.s.d. bond angles (°)1.992.6Estimate of the coordinate error based on SigmaA plot (Å)0.110.16 Open table in a new tab The program Xtalview/Xfit (21McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2023) Google Scholar) was used for molecular rebuilding and visualization for both structures. The stereochemical quality was assessed using the program PROCHECK (22Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The program DALI (23Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1296) Google Scholar) was used to search the Protein Data Bank for proteins with a similar structure to CutA1. X-ray Absorption Spectroscopy (XAS) Data Collection and Analysis— XAS data were collected at DESY (Hamburg, Germany) at the European Molecular Biology Laboratory bending magnet beam line D2 using a silicon (111) double monochromator for the measurement at the copper edge, with the DESY storage ring operating under normal conditions (4.5 GeV, 90-140 mA). The XAS data were recorded by measuring the Cu-Kα fluorescence using a Canberra 13-element solid-state detector over the energy range from 8735 to 9875 eV using variable energy step widths. In the x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) regions steps of 0.3 and 0.5-1.2 eV were used, respectively. 10 scans were recorded for a total of more than 1.0/0.6 million counts per experimental point and then averaged. The full, k3 weighted, EXAFS spectrum (17-750 eV above E 0 and its Fourier transform calculated over the range 3.0-14.0 Å-1 were compared with theoretical simulations obtained using the set of programs EXCURVE9.20 (24Binsted N. Hasnain S.S. J. Synchrotron Rad. 1996; 3: 185-196Crossref PubMed Scopus (124) Google Scholar). The quality of the fit was assessed by the fit function through the parameter ϵ2 (25Lytle F.W. Sayers D.E. Stern E.A. Phys. Rev. B. 1989; 11: 2795-2801Google Scholar) and by the R-factor as defined within EXCURVE9.20 (24Binsted N. Hasnain S.S. J. Synchrotron Rad. 1996; 3: 185-196Crossref PubMed Scopus (124) Google Scholar). Ortholog and Paralog Analysis of CutA1 Sequences—In E. coli CutA1 is encoded by one of the three genes of the cutA locus, which is involved in tolerance to Cu2+ and other heavy metal ions (1Fong S.T. Camakaris J. Lee B.T. Mol. Microbiol. 1995; 15: 1127-1137Crossref PubMed Scopus (98) Google Scholar). A search of gene data banks located numerous sequences similar to CutA1 from E. coli in a large variety of organisms (36 bacteria, 13 Archaea, and 9 eukaryotes). They are reported in Supplemental Fig. 1. The second gene of the cutA locus encodes the transmembrane electron transporter CutA2. A gene homologous to CutA2 is found adjacent to CutA1 in a group of bacterial species (Yersinia pestis, Salmonella typhimurium, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas axonopodis, Xylella fastidiosa). For some of them (Y. pestis and S. typhimurium) the CutA1 sequence is highly similar to the E. coli protein (>60% identity). Moreover, in the same group of organisms a third gene, called CutA3 and belonging to the cutA locus, is found next to CutA2. In E. coli, CutA1 and CutA2 are in different operons but implicated together in divalent cation tolerance (1Fong S.T. Camakaris J. Lee B.T. Mol. Microbiol. 1995; 15: 1127-1137Crossref PubMed Scopus (98) Google Scholar). The function of CutA3 is not known, but a search of similar sequences suggests it is a putative transcriptional regulator. CutA1 also exists in all the eukaryotic genomes sequenced up to now, except yeast, with all the sequences sharing a high degree of identity except in the N-terminal part. In CutA1 of mammals the N-terminal sequence is hydrophobic and might represent either a cleavable secretion signal, a mitochondrial import signal, or a transmembrane anchor (2Perrier A.L. Cousin X. Boschetti N. Haas R. Chatel J.M. Bon S. Roberts W.L. Pickett S.R. Massoulie J. Rosenberry T.L. Krejci E. J. Biol. Chem. 2000; 275: 34260-34265Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The bacterial protein is devoid of this hydrophobic domain, and CutA1 is cytoplasmic in E. coli. The rat sequence contains an additional Pro-rich stretch of 12 residues (to which we assign negative residue numbers) at the N terminus, and 6 more residues at the C terminus. Excluding the N-terminal sequence, CutA1 from E. coli and rat have 35% residue identity, which increases to 55% when conservative substitutions are considered. Interestingly, in rat and mouse two further genes are present whose sequences are very similar (80% residue identity). These proteins lack the highly conserved Cys-39, which is replaced by a Ser, and share only 40% residue identity with their paralog sequences containing Cys-39. Protein Characterization—The expressed CutA1 proteins from both organisms turned out soluble and stable for concentrations up to 3 mm in various buffers. The extinction coefficient at 280 nm was determined on the basis of the amino acidic compositions of the proteins and was 17,900 cm-1m-1 for E. coli and 16,500 cm-1m-1 for rat CutA1. The molecular weight, as measured from electrospray mass spectra in denaturing conditions, was 11,841 ± 1 Da for E. coli CutA1, corresponding to residues 5-112, and 13,893 ± 1 Da for rat CutA1, corresponding to residues -12 to 118. On the other hand gel filtration chromatography at neutral pH gives an apparent molecular mass of 35,300 ± 400 Da for E. coli CutA1, which indicates that the protein is in a trimeric state (Fig. 1). The foldedness and the aggregation state were assessed through 1H,15N heteronuclear single quantum coherence NMR spectra, which showed well dispersed signals in both dimensions, indicative of a well folded protein (Supplemental Fig. 2). Furthermore, a signal line width analysis carried out on the 1H,15N heteronuclear single quantum coherence spectrum of CutA1 indicates that line widths are essentially the same for all the signals, with an average value of 32 ± 3Hz, which is comparable with a value of 30 ± 4Hz found for dimeric human Cu,Zn-superoxide dismutase (153 amino acids, ∼32 kDa) and different, outside the experimental error, from that of monomeric superoxide dismutase (∼16 kDa), which is found to be 17 ± 2 Hz. All these data are consistent beyond any uncertainty with a trimeric state of the protein in solution. Structure Analysis, a Trimeric Assembly of Ferredoxin-like Subunits—The structure of E. coli CutA1 consists of homotrimers displaying approximately a c3v symmetry. In the crystal asymmetric unit, two homotrimers are present that make extensive contacts leading to a dimer of trimers. One trimer is rotated by 60° with respect to the other one around the axis perpendicular to the trimer plane (Fig. 2A). The interface between the two trimers is about 3000 Å2, whereas the total interacting surface between each pair of monomers in the trimer is greater than 6000 Å2, giving some evidence that the interactions within the trimer are more specific than those between the two trimers. This is consistent with the observation that in solution the protein exists as a trimer (Fig. 2C). The overall structure of rat CutA1 clearly resembles that of CutA1 from E. coli; the space group is the same, and the asymmetric unit contains six subunits in both cases. However, in rat CutA1 the second trimer is rotated by 25° with respect to the first one around the ternary axis (Figs. 2, B and D). The a axis of the cell is about 15 Å larger in the case of rat CutA1, consistent with the rat protein construct having 18 residues more than the E. coli protein. However, the longer N terminus in the rat protein cannot be resolved as the interpretable electron density of rat CutA1 crystals starts at residue Gly-3, whereas for E. coli CutA1 starts at residue Ser-7. The r.m.s.d. of Cα atoms between E. coli and rat proteins as trimers is 0.95 Å, which confirms a high degree of homology between them, with the larger deviations located in the N- and C-terminal regions. The following analysis applies to both E. coli and rat CutA1. Each monomer exhibits the same overall structure, adopting a ferredoxin-like fold made of an α-β sandwich with antiparallel β-sheet (SCOP classification) (26Lo Conte L. Ailey B. Hubbard T.J. Brenner S.E. Murzin A.G. Chothia C. Nucleic Acids Res. 2000; 28: 257-259Crossref PubMed Scopus (544) Google Scholar) and containing an additional short strand (β5) and a C-terminal helix (α3). In the β-sheet, alternate strands are connected by helices with positive crossovers, resulting in a double βαβ motif where the antiparallel β-sheet packs against antiparallel α-helices. The C-terminal helix packs orthogonal to the N terminus. The β-strands 2 and 3 are connected by an extended β-hairpin (residues Gly-45-Glu-61 in E. coli and Ile-46-Glu-60 in rat CutA1) with a Gly at the apex (Gly-54 in E. coli and in rat CutA1). The β-hairpin includes a number of residues conserved among all the species (Ser-48, Glu-61, and two aromatic residues, Tyr-50 and Trp-52; residue numbering refers to the E. coli CutA1 sequence). Mapping of residue conservation on the structure of the CutA1 monomer, shown in Fig. 3A, also highlights a loop region between α2 and β4, spatially close to the β-hairpin and encompassing conserved residues His-84, Tyr-86, and Glu-90. The other highly conserved amino acids are Ala-40 and Cys-39 on β2, Lys-67 on β3, and Trp-106 on α3. These four residues are clustered at the other end of the scaffold. Least squares superposition of the three E. coli CutA1 monomers shows that there are small but significant differences among them, mainly located in the β-hairpin loop (r.m.s.d. of all Cα atoms 0.43-0.56 Å, compared with an estimated error on the coordinates of 0.11 Å). The same occurs for the rat CutA1 monomers (r.m.s.d. of all Cα atoms is 0.32-0.77 Å compared with an estimated error of 0.16 Å). The formation of inter-subunit β-sheets is the primary force driving trimer assembly. Hydrogen-bond pairing occurs between the N-terminal half of strand β2 from one subunit and the C-terminal half of strand β2 from another subunit and between strand β4 from one subunit and the short strand β5 from another subunit. These interactions result in three six-stranded anti-parallel β-sheets in the trimer. The β-sheets pack around the 3-fold axis and form 3 concave surfaces (see Fig. 3B). The β-hairpin (β2-Loop3-β3) acts as a recognition/oligomerization site because it protrudes from the body of the structure, thus favoring inter-subunit hydrogen bond formation. In addition to the inter-subunit β-sheets, some conserved contacts between two CutA1 subunits (e.g. a salt bridge between Lys-67 and Glu-90) stabilize the trimer. The three monomers assemble such that the two highly conserved regions in the monomers are brought together, thus forming three potential functional sites per trimer (Fig. 3, B and C). A similar trimeric organization is observed in the crystal structure of copper(II)-nitrosocyanin, a trimer of single domain cupredoxins, where each copper center was partially covered by an unusual extended β-hairpin structure from an adjacent monomer (27Lieberman R.L. Arciero D.M. Hooper A.B. Rosenzweig A.C. Biochemistry. 2001; 40: 5674-5681Crossref PubMed Scopus (71) Google Scholar). The electrostatic potential surface of the E. coli CutA1 monomer is shown in Fig. 4A, top panel. One face of the protein is largely neutral, whereas the other contains several charged residues producing regions either with negative or positive electrostatic potential. In particular, four Glu residues, also present in the rat protein, produce a large negative potential on the β-hairpin. The rest of the surface shows some scattered negative and positive charges. The trimer-trimer interface is mainly non-polar and hydrophobic, whereas the opposite side of the trimers is negatively charged and exposed to the solvent (Fig. 4A, bottom panel). The clefts occurring at the trimer interface are lined with conserved acidic residues because they are partially formed by the negatively charged β-hairpins. Sideways, the trimer has a ridge of both positive and negative charges. In rat CutA1 monomers, the neutral region characterizing one face of the protein is somewhat reduced compared with E. coli CutA1. The other face shows a more marked separation between a negative and a positive half, with the latter more extended than in E. coli CutA1 (Fig. 4B, top panel). In the trimer of rat CutA1, the non-polar face has a negative cavity not present in the E. coli protein, whereas the other face is entirely charged (Fig. 4B, bottom panel). As found in E. coli CutA1, negative and positive regions from different subunits are in contact, thus stabilizing the trimer through electrostatic interactions throughout its perimeter. The properties of the electrostatic potential surface of CutA1 trimers suggest very different interaction mechanisms for the apolar face, which interact with the same face of a second trimer, and for the negatively charged face, which is exposed to the solvent. The interaction among the trimers observed in the crystals might mimic a functional property of the hydrophobic surface. Metal Binding
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