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• W1999660388 abstract "The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal α-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX18H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate. The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal α-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX18H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate. It is generally accepted that thermal adaptation of proteins is correlated with changes in their overall or local structure flexibility. Indeed, although thermophilic enzymes are characterized by a relatively high rigidity, their psychrophilic homologues display molecular characteristics enhancing their plasticity. The current understanding is that this increased flexibility, by enhancing accommodation and transformation of their substrates, allows psychrophilic enzymes to be active at low temperature. To identify features that may be important for cold adaptation, the ColAP 2The abbreviations used are:ColAPColwellia psychrerythraea aminopeptidaseLTleukotrieneLTA4Hhuman leukotriene A4 hydrolaseAPNaminopeptidase Nbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolr.m.s.d.root mean square deviationGRAVYgrand average of hydropathy. 2The abbreviations used are:ColAPColwellia psychrerythraea aminopeptidaseLTleukotrieneLTA4Hhuman leukotriene A4 hydrolaseAPNaminopeptidase Nbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diolr.m.s.d.root mean square deviationGRAVYgrand average of hydropathy. structure was analyzed and compared in detail with one of its closest structural homologues, the human mesophilic leukotriene A4 hydrolase (LTA4H).A recent study (1Huston A.L. Methe B. Deming J.W. Appl. Environ. Microbiol. 2004; 70: 3321-3328Crossref PubMed Scopus (138) Google Scholar) revealed a high sequence similarity (34% identity and 56% similarity) between cold-active aminopeptidase ColAP and LTA4H. Both enzymes belong to the M1 metallopeptidase family that includes enzymes such as aminopeptidase N (APN), pyroglutamyl-peptidase II, and aminopeptidase A. Interestingly, human LTA4H has the particularity of being bifunctional. Indeed, this enzyme is also an epoxide hydrolase and catalyzes the conversion of leukotriene A4 (LTA4) in LTB4, a chemoattractant implicated in inflammatory mechanisms. Although it is well known that both reactions occur in the same unique active site (2Rudberg P.C. Tholander F. Thunnissen M.M. Haeggstrom J.Z. J. Biol. Chem. 2002; 277: 1398-1404Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the molecular features that make LTA4H bifunctional, even though their homologues are not, are still unknown. In this article, we report the crystal structure of cold-adapted ColAP. This structure has been compared with the three-dimensional structure of its close mesophilic homologue LTA4H, solved at 1.95 Å (3Thunnissen M.M. Nordlund P. Haeggstrom J.Z. Nat. Struct. Biol. 2001; 8: 131-135Crossref PubMed Scopus (253) Google Scholar). Such a study may help not only to improve our understanding of molecular properties leading to cold-adaptation of this enzyme but also to indicate the structural differences that allow the human enzyme to have a second catalytic function.EXPERIMENTAL PROCEDURESProtein Purification—Recombinant ColAP was overproduced in Escherichia coli and purified as described previously (4Huston A.L. Haeggstrom J.Z. Feller G. Biochim. Biophys. Acta. 2008; (in press)PubMed Google Scholar).Crystallization—Crystals of ColAP were obtained after a 1-week incubation at 18 °C by using the hanging drop method. Optimal crystallization conditions were obtained by mixing the protein with an equal volume of reservoir solution composed of polyethylene glycol 3350 (23% w/v), NaCl 0.5 m, and bis-Tris 0.1 m, pH 6.5.Structure Determination and Refinement—Diffraction data were collected at the FIP beamline (French beamline for Investigation of Proteins; BM30A) (5Roth M. Carpentier P. Kaikati O. Joly J. Charrault P. Pirocchi M. Kahn R. Fanchon E. Jacquamet L. Borel F. Bertoni A. Israel-Gouy P. Ferrer J.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 805-814Crossref PubMed Scopus (78) Google Scholar) of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) at 100 K. The data set was processed using the XDS program (6Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3214) Google Scholar). The precision-indicating merging R-factor (Rpim) and the redundancy-independent merging R-factor (Rrim) were calculated using RMERGE (7Weiss M. J. Appl. Crystallogr. 2001; 34: 130-135Crossref Scopus (576) Google Scholar, 8Evans P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3694) Google Scholar) (Table 1).TABLE 1Crystallographic data and refinement statisticsX-ray diffraction dataSpace groupP1Resolution range (Å)46.7-2.7 (2.87-2.7)Cell parametersa = 81.37 Å, b = 87.1 Å, c = 116.4 Å, α = 88.8°, β = 70.7°, γ = 88.4°Unique reflections82,772Completeness (%)97.28 (96.7)I/σ7.27 (2.61)Rpim (%)aRpim = ∑hkl[1/(n - 1)]½ ∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl) (7, 8).9.3 (38.1)Rrim (%)bRrim = ∑hkl[n/(n - 1)]½ ∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl) (7, 8).14.2 (57.1)RefinementProtein atoms18,697Solvent molecules919Rcryst (%)cRcryst = ∑∥Fobs| - |Fcalc∥/∑|Fobs|./Rfree (%)d5% of the data were set aside for the Rfree calculation.24.8/26.7Average B-values for protein (Å2)32.2r.m.s.d. of bonds (Å)/angles (degrees)0.026/2.28Ramachandran plotePercentage of residues in favored/allowed/disallowed regions of the Ramachandran plot. Ramachandran results were determined by using MOLPROBITY (14).86.4/98.8/1.2a Rpim = ∑hkl[1/(n - 1)]½ ∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl) (7Weiss M. J. Appl. Crystallogr. 2001; 34: 130-135Crossref Scopus (576) Google Scholar, 8Evans P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3694) Google Scholar).b Rrim = ∑hkl[n/(n - 1)]½ ∑i|Ii(hkl) - 〈I(hkl)〉|/∑hkl∑iIi(hkl) (7Weiss M. J. Appl. Crystallogr. 2001; 34: 130-135Crossref Scopus (576) Google Scholar, 8Evans P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3694) Google Scholar).c Rcryst = ∑∥Fobs| - |Fcalc∥/∑|Fobs|.d 5% of the data were set aside for the Rfree calculation.e Percentage of residues in favored/allowed/disallowed regions of the Ramachandran plot. Ramachandran results were determined by using MOLPROBITY (14Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3790) Google Scholar). Open table in a new tab The native data set was used for molecular replacement, with the structure of LTA4H as a starting model (Protein Data Bank code 1HS6). Four molecules were found in the asymmetric unit. The electronic density was good enough to build a first model manually using the programs O and COOT (9Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar, 10Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22799) Google Scholar). Then, this first model was subjected to standard simulated annealing using a protocol of torsional dynamics refinement, as implemented in the CNS suite (11Brunger 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 (16929) Google Scholar), followed by energy minimization using the REFMAC5 program (12Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13776) Google Scholar).Structure and Sequence Analysis—Sequence analysis and GRAVY and aliphatic indexes were computed with ProtParam (13Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3312) Google Scholar) tools from the ExPASy proteomics server. Structure quality was evaluated with MOLPROBITY (14Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3790) Google Scholar) and WHAT CHECK (15Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1792) Google Scholar). Polar and apolar exposed surfaces were calculated with the DSSP program (16Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12100) Google Scholar), and hydrogen bonds were defined using HBPLUS software (17McDonald I.K. Thornton J.M. J. Mol. Biol. 1994; 238: 777-793Crossref PubMed Scopus (1858) Google Scholar). Ion pairs were identified as two oppositely charged residues (considering Asp, Glu, Lys, and Arg) having their charges located within 4 Å. Structure superimpositions were performed using DEEPVIEW (18Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9467) Google Scholar). All figures have been generated using PyMOL.RESULTS AND DISCUSSIONOverall Structural Comparisons—As expected from the high sequence similarity, the overall fold and the main secondary structures of ColAP are very similar to those of LTA4H (Fig. 1, b and c). The backbone structures of both enzymes can be superimposed with a root mean square deviation (r.m.s.d.) of 1.21 Å for 509 Cα. These monomeric enzymes are folded into three distinct domains (N-terminal, catalytic, and C-terminal domains) in which the topologies are largely conserved. The N-terminal domain comprises three β-sheets, the major one consisting of seven mixed β-strands and two minor ones consisting of three and four antiparallel β-strands, respectively. Small β-sheets are located under the large sheet at opposite extremities. On the whole, N-terminal domain β-sheets resemble a kind of saddle, presenting its large concave surface to the solvent. As already observed for LTA4H, the architecture of the ColAP catalytic domain is similar to that of thermolysin. It comprises two lobes: one is composed of α-helices only, whereas the other is a mix of α-helices and β-strands. Between the lobes, a depression contains the zinc binding site. The C-terminal domain of ColAP is α-helical. In this domain, eight successive helices are arranged in a right-handed flat spiral. Helices are situated in two layers, five in the inner layer and three in the outer layer, respectively. In the LTA4H enzyme, there is a Pro-rich loop located between the catalytic and C-terminal domains. The sequence of this loop (453LPPIKP458) resembles the Src homology 3-binding domains (XPpXP, where X is an aliphatic residue, P is a conserved proline, and p is sometimes a proline) (19Weng Z. Rickles R.J. Feng S. Richard S. Shaw A.S. Schreiber S.L. Brugge J.S. Mol. Cell. Biol. 1995; 15: 5627-5634Crossref PubMed Scopus (109) Google Scholar). In ColAP, the corresponding sequence is 480LPSYAP485 and thus is moderately conserved. Of the three domains of ColAP and LTA4H, the least conserved is the C-terminal domain. The greatest sequence variations are indeed found in this domain (18% identity for the C-terminal domain versus 38 and 41% for the N-terminal and catalytic domains, respectively) (supplemental Table 1). These differences are reflected in the r.m.s.d. between Cα atoms of the domain structures. Superimposition of the different domains of ColAP and LTA4H gives r.m.s.d. values of 0.83, 1.15, and 1.63 Å based on 716, 920, and 440 involved atoms for the N-terminal, catalytic, and C-terminal domains (supplemental Table 1).Other enzymes share a similar overall fold, such as the Tricorn protease-interacting factor 3 from Thermoplasma acidophilum (20Kyrieleis O.J. Goettig P. Kiefersauer R. Huber R. Brandstetter H. J. Mol. Biol. 2005; 349: 787-800Crossref PubMed Scopus (69) Google Scholar) and the APN from E. coli (21Ito K. Nakajima Y. Onohara Y. Takeo M. Nakashima K. Matsubara F. Ito T. Yoshimoto T. J. Biol. Chem. 2006; 281: 33664-33676Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although APN shares the three equivalent domains described for ColAP and LTA4H, Tricorn protease contains an additional small barrel-like β-structure domain located between the catalytic and C-terminal domains. Interestingly, in these two enzymes the major differences are also found in the C-terminal domains. The discrepancies are so significant that backbone superimposition could not be performed with ColAP.Amino Acid Distribution and Cold Adaptation—A comparison of the amino acid composition of the psychrophilic ColAP with its mesophilic human homologue is shown in Table 2. Although the hydrophobic amino acid content is similar for both enzymes, the number of Ala residues is much higher in ColAP. Interestingly, this increase (∼3.2%) seems to compensate for the decrease observed for Ile, Leu, and Pro residues (∼3.1%). Ala is less hydrophobic than Ile and Leu and less constraining for the backbone than Pro, which suggests a global gain in structure flexibility. The high Ala content and the small side chain of this residue may also contribute to the reduction of the global volume of the psychrophilic ColAP.TABLE 2Proportion of residues and computed chemical parameters in the psychrophilic ColAP and in human LTA4HAmino acid compositionAmino acidsColAPaPeptide signal residues were omitted for calculation.LTA4HFrequency in Swiss-ProtbSee ExPASy Web site.No.FrequencyNo.Frequency%%%Asp467.6345.65.3Glu345.6416.76.7Negatively charged residues80cValues in italic type correspond to the sum of amino acids gathered by type.13.27512.312Arg223.6233.85.4Lys386.3406.65.9His152.5162.62.3Positively charged residues75cValues in italic type correspond to the sum of amino acids gathered by type.12.4791313.6Total charged residues155cValues in italic type correspond to the sum of amino acids gathered by type.25.615425.325.6Asn325.3213.44.1Gln264.3264.33.9Ser345.6457.46.8Thr345.6406.65.4Cys00111.81.5Tyr305.0223.63.0Polar residues156cValues in italic type correspond to the sum of amino acids gathered by type.25.816527.024.7Met132.1101.62.4Leu6310.46911.39.6Ile274.5315.15.9Val396.4386.26.7Pro254.1355.74.8Ala579.4386.27.9Phe264.3274.43.9Trp132.1132.11.1Nonpolar residues263cValues in italic type correspond to the sum of amino acids gathered by type.43.526142.842.3Gly315.1304.97.0Total number of residues605610Computed chemical parametersMolecular mass (Da)68,59369,154Theoretical pI5.255.80Aliphatic index86.288.2GRAVY indexTotal−0.339−0.259Strands0.280.17Helices−0.08−0.21Loops−0.75−0.59a Peptide signal residues were omitted for calculation.b See ExPASy Web site.c Values in italic type correspond to the sum of amino acids gathered by type. Open table in a new tab Table 2 also reveals the substitution of glutamic acid by aspartic acid in ColAP. This is in agreement with the trend observed in the whole genome of Colwellia psychrerythraea (22Methe B.A. Nelson K.E. Deming J.W. Momen B. Melamud E. Zhang X. Moult J. Madupu R. Nelson W.C. Dodson R.J. Brinkac L.M. Daugherty S.C. Durkin A.S. DeBoy R.T. Kolonay J.F. Sullivan S.A. Zhou L. Davidsen T.M. Wu M. Huston A.L. Lewis M. Weaver B. Weidman J.F. Khouri H. Utterback T.R. Feldblyum T.V. Fraser C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10913-10918Crossref PubMed Scopus (402) Google Scholar). By contrast, the Asp content in thermophilic proteins is low, and it has been shown that Asp has less favorable conformational entropy in stability than Glu (23Lee D.Y. Kim K.A. Yu Y.G. Kim K.S. Biochem. Biophys. Res. Commun. 2004; 320: 900-906Crossref PubMed Scopus (22) Google Scholar). Accordingly, the high Asp content in ColAP may contribute to its lower stability and higher flexibility. The Pro content is also significantly lower in ColAP. Five of these substituted Pro residues are located in loops, three in α-helices and two in β-strands in the mesophilic homologue LTH4A. In the latter structure, Pro-511 adopts the cis-conformation whereas in ColAP all Pro are in trans-conformation. The replacement of these Pro by residues possessing a larger dihedral angle probably contributes to the increase in backbone flexibility in ColAP.Other variations in the amino acid content can be also influenced by the cold environment. Indeed, ColAP is characterized by a higher Asn content. A similar trend has been observed in the whole genome of another psychrophilic bacterium, Pseudoalteromonas haloplanktis TAC125 (24Medigue C. Krin E. Pascal G. Barbe V. Bernsel A. Bertin P.N. Cheung F. Cruveiller S. D'Amico S. Duilio A. Fang G. Feller G. Ho C. Mangenot S. Marino G. Nilsson J. Parrilli E. Rocha E.P. Rouy Z. Sekowska A. Tutino M.L. Vallenet D. von Heijne G. Danchin A. Genome Res. 2005; 15: 1325-1335Crossref PubMed Scopus (312) Google Scholar). This residue is involved in protein aging, as the Asn side chain is heat-labile and prone to deamination at high temperatures (23Lee D.Y. Kim K.A. Yu Y.G. Kim K.S. Biochem. Biophys. Res. Commun. 2004; 320: 900-906Crossref PubMed Scopus (22) Google Scholar). In a cold environment, this feature is not under strong selective pressure, which may explain the increased Asn content in the cold-adapted enzyme. The absence of Cys in ColAP may also be related to the environment of C. psychrerythraea. Indeed, cold aerobic environments are more oxidative, as oxygen solubility increases at low temperatures. As ColAP enzyme is secreted in the external medium in a temperature range of -1 to 10 °C, natural selection may have led to the lack of Cys.The GRAVY and aliphatic indexes are computed parameters related to global hydrophilicity and hydrophobicity of proteins. These indexes are often lower for cold-adapted enzymes as compared with their mesophilic or thermophilic homologues. Actually, the values obtained from the GRAVY index, isoelectric point, and aliphatic index of ColAP are lower than those for LTA4H. Both of the former parameters can be tentatively related to better interactions with the solvent (25Schiffer C.A. Dotsch V. Curr. Opin. Biotechnol. 1996; 7: 428-432Crossref PubMed Scopus (64) Google Scholar), whereas the latter (the aliphatic index) suggests a lower hydrophobic effect stabilizing the core of ColAP.Loop and Secondary Structure Variations—An extension of the enzyme loop regions has been suggested as a possible determinant in cold-adaptation, as their increased lengths and less constrained conformations should lead to an increase in the conformational entropy of psychrophilic proteins (26Siddiqui K.S. Cavicchioli R. Annu. Rev. Biochem. 2006; 75: 403-433Crossref PubMed Scopus (617) Google Scholar). However, one should keep in mind that the delimitation of secondary structures is also related to the preciseness of the atomic position and, as a consequence, is linked to the resolution of the crystallographic data. From our point of view, such an analysis needs to be considered carefully when comparing two different structures, and only general trends should be interpreted as thermally related parameters. An analysis of ColAP secondary structures revealed a striking increase in the loop content as compared with LTA4H. Indeed, the relative residue content in the loop, helices, and strand was found to be 47 versus 38%, 31 versus 38%, and 22 versus 24% in ColAP versus LTA4H. The additional 9% in loop content, 2, 3, and 4% was found in the N-terminal, catalytic, and C-terminal domains, respectively. Interestingly, although globally the regular secondary structures are shorter in ColAP (with 68 additional residues adopting a loop conformation), the loops conserved in both enzymes are shorter in the human enzyme. This clearly indicates that the increased loop content observed in ColAP corresponds mainly to a shortening of the regular secondary structures and not to insertions in exposed loops. The α-helix content is more affected than the β-strand content; about two-thirds of the residues concerned are situated in α-helices, whereas only one-third are located in β-strands in LTA4H. Interestingly, based on structural genomics studies, the inverse effect has been observed in thermophilic proteins, as a decrease in the overall loop content was found to be correlated to an increase in helical content (27Chakravarty S. Varadarajan R. Biochemistry. 2002; 41: 8152-8161Crossref PubMed Scopus (227) Google Scholar). As seen from Table 3, the global higher loop content of ColAP is correlated with a larger proportion of loops exposed to the solvent. In ColAP, the total accessible surface of the loops is 9% higher as compared with LTA4H. The GRAVY index of secondary structures (Table 2) indicates that loops tend to be more hydrophilic in ColAP. In addition, the backbones of the exposed loops in ColAP are generally less implicated in hydrogen bonding to each other and therefore favor interactions with water molecules. Altogether, these observations suggest that ColAP, by having a higher loop content exposed to the solvent, has improved the breathing (or microunfolding) of its external shell in comparison with its mesophilic counterpart.TABLE 3Accessible surface area statisticsSolvent-accessible surface areaColAPLTA4HÅ2%Å2%Primary structuresHydrophobicAla, Ile, Phe, Leu, Met, Pro, Val5,21222.855,26322.00PolarNeutral polarAsn, Gln, Cys, Ser, Thr, Trp, Tyr6,34027.806,13225.63Basic residuesArg, Lys, His5,54624.326,11725.57Acidic residuesAsp, Glu5,13722.525,87724.56Charged residues10,68346.8411,99450.13Total17,02374.6418,12675.76Glycine5722.515372.24AromaticPhe, Trp, Tyr1,8918.291,3595.68Secondary structuresα-Helices6,44528.267,81232.65β-Strands3,64715.994,96520.75Loops12,71555.7511,14946.60Total accessible surface22,80723,926 Open table in a new tab In addition to the above mentioned size reduction of α-helices in ColAP, charge stabilization of the helix macrodipole, generated by the helical alignment of polarized peptide bonds, is weakened in the psychrophilic enzyme. Considering for instance the charges present in the first turn and on the N-cap of all helices, ColAP displays nine favorable (negative) and four unfavorable (positive) charges, whereas the human enzyme possesses 11 favorable and only two unfavorable charges at the helix N termini. Such weakening of the macrodipole in ColAP is thought to reduce the compactness of helices, originating from local microdipole alignment.Finally, it should be noted that no electron density was found for the first 13 amino acid residues of ColAP. This suggests that the N terminus of the psychrophilic enzyme has a weakly defined structure in the solvent. As a matter of fact, the N- and C-terminal extremities of cold-adapted proteins are frequently less constrained when compared with their mesophilic or thermophilic homologues (28Riise E.K. Lorentzen M.S. Helland R. Smalas A.O. Leiros H.K. Willassen N.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 135-148Crossref PubMed Scopus (25) Google Scholar). As these relaxed extremities are preferential sites for unfolding, they may contribute to the global destabilization strategy in psychrophilic enzymes.Molecular Surface Properties—In cold-adapted enzymes, the accessible surface area is frequently characterized by a higher proportion of hydrophobic groups and an excess of negative charges. Although exposed hydrophobic groups are supposed to be entropically unfavorable, the excess of charge may improve interactions with the surrounding medium. However, there are few significant differences in the accessible surface properties of ColAP in terms of global hydrophobicity, polarity, or charge when compared with its mesophilic homologue (Table 3). Although theses surface properties display little correlation with those of other psychrophilic enzymes, it should be noticed that these results are in agreement with global trends observed for proteins modeled from the genome of C. psychrerythraea (22Methe B.A. Nelson K.E. Deming J.W. Momen B. Melamud E. Zhang X. Moult J. Madupu R. Nelson W.C. Dodson R.J. Brinkac L.M. Daugherty S.C. Durkin A.S. DeBoy R.T. Kolonay J.F. Sullivan S.A. Zhou L. Davidsen T.M. Wu M. Huston A.L. Lewis M. Weaver B. Weidman J.F. Khouri H. Utterback T.R. Feldblyum T.V. Fraser C.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10913-10918Crossref PubMed Scopus (402) Google Scholar). By contrast, the nature of the surface residues differs from those of the human enzyme, as ColAP exposes a higher proportion of aromatic residues to the solvent, as seen in several other cold-adapted proteins. Although it is generally accepted that a greater exposure of these groups results in entropic destabilization by weakening the hydrophobic effect, this may be also related to a lower entropic penalty cost at low temperatures. Indeed, at low temperatures, the cost of water structure ordering is reduced, which may favor the enhancement of aromatic and hydrophobic group exposure to the solvent (29Arnorsdottir J. Kristjansson M.M. Ficner R. FEBS J. 2005; 272: 832-845Crossref PubMed Scopus (71) Google Scholar).Ion Pair Distribution and Complexity—Among the various molecular features suggested as determinants for protein thermal adaptation, electrostatic interaction optimization is considered a key mechanism. Several studies have shown that with increasing environmental temperature, ions pairs become more abundant in proteins and, in parallel, form networks of rising complexity (30Aghajari N. Feller G. Gerday C. Haser R. Structure (Lond.). 1998; 6: 1503-1516Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 31Bell G.S. Russell R.J. Connaris H. Hough D.W. Danson M.J. Taylor G.L. Eur. J. Biochem. 2002; 269: 6250-6260Crossref PubMed Scopus (54) Google Scholar, 32Karshikoff A. Ladenstein R. Trends Biochem. Sci. 2001; 26: 550-556Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 33Violot S. Aghajari N. Czjzek M. Feller G. Sonan G.K. Gouet P. Gerday C. Haser R. Receveur-Brechot V. J. Mol. Biol. 2005; 348: 1211-1224Crossref PubMed Scopus (84) Google Scholar). This aspect was investigated here by a survey of the electrostatic interactions in the psychrophilic ColAP and in the mesophilic LTH4A enzyme. Ions pairs were determined using three different criteria: (i) only Asp, Glu, Lys, and Arg side chains were considered; (ii) the maximal distance cutoff between charges was 4 Å; and (iii) adjacent residues in the primary structure were not retained if they formed a single ionic pair. Table 4 indicates that, as the number of ion pairs is similar in both enzymes, more difference can be seen in the nature of the residues implicated in these interactions. Although ions pairs are formed mainly by the interaction of Asp with Arg in ColAP, Asp tends to be replaced by Glu residues in the mesophilic enzyme. One should also note that for both enzymes, more than 60% of salt bridges include an Arg residue as the cation partner. 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• W1999660388 title "Crystal Structure of the Cold-active Aminopeptidase from Colwellia psychrerythraea, a Close Structural Homologue of the Human Bifunctional Leukotriene A4 Hydrolase" @default.
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