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• W2059606622 abstract "Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35Å of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*. Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35Å of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*. Copper is an essential nutrient for all living organisms, and molecular systems have been developed by all cells to maintain adequate supplies of copper. Copper-containing proteins play a key role in cellular respiration and are involved in biological processes such as pigment formation, neurotransmitter synthesis, and antioxidant defense. Recently, they have received increasing attention due to their role in human pathogenesis, in particular in neurodegenerative diseases such as Alzheimer, Parkinson, and prion diseases (1Gaggelli E. Kozlowski H. Valensin D. Valensin G. Chem. Rev. 2006; 106: 1995-2044Crossref PubMed Scopus (1366) Google Scholar).In Methylococcus capsulatus (Bath) and other methane-oxidizing bacteria, copper is important for both regulation and catalytic activity of the particulate methane-monooxygenase (2Hakemian A.S. Rosenzweig A.C. Annu. Rev. Biochem. 2007; 76: 223-241Crossref PubMed Scopus (275) Google Scholar). An equivalent of this enzyme is used by almost all methanotrophs to catalyze the oxidation of methane to methanol, the initial and obligate step for all carbon fixation and energy production in these bacteria. A subset of methanotrophs, including M. capsulatus, produces a second soluble monooxygenase. Soluble monooxygenase does not require copper for activity and is produced only when the level of copper in the growth medium is very low (3Choi D.W. Kunz R.C. Boyd E.S. Semrau J.D. Antholine W.E. Han J.I. Zahn J.A. Boyd J.M. de la Mora A.M. DiSpirito A.A. J. Bacteriol. 2003; 185: 5755-5764Crossref PubMed Scopus (162) Google Scholar, 4Murrell J.C. McDonald I.R. Gilbert B. Trends Microbiol. 2000; 8: 221-225Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The particulate methane-monooxygenase is the most growth effective of the monooxygenases, and hence, copper is required at relatively high levels for methanotrophs to grow optimally (3Choi D.W. Kunz R.C. Boyd E.S. Semrau J.D. Antholine W.E. Han J.I. Zahn J.A. Boyd J.M. de la Mora A.M. DiSpirito A.A. J. Bacteriol. 2003; 185: 5755-5764Crossref PubMed Scopus (162) Google Scholar). Copper is most likely actively accumulated from the growth medium (3Choi D.W. Kunz R.C. Boyd E.S. Semrau J.D. Antholine W.E. Han J.I. Zahn J.A. Boyd J.M. de la Mora A.M. DiSpirito A.A. J. Bacteriol. 2003; 185: 5755-5764Crossref PubMed Scopus (162) Google Scholar), and thus, the copper-homeostatic activity of methanotrophs differs from that of other prokaryotes in which systems handling extracellular copper is mainly involved in detoxification and elimination (5Rensing C. Grass G. FEMS Microbiol. Rev. 2003; 27: 197-213Crossref PubMed Scopus (532) Google Scholar).Both methanobactin and the MopE protein have been postulated to have a role in copper uptake in M. capsulatus (6Karlsen O.A. Berven F.S. Stafford G.P. Larsen O. Murrell J.C. Jensen H.B. Fjellbirkeland A. Appl. Environ. Microbiol. 2003; 69: 2386-2388Crossref PubMed Scopus (30) Google Scholar, 7Kim H.J. Graham D.W. DiSpirito A.A. Alterman M.A. Galeva N. Larive C.K. Asunskis D. Sherwood P.M. Science. 2004; 305: 1612-1615Crossref PubMed Scopus (247) Google Scholar). Methanobactin is a small, siderophore-like compound that binds a single copper ion with high affinity (7Kim H.J. Graham D.W. DiSpirito A.A. Alterman M.A. Galeva N. Larive C.K. Asunskis D. Sherwood P.M. Science. 2004; 305: 1612-1615Crossref PubMed Scopus (247) Google Scholar). When copper is present in the growth medium, methanobactin is mainly associated with the membranes, possibly in direct association with the particulate methane-monooxygenase, whereas at copper-limited growth conditions, methanobactin accumulates in the growth medium. The MopE protein was originally identified as one of five outer membrane-associated proteins, designated MopA-E, Mop being short for M. capsulatus outer membrane protein (8Fjellbirkeland A. Kleivdal H. Joergensen C. Thestrup H. Jensen H.B. Arch. Microbiol. 1997; 168: 128-135Crossref PubMed Scopus (24) Google Scholar). Later it was discovered that an N-terminal-truncated version (MopE*) of the cell surface-associated protein (MopEc) was secreted in large amounts to the growth medium under copper-limited growth conditions (6Karlsen O.A. Berven F.S. Stafford G.P. Larsen O. Murrell J.C. Jensen H.B. Fjellbirkeland A. Appl. Environ. Microbiol. 2003; 69: 2386-2388Crossref PubMed Scopus (30) Google Scholar, 9Fjellbirkeland A. Kruger P.G. Bemanian V. Hogh B.T. Murrell J.C. Jensen H.B. Arch. Microbiol. 2001; 176: 197-203Crossref PubMed Scopus (18) Google Scholar). MopEc is composed of 512 amino acids, whereas MopE* represents the 336 C-terminal amino acids of MopEc. A MopE* homologue, designated CorA, has been isolated from the membranes of the methanotroph Methylomicrobium album BG8 (10Berson O. Lidstrom M.E. FEMS Microbiol. Lett. 1997; 148: 169-174Crossref PubMed Google Scholar). The expression of both MopE and CorA is negatively regulated by copper, indicating an involvement in copper-homeostatic activities. Importantly, a copper binding motif could not be predicted with significance from the primary sequence of the two proteins (9Fjellbirkeland A. Kruger P.G. Bemanian V. Hogh B.T. Murrell J.C. Jensen H.B. Arch. Microbiol. 2001; 176: 197-203Crossref PubMed Scopus (18) Google Scholar, 10Berson O. Lidstrom M.E. FEMS Microbiol. Lett. 1997; 148: 169-174Crossref PubMed Google Scholar).In the present study we determined the crystal structure of MopE* to 1.35 Å of resolution and demonstrate that the protein has a novel protein fold and contains a mononuclear copper site. Importantly, we present evidence that one of amino acids in the copper-binding site is kynurenine, a tryptophan derivate not previously described in protein metal-binding sites.EXPERIMENTAL PROCEDURESGrowth Conditions and Purification of MopE*—MopE* was obtained from spent medium of M. capsulatus (Bath) strain NCIMB 11132 grown in continuous cultures in nitrate mineral salts medium containing no added copper, as described previously (6Karlsen O.A. Berven F.S. Stafford G.P. Larsen O. Murrell J.C. Jensen H.B. Fjellbirkeland A. Appl. Environ. Microbiol. 2003; 69: 2386-2388Crossref PubMed Scopus (30) Google Scholar), and purified as described in the supplemental information.Protease Treatment and Mass Spectrometry Analyses—MopE* was digested with Lys-C endoproteinase (Roche Applied Science) (11Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7765) Google Scholar, 12Gobom J. Nordhoff E. Mirgorodskaya E. Ekman R. Roepstorff P. J. Mass Spectrom. 1999; 34: 105-116Crossref PubMed Scopus (627) Google Scholar). Mass spectrometric analyses (MALDI-TOF 2The abbreviations used are: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectroscopy; ICP, inductively coupled plasma. MS and MS/MS) were performed at PROBE, University of Bergen. A detailed description is given in the supplemental information.Metal Determination—The amount of copper bound per MopE* molecule was determined by inductively coupled plasma mass spectrometry (ICP-MS) at the Center for Element and Isotope Analyses, University of Bergen, Norway (see the supplemental information).The Copper Binding Affinity of MopE—Information on the dissociation constant of copper binding to MopE* was obtained using the Cu(I) chelator bathocuproine. Bathocuproine disulfonic acid (Sigma) and ascorbate were added to MopE* (∼10 μm) in 20 mm Tris-HCl (pH 7.5) and 1 mm CaCl2 to final concentrations of 0.5 and 1 mm, respectively. Ascorbate was included in the assay to ensure that the copper ions were maintained in the Cu(I) state. Samples were incubated either at room temperature or at 45 °C for 1 h with gentle shaking every 5 min. The protein was subsequently isolated using a 5-ml Hitrap desalting column (GE Healthcare), and the concentration of copper in the protein fraction was determined by ICP-MS analyses.Cloning, Expression, and Purification of MopE* in Escherichia coli—MopE* was amplified by PCR and cloned into the pETM41 vector using BamHI and NcoI restriction sites. Large-scale protein expression was performed using E. coli BL21 Star™ (DE3) and the pETM41 vector. Purification of MopE* is described in the supplemental information.Crystallization, Data Collection, Structure Determination, and Refinement—All variants of MopE* were crystallized from ammonium sulfate in the pH range 7.0–7.75. Data were collected at 100–120 K, and the data were processed in XDS (14Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3213) Google Scholar), MOSFLM (13Powell H.R. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 1690-1695Crossref PubMed Scopus (304) Google Scholar), and SCALA and TRUNCATE of the CCP4 suite (15Collaborative Computational ProjectActa Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19702) Google Scholar). The structure was phased by single anomalous dispersion techniques using SHELXD (16Schneider T.R. Sheldrick G.M. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1574) Google Scholar) on a mercury derivative. The phases were improved by SHARP (17De la Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1796) Google Scholar) and solvent flattening using SOLOMON (18Abrahams J.P. Leslie A.G. Acta Crystallogr. D Biol. Crystallogr. 1996; 52: 30-42Crossref PubMed Scopus (1140) Google Scholar). ARP/wARP (19Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2562) Google Scholar) was used for automatic tracing of the protein, and the model was further improved by refinement using Refmac5 (21Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13774) Google Scholar) and manual refitting of the model using O (20Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar). The structures of MopE* and recombinantly expressed in E. coli (Rec-MopE*) were solved by molecular replacement using MOLREP (22Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4118) Google Scholar). A more detailed description of the MopE* structure determination is presented in the supplemental information, and data collection and refinement statistics are summarized in Table 1.TABLE 1Data collection and refinement statistics Values for outer shell are indicated in parenthesisHg-MopE*MopE*Rec-MopE*Data collection BeamlineBessy, BL14ESRF, BM01Bessy, BL14 Wavelength1.00850.87220.9184 DetectorMAR CCD165MAR CCD165MAR CCD225 Space groupC2I222C2 Diffraction limit1.91.351.65 No. molecules in asymetric unit111 Unit cell parameters a axis (Å)65.9272.9965.28 b axis (Å)101.2288.57100.67 c axis (Å)54.78101.4354.65 β-Angle (°)98.2490.0097.56 Total no. of reflections367,401 (53,094)368,670 (52,775)151,184 (15,832) No. of unique reflections27,480 (3,963)71,009 (10,134)41,358 (5,489) Completeness (%)98.2 (97.3)98.5 (97.3)98.4 (89.9) I/σ(I)8.4 (1.3)8.6 (1.6)7.5 (2.3) Mean ((I)/S.D. (I))24.7 (5.5)19.4 (3.0)13.6 (3.3) Rmerge (%)6.9 (55.4)4.5 (47.6)5.3 (33.4) Multiplicity13.4 (13.4)5.2 (5.2)3.7 (2.9) Wilson B (Å2)26.7714.1020.14 Outer shell2.00-1.901.42-1.351.74-1.65Structure determination Phasing power0.91 RCullis0.85 Figure of merit0.25Refinement Rwork (%)22.1119.2718.14 Rfree (%)24.4821.1320.39 Average B factors (Å2)31.0016.7021.38 Root mean square deviations Bond lengths (Å)0.0220.0070.013 Bond angles (°)2.3471.1411.348 Diffraction-component precision indicator0.13440.05550.0834 Ramachandran plot In most favored regions (%)85.287.387.3 In additional allowed regions (%)13.912.212.2 In generously allowed regions (%)0.80.40.4 Open table in a new tab RESULTSThe MopE* Structure—Crystals suitable for x-ray diffraction studies, with different morphology and cell parameters belonging to monoclinic (C2) and orthorhombic (I222) space groups, were obtained (Table 1 and supplemental Fig. S1). The crystal structure of MopE* was determined using single anomalous dispersion data to 1.9 Å collected on a HgCl2 derivative (Hg-MopE*). The structures of the wild-type protein (MopE*) and MopE* recombinantly expressed in E. coli (Rec-MopE*) were determined to 1.35 and 1.65 Å, respectively, by molecular replacement using the mercury derivative as starting model. Data collection and refinement statistics are provided in Table 1. The three structures of MopE* are essentially similar but with a significant difference in the metal-binding site.The present structure (Fig. 1) includes the 290 C-terminal residues of the protein, and the structures have been refined to crystallographic R-factors of about 18–21% (Rwork) and Rfree of 20–24% (Table 1). Main chain atoms of MopE* and Rec-MopE* residues 47–336 superimpose on each other with a root mean square deviation value of 0.57 Å. The differences are caused by the loops formed by residues 75–80 (β2–β3 loop), 105–110 (β3–β4 loop), 115–119 (β4–β5 loop), and 308–312 (β19–β21 loop). Different crystal packing environments in the β2–β3 and β4–β5 loops and poorly defined electron density in the solvent-exposed β19–β20 loops of both proteins are expected to cause the different conformations in three of the four loops. Only the conformation of the β3–β4 loops is expected to be caused by differences in the metal-binding sites of the wild-type and recombinant proteins. The 46 N-terminal amino acids of MopE* were not identified in the electron density maps in any of the structures.The protein forms a nest-like structure with dimensions of about 40 × 45 × 55 Å3 (Fig. 1, A and C). The majority of the MopE* structure folds into a coiled structure where only a third of the residues forms 21 β-strands (Fig. 1B). Eight of these build the only extensive secondary structure motif, an antiparallel β-sandwich. The MopE* structure also contains four 310 helical segments. The polypeptide is folded such that regions of the N-terminal, the middle, and C-terminal parts all are included in the β-sandwich (Fig. 1). One of the strands of the sandwich extends into a second 3-stranded sheet such that the central core of the protein is a 6-stranded sheet. Of the remaining β-strands, seven consist of only two residues each being involved in stabilizing the MopE* structure. The structure is also stabilized by a calcium ion octahedrally coordinated by three aspartic acid residues (Asp-250, Asp-252, Asp-274), two main chain carbonyl oxygens (Thr-276, Ala-279), and a solvent molecule (Fig. 1).The copper-binding site contains a single partially buried copper ion (Fig. 1C) located in a trigonal arrangement by the side groups of His-132, His-203, and intriguingly, Trp-130. Tryptophan has been shown to bind various metals in chemical systems (23Kaminskaia N.V. Kostic N.M. Inorg. Chem. 2001; 40: 2368-2377Crossref PubMed Scopus (52) Google Scholar, 24Takani M. Takeda T. Yajima T. Yamauchi O. Inorg. Chem. 2006; 45: 5938-5946Crossref PubMed Scopus (22) Google Scholar); the indole side chain has not been reported to coordinate metal ions in biological systems. The electron density maps revealed a broken bond between the CD1 and NE1 atoms of Trp-130 (Fig. 2). In addition, the planar arrangement around the CG atom and the distances from the atom at the CD1 position to the main chain amine nitrogen atoms of residues 130, 131, and 132 suggested oxidation of the tryptophan to kynurenine. Kynurenines are formed by oxidative cleavage of the tryptophan indole ring with subsequent hydrolysis of the CD1 carbon, resulting in a mass increase of 4 Da compared with the unmodified tryptophan (Fig. 3B and supplemental Fig. S2).FIGURE 2Electron density maps of the copper-binding site of MopE*. The copper and the coordinating water, or hydroxyl, are illustrated as yellow and red spheres, respectively. The 2fofc electron density is contoured at 1σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3MALDI-MS spectra. A, MALDI-MS spectra of Lys-C produced peptides from MopE* (upper) and Rec-MopE* (lower) isolated from M. capsulatus and E. coli, respectively. Monoisotopic peaks are labeled with their respective m/z ratio. B, MALDI spectra (mass range 2783–2796 Da) of A) showing the m/z 2789 (upper) and m/z 2785 (lower) ions. Right-hand side, chemical structures of kynurenine and tryptophan. C, tandem mass spectra of the m/z 2789 ion (left panel) and the m/z 2785 ion (right panel), indicating the observed fragmentation pattern and the sequence ion assignments. Predicted ions are shown at top of the figures, where a 4-Da increase in mass at Trp-130 is considered for the m/z 2789 ion. The oxidized tryptophan is displayed as kyn in the amino acid sequence of the 2789-Da peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The copper ion is also coordinated by an axial water molecule, forming a distorted tetrahedral arrangement of the copper-binding site with the copper ion located almost in the middle of the base of the pyramid and the solvent molecule in the apical position (Fig. 2). Trigonal, tetrahedral, and trigonal bipyramidal are all geometries previously observed for copper-binding proteins (25Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2325) Google Scholar). The distances between the copper ion and the ND1 atoms of the histidines are in the range 1.99–2.17 Å, indicating covalent/ionic interactions. This is also in the same order as copper-histidine distances found in most other copper-binding proteins (26Harding M.M. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 678-682Crossref PubMed Scopus (203) Google Scholar, 27Rulisek L. Vondrasek J. J. Inorg. Biochem. 1998; 71: 115-127Crossref PubMed Scopus (328) Google Scholar). The distance between the copper ion and the nitrogen atom of the kynurenine is about 3 Å, somewhat longer than what is usually considered a copper-nitrogen interaction. The amino group of the kynurenine side chain would generally be considered a poor cooper ligand. The amino group is ∼6 degrees off a linear phenyl-amino-copper interaction and, as such, does not coincide with a possible amino-metal interaction previously described (25Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2325) Google Scholar). The copper to solvent distance in MopE* refines to about 2.5 Å, similar to what is found in other copper proteins. The occupancy of the copper ion is refined to about 0.65, which corresponds well with the ICP-MS analyses on purified MopE* revealing a copper-to-protein ratio between 0.5 and 0.6.Identification of Kynurenine by Mass Spectrometry—To verify the oxidation of tryptophan to kynurenine, purified MopE* was digested with Lys-C, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. Computer-generated Lys-C peptide maps of MopE* calculated theoretical m/z ions of 2785 and 2789 for tryptophan and kynurenine, respectively. The MS analyses revealed a MALDI molecular ion at m/z 2789, consistent with the theoretically predicted ion for the kynurenine-containing peptide (Fig. 3, A and B, upper panels). Subsequent fragmentation of the m/z 2789 ion produced an MS/MS spectrum corresponding to the amino acid sequence Trp-112—Lys-135 of MopE* (Fig. 3C, left panel). From both the y-ion and b-ion series it was possible to identify a mass increase of 4 Da at residue 130. No distinct peak was observed at m/z 2785 (Fig. 3, A and B, upper panels).Analyses of Recombinant MopE*—Crystallization and MS studies were also carried out on MopE* heterologously expressed and purified from E. coli (Rec-MopE*). The MS spectrum and MS/MS fragmentation pattern on peptides derived from Rec-MopE* revealed solely the unmodified m/z 2785 peptide, demonstrating that the post-translational formation of kynurenine in MopE* had not occurred when expressed in E. coli (Fig. 3, A and B, lower panels, and C, right panel). This was confirmed by crystal structure analysis of Rec-MopE*. The electron density clearly displayed an intact CD1–NE1 bond (Fig. 4A). In addition, the side chain was rotated about 60 degrees in χ1 and 180 degrees in χ2 relative to kynurenine in wild-type MopE* (Fig. 4B). To accommodate the tryptophan side chain in this position, the β3–β4 loop (residues 105–110) is displaced about 1.5 Å. Thus, the metal-binding site is sufficiently disrupted to prevent binding of copper in Rec-MopE*, as demonstrated by the lack of electron density at the wild-type copper position. The latter was confirmed by ICP-MS; Rec-MopE* did not bind detectable levels of copper. These findings substantiate that the conversion of tryptophan to kynurenine is an endogenous modification that specifically takes place in M. capsulatus and that the oxidation of Trp-130 to kynurenine is a prerequisite for copper binding in wild-type MopE*.FIGURE 4A, electron density maps covering the residues forming the copper-binding site of Rec-MopE*. The 2fofc electron density is contoured at 1σ and shows that Trp-130 is in its unoxidized state. B, MopE* (yellow) superimposed on Rec-MopE* (green). Trp-130 in Rec-MopE* is rotated relative to Kyn130 in MopE. The copper found in MopE* is illustrated as a yellow sphere.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Copper Binding Affinity of MopE*—Bathocuproine disulfonic acid was used to obtain information on the copper binding affinity of MopE*. Bathocuproine disulfonic acid forms a stable 2:1 complex with Cu(I) with an association constant of ∼1020 (28Xiao Z. Loughlin F. George G.N. Howlett G.J. Wedd A.G. J. Am. Chem. Soc. 2004; 126: 3081-3090Crossref PubMed Scopus (219) Google Scholar). No significant decrease in the amount of copper bound to MopE* could be observed after treatment with the copper chelator either at room temperature or at 45 °C, thus indicating a high affinity binding (Kd < 10-20 m).DISCUSSIONWe present here x-ray diffraction and mass spectrometry data on the M. capsulatus-secreted protein, MopE*. The protein presents a unique kynurenine-containing copper-binding site and a novel protein fold, of which only about a third of the MopE* structure displays similarity to known proteins. The only extensive secondary structure element previously observed is an 8-stranded antiparallel β-sandwich formed by about 25% of the residues. This motif is often found in virus capsid proteins and sugar binding and hydrolyzing proteins as well as in oxidoreductases, cupredoxins (proteins with Type I copper center), and proteins involved in electron transport (DALI data base (29Holm L. Sander C. Science. 1996; 273: 595-603Crossref PubMed Scopus (1283) Google Scholar), ProFunc (30Laskowski R.A. Watson J.D. Thornton J.M. Nucleic Acids Res. 2005; 33: W89-W93Crossref PubMed Scopus (496) Google Scholar), and CATH protein classification data base (31Orengo C.A. Michie A.D. Jones S. Jones D.T. Swindells M.B. Thornton J.M. Structure. 1997; 5: 1093-1108Abstract Full Text Full Text PDF PubMed Google Scholar)). The remaining residues form an extensive coil-like structure interspersed with 13 short β-strands. Particularly interesting is the copper-binding site located in a depression on the molecular surface about 8–10 Å from the β-sandwich and relatively distant from any secondary structure element. The partially buried copper ion is located between two histidines and an oxidized tryptophan (kynurenine) in a planar trigonal arrangement and displays, therefore, similarities to Type I (blue copper proteins) and CuB copper centers (25Holm R.H. Kennepohl P. Solomon E.I. Chem. Rev. 1996; 96: 2239-2314Crossref PubMed Scopus (2325) Google Scholar, 32Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2606Crossref PubMed Scopus (3133) Google Scholar). Type I copper centers are characterized by two histidines and a cysteine in a planar trigonal arrangement and a variable axial ligand. CuB copper centers coordinate copper by three histidines in a trigonal pyramidal geometry, and the axial ligand forms a bridge to the iron ion of the cytochrome heme. Two and three histidine ligands are frequently observed in copper-binding proteins, but to our knowledge this is the first observation of a tryptophan or a tryptophan metabolite involved in metal binding.When previously detected in proteins, kynurenine was considered the result of oxidative damage, and in most studies on proteins containing modified tryptophan residues, the kynurenine modification was formed by exposing the proteins to oxidative stress in vitro (33Yang C. Gu Z.W. Yang M. Lin S.N. Siuzdak G. Smith C.V. Biochemistry. 1999; 38: 15903-15908Crossref PubMed Scopus (34) Google Scholar, 34Zhang H. Joseph J. Crow J. Kalyanaraman B. Free Radic. Biol. Med. 2004; 37: 2018-2026Crossref PubMed Scopus (31) Google Scholar, 35Manzanares D. Rodriguez-Capote K. Liu S. Haines T. Ramos Y. Zhao L. Doherty-Kirby A. Lajoie G. Possmayer F. Biochemistry. 2007; 46: 5604-5615Crossref PubMed Scopus (52) Google Scholar). There is accumulating evidence that oxidation of specific tryptophan residues takes place in vivo (36Anderson L.B. Maderia M. Ouellette A.J. Putnam-Evans C. Higgins L. Krick T. MacCoss M.J. Lim H. Yates III, J.R. Barry B.A. Proc. Natl. Acad. Sci. U S A. 2002; 99: 14676-14681Crossref PubMed Scopus (50) Google Scholar, 37Ball L.J. Goult C.M. Donarski J.A. Micklefield J. Ramesh V. Org. Biomol. Chem. 2004; 2: 1872-1878Crossref PubMed Scopus (88) Google Scholar, 38Jung D. Rozek A. Okon M. Hancock R.E. Chem. Biol. 2004; 11: 949-957Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 39Moller I.M. Kristensen B.K. Free Radic. Biol. Med. 2006; 40: 430-435Crossref PubMed Scopus (61) Google Scholar, 40Taylor S.W. Fahy E. Murray J. Capaldi R.A. Ghosh S.S. J. Biol. Chem. 2003; 278: 19587-19590Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), but evidence for biological functions of kynurenines in proteins has to our knowledge not been presented. Structural evidence by NMR for naturally occurring kynurenine has been provided only for the antibiotic peptide daptomycin (37Ball L.J. Goult C.M. Donarski J.A. Micklefield J. Ramesh V. Org. Biomol. Chem. 2004; 2: 1872-1878Crossref PubMed Scopus (88) Google Scholar, 38Jung D. Rozek A. Okon M. Hancock R.E. Chem. Biol. 2004; 11: 949-957Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) from Streptomyces roseosporus. In other studies on kynurenine-containing proteins, kynurenine was detected by MS analyses, and for all proteins examined the MS spectra revealed two distinct peaks with m/z corresponding to oligopeptides containing both unmodified and modified tryptophan residues (35Manzanares D. Rodriguez-Capote K. Liu S. Haines T. Ramos Y. Zhao L. Doherty-Kirby A. 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