Tài liệu Báo cáo khoa học: ˚ The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species docx

Œ ⁄ RI, where I is the observed intensity and is the average intensity) was about 11%, I ⁄ rI was about and multiplicity was about 3.7 The final R-factors are 16.0% (Rwork) and 19.3% (Rfree) The refined structure contains about 3900 protein atoms, including a tripeptide found in the active site binding cleft, and 225 solvent atoms including two SO42– ions and one Ca2+ ion The average B-factor is ˚ ˚ 8.86 A2 for all enzyme atoms and 10.05 A2 for all atoms The electron density is well defined for the whole molecule and only a small number of side chains on the molecular surface could not be fitted into electron density One residue was refined with alternate conformations The structure of the Serratia peptidase Overall fold The sequence (Fig 1) identity between SPRK and the two other peptidases is about 40 and 64% (PRK and VPRK, respectively), but still the fold of SPRK is virtually identical to PRK and VPRK (Fig 2) The structures of the two bacterial peptidases are more similar to each other than to PRK SPRK superimposes on PRK and VPRK with an root mean square deviation (rmsd) ˚ value of 1.47 and 0.66 A, respectively, for all main chain atoms, excluding the inserted or deleted regions FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS R Helland et al Structure of proteinase K from a Serratia sp Fig Structural alignment of SPRK, PRK and VPRK Helices (red tubes) and sheets (yellow arrows) are according to SPRK Residues belonging to the S1 site are shaded blue and residues belonging to the S4 site are shaded light blue *represents residues that are involved both in the S1 and S4 sites Residues forming the calcium binding site found in SPRK (and VPRK) are shaded green, residues forming the ‘strong’ calcium binding site in PRK (and in VPRK) are shaded khaki, and residues forming the calcium sites unique for either PRK or VPRK are shaded pale green Fig Stereo plot of SPRK (red), PRK (green) and VPRK (blue) The catalytic triad residues of SPRK are represented as ball-and-stick models, and calcium ions are represented as spheres and follow the coloring of enzymes Sulfate ions found in SPRK are drawn as yellow spheres Cyan and magenta ball-and-stick models represent disulfide bridges in SPRK and VPRK, and yellow represent disulfide bridges in PRK Selected loop legions with significant structural differences are labeled Eight regions in the three proteinase K structures can be considered to display differences in the fold of the main chains None of these includes any of the binding sites, but some are located in the vicinity Three of the regions with different conformation involve calcium binding and four of them involve insertion or deletion of amino acids This can probably influence the flexibility and stability of the enzymes SPRK and VPRK have an insertion of three residues in the loop comprising residues 58–65 after residue 60 At the end of this loop SPRK and VPRK have a cysteine residue, Cys66, which forms a disulfide bridge to residue 98, which is in the proximity of the S2 binding site Cys66 is also close to the catalytic triad residue His69 The potential effect of the disulfide bridges will be discussed further below The region FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS 63 Structure of proteinase K from a Serratia sp R Helland et al comprising residues 58–63 (56–63 in VPRK) also binds a calcium ion in VPRK and this will be further discussed below Five residues are deleted in the loop comprising the residues 120–127 in SPRK and VPRK PRK has one of its two disulfide bridges (Cys123– Cys34) located in this region The loop formed by residues 240–245 in the bacterial species has conformation slightly different from PRK due to an insertion of one residue The only region where SPRK is different from both of the other two enzymes is after residue 213, where two residues are inserted The length of the loop comprising residues 16–29 is identical in the three enzymes, but the conformation is slightly different A calcium ion is present in SPRK and VPRK in this loop, but is absent in PRK This will be discussed further below The helix from residues 140–151 is slightly translated in SPRK and VPRK relative to PRK, possibly due to Val and Asn at positions 145 and 146 in SPRK (both Ala in PRK) and Val and Glu in VPRK Small differences are also observed for residues 175– 177 These are two of the residues comprising the ‘strong’ calcium binding site in PRK and will be discussed below Position 178 in PRK is occupied by a cysteine which forms a disulfide bridge to residue 249 Minor differences are observed in the region 263–270 This is a region where VPRK has a deletion of one residue Calcium binding site Binding of calcium ions in subtilases has been shown to be essential for correct folding and stability [15] A total of four calcium binding sites have been reported for the proteinase K-like enzymes, and we define the sites as follows Ca1 constitute the new binding site reported by [21] and is formed by residues Asp11, Asp14, Gln15, Asp21 and Asp23 (SPRK numbering), and is involved in stabilizing the N-terminal region of the enzyme Ca2 is the site observed in VPRK and thermitase [22] which is formed by residues Asp58, Asp60D and Asp62 (SPRK numbering) This site stabilizes the loop between strand b4 and helix a2 where VPRK and SPRK have an insert of three residues Ca3 is the so-called strong binding site found in PRK and formed by the side chain of Asp200 and the carbonyl oxygen atoms of residues 175 and 177 and is involved in stabilizing strands b8 and b9 Ca4 is the weak binding site in PRK, which is formed by the side chain of Asp260 and the carbonyl oxygen of Thr16, and stabilizes the N- and C-terminal regions of the molecule Only one calcium binding site is found in SPRK, in contrast to PRK which has two and VPRK which has three The Ca2+ ion in SPRK binds at the Ca1 site, and it is coordinated to the carboxyl oxygen atoms of the side chains of Asp11, Asp14 and Asp21, the amide oxygen atom of Gln15 and the carbonyl oxygen atoms of Asp11 and Asn23 (Fig 3) in an arrangement similar to what is observed in VPRK Both PRK and VPRK have calcium bound at Ca3 SPRK also has an aspartic acid residue at position at 200, and with the same conformation as in the two other structures, but Asp200 forms a salt bridge to Lys253 (Ala253 and Asn250 in PRK and VPRK, respectively), and probably loses some of its potential as calcium ligand The Fig Stereo plot of the calcium binding site in SPRK 64 FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS R Helland et al amine group of the lysine side chain is about 4.6–4.8 ˚ A from the position of calcium in PRK and VPRK Position 260 at the second calcium binding site in PRK, Ca4, is occupied by an aspartic acid This position is a lysine in SPRK and VPRK, and thus prevents binding of calcium The side chain of this lysine forms a hydrogen bond to Asn17 in SPRK (15 in VPRK) In addition, Arg16 in SPRK forms hydrogen bonds to residues 255 and 257, thus stabilizing the N- and C-terminal regions which are stabilized by calcium in PRK The residues forming the second calcium binding site in VPRK, Ca2, are identical in SPRK and VPRK, so there is no obvious explanation to why calcium is absent in this site in SPRK Calcium binding in SPRK could be prevented by the side chain of Arg94 which is ˚ about 4.0 A from the position occupied by calcium in VPRK However, both VPRK and thermitase also have an arginine at position 94 SPRK loses about 40% of its activity when incubated at 37 °C for h in the presence of EDTA [18], while PRK is essentially unaffected At 50 °C SPRK was totally inactivated, while PRK still had approximately 50% activity remaining The crystal structure of SPRK shows that the calcium ion is tightly bound with six protein atom binding partners, while the two calcium ions are bound weaker to the protein in PRK, with four and three protein ligands for binding sites Ca1 and Ca2, respectively This could probably explain why SPRK is less stable than PRK towards EDTA, and suggests that the calcium ion is more crucial for stability in SPRK VPRK has two binding sites that bind calcium stronger than PRK, and this could probably explain why also the stability of VPRK depends strongly on calcium [21] Disulfide bridges Two disulfide bridges are found in the crystal structure of SPRK, as in PRK, whereas VPRK has three The disulfide bridges in SPRK occupy the same positions as two of the bridges in VPRK (Figs and 2), and sequence alignment [18] suggests that the third disulfide bridge found at the C-terminal in VPRK could in principle be present in SPRK too if the C-terminal was not cleaved off The bridges are formed by residues 66–98 and 167–198 in SPRK, 67–99, 163–194 and 277– 281 in VPRK (VPRK numbering) and 34–123 and 178–249 in PRK One of the disulfide bridges in SPRK and VPRK is close to the S2 binding site It anchors a tight loop (see below) which is part of the S2 site, to the rest of the molecule, and may therefore affect the catalytic activity of the enzyme by reducing the flexibility of the protein Reduced flexibility may prevent effi- Structure of proteinase K from a Serratia sp cient binding of a substrate, something which can be illustrated by the lower binding affinity SPRK has for Suc-AAPF-pNA than PRK does [18] Docking this substrate into the active site of SPRK shows that the Cd atom of the P2 residue (here Pro) would be only ˚ 2.4 A away from the carbonyl oxygen atom of residue 100 (data not shown) The structure of proteinase K in complex with the inhibitor methoxysuccinyl-AAPAchlorometylketone (PDB 3prk [23]) shows that the main chain atoms of the residues forming the S2 site ˚ (residues 96 and 100) move more than A to accommodate a proline residue at the P2 position The second disulfide in SPRK (167–198) and VPRK (163–194) is located in a region close to the bottom of the S1 pocket This bridge can be considered to be involved in the stabilization of the region which is stabilized by calcium (Ca3) in PRK (residues 200 and 175–177) Both disulfide bridges in PRK are further away from the active site than in SPRK and VPRK, and are expected to influence the activity to a lesser degree They could, however, influence stability Salt bridges The three enzymes have 18, 19 and 15 residues forming ˚ 24, 23 and 18 interactions less than A (SPRK, PRK and VPRK, respectively) between atoms of positively and negatively charged residues, of which 6, and include histidine The average length is quite similar in ˚ ˚ the three enzymes, 3.15 A in SPRK and 3.21 A in PRK and VPRK None of enzymes have extended salt bridge networks but both SPRK and PRK have salt bridges involving three residues [Lys265–Asp263– Arg189 in SPRK (Pro, Asn, Arg in PRK, Arg, Asp, Arg in VPRK) and Asp187–Arg12–Asp260 in PRK (Asp, Arg, Lys in SPRK and VPRK)] The three-residue network observed in PRK is not possible in SPRK and VPRK due to the Lysine residue in position 260 A reduced number of salt bridges are generally accepted to be one of the properties of enzymes adapted to cold environments The reduced number is believed to increase the flexibility of an enzyme, and hence, the catalytic efficiency, but at the expense of the stability towards temperature (for reviews see [19,24,25]) SPRK from the psychrotrophic Serratia sp is not found to be considerable less stable than PRK [18], but VPRK is [26,27] This could possibly in part be related to the number of salt bridges in the three enzymes Position 98 close to the S2 site is involved in a disulfide bridge in SPRK and VPRK The Asp residue in this position in PRK forms a salt bridge to Lys94 It is not yet clear to what extent this interaction would affect PRK specificity at the S2 site FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS 65 Structure of proteinase K from a Serratia sp R Helland et al Structure in the binding region The electrostatic surface calculated by icm (Fig [28]) shows that the bacterial peptidases have a more anionic character than the fungal species The amino acid sequence in the binding region is very similar in the three enzymes, and so is the conformation of the side chains, but some differences exist The amino acid sequence in the S1 site is conserved, except at positions 162 and 169 located at the bottom of the pocket The neutral Asn162 in PRK is replaced by the polar and smaller Ser residue in VPRK, and by the acidic Asp in SPRK The conformation of Asn162 and Asp162 is practically identical in the two enzymes, but the acidic residue would contribute to the more anionic character of the S1 site in SPRK Asn or Asp at position 162 ˚ would be about A from the position occupied by a docked P1 Phe residue (data not shown) Position 169 is occupied by Tyr in SPRK and PRK and Thr in VPRK The residue at this position in subtilisin BPN has been subjected to extensive mutational studies in order to modify specificity [4,5] Tyr169 in SPRK and PRK is not likely to interact directly with a P1 residue of a substrate For such interactions to take place, the side chain will have to rotate, and hence, cause steric clashes with the enzyme main chain that will require energy to resolve The most obvious difference between the binding sites of the three enzymes is found in the S4 site at residues 103, 104, 107 and 141 where all three enzymes have different amino acid sequence This segment is Gln, Tyr, Ile and Val in PRK, Thr, Thr, Val and Leu in VPRK and Ser, Asn, Val and Thr in SPRK The fungal enzyme thus has a smaller and more hydrophobic binding site than the two other enzymes One of the most interesting differences between the bacterial and fungal enzymes is located at the periphery of the S2 site As mentioned above, Asp98 in PRK is replaced by a cysteine in the other two proteins, but the residues on both sides of this position are also of interest because the region 97–101 forms a tight loop which is stabilized differently in the three enzymes The side chains of residues Asn97, Ser99 and Ser101 in SPRK (Asp, Asn, Ser and Ser, Ser, Ser in PRK and VPRK, respectively) form a strong hydrogen bond network which brings the conformation of the loop almost into a ‘hub and spokes’ arrangement (Fig 5) The side chain of Asn99 in PRK is too long to participate efficiently in a similar arrangement, and the side chain of Ser97 in VPRK is too short One side of the loop is involved in the binding of the P2–P4 residues of a substrate The other side of the loop is close to Arg94 (Lys in PRK), which in SPRK and VPRK are further stabilized by salt bridges 66 Fig Electrostatic surface potential of SPRK (A), PRK (B) and VPRK (C) The surfaces are contoured at 5.0 kcalỈe.u.)1 charge where red regions represent negative potential and blue regions represent positive potential to residues in the 58–65 loop Hence, the region containing residues 97–101 is likely to be more rigid in SPRK than in the other two enzymes It is therefore FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS R Helland et al Structure of proteinase K from a Serratia sp Fig Stereo plot illustrating the tight loop forming the S2 binding site Red is SPRK, green is PRK and blue is VPRK Labels and distances in the loop refer to SPRK The stabilizing hydrogen bonding network formed by residues Asn97, Ser99 and Ser101 in SPRK is displayed as ball-and-stick models A similar network is not as strong in PRK and VPRK due to a shorter Ser97 in VPRK and a too-long Asn99 in PRK The loop is anchored to the rest of the molecule in SPRK and VPRK by a disulfide bridge between Cys98 and Cys66 (Asp98 in PRK) The catalytic His69 is displayed as ball-and-stick model in order to illustrate the orientation of the loop relative to the binding site not unreasonable to believe that the three enzymes will have a different specificity profile not only for the S4 site, but possibly also in the S2 site Tripeptide in the active site cleft Extra electron difference density was observed in the prime site of the substrate binding region of SPRK This was initially interpreted as a Pro-Ser dipeptide, but was after a few cycles of refinement interpreted as an Ala-Pro-Thr tripeptide (Fig 6) Attempts were made to fit the ligand into the initial difference density as a buffer or cryprotectant (glycerol) molecule, but it did not fit the electron density maps as well as a pep˚ tide Average B-factors for the peptide is about 20 A2, which is higher than the average for the enzyme (about Fig Stereo plot illustrating the electron density surrounding the Ala-Pro-Thr tripeptide found in the S1¢ and S2¢ binding site of SPRK The 2fofc electron density is contoured at 1.0 r FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS 67 Structure of proteinase K from a Serratia sp R Helland et al ˚ A2), indicating reduced occupancy The B-factor is, however, in the same order as the average of the solvent molecules The tripeptide is most likely a proteolysis product from the expression or purification process The gene for SPRK codes for a pre-pro enzyme including an N-terminal chaperone like domain, the catalytic domain and two similar C-terminal domains with unknown structure and function The N-terminal pro domain is autocatalytically cleaved off when the enzyme has obtained its active conformation, and the two C-terminal domains are cleaved off by heat treatment at 50 °C An Ala-Pro-Thr sequence is located in the first C-terminal domain The tripeptide is not located in the primary binding site (the S1 site), where most inhibitors, substrates and substrate analogues are observed in the structures of serine peptidase complexes Instead it is located in the S1¢ and S2¢ sites, and Thr3 and Pro2 occupy the same space as P1¢ and P2¢ residues found for naturally occurring protein inhibitors in complex with other subtilases (Fig 7); subtilisin BPN in complex with chymotrypsin inhibitor (CI2; PDB 1lw6, 1tm1 and 2sni [29–31]) or eglinC (PDB 1sbn [32]) and subtilisin Carlsberg in complex with eglinC (PDB 1cse [33]), ovomucoid turkey inhibitor (OMTKY3; PDB 1r0r [34]) or the double-headed tomato inhibitor II (TI2; PDB 1oyv [35]) The tripeptide is hydrogen bonded to the enzyme by interactions only through Thr3 The amine atom (Thr3 N) forms a hydrogen bond to Ser221 O ˚ (2.75 A) One of the terminal carboxyl oxygen atoms of Thr3 is hydrogen bonded to the catalytic His69 Ne2 ˚ ˚ (2.88 A) and Ser224 Oc (2.68 A) and the second terminal carboxyl oxygen atom occupy a position in the so-called oxyanion hole, with hydrogen bonds to ˚ ˚ Asn161 Od1 (2.90 A) and Ser224 Oc (3.16 A) The position of the second terminal oxygen atom of Thr3 ˚ atom is about 0.8–1.5 A from the position occupied by the carbonyl oxygen atom of the scissile bond in other subtilase–inhibitor complexes (data not shown) The Ca positions of the tripeptide occupy almost the same positions as the Ca positions of the inhibitors Introduction of the tripeptide causes a slight rotation of Ser224, such that the hydroxyl group of the side chain weakens its catalytic triad interaction with His69 Ne2 and instead forms hydrogen bonds to both terminal oxygen atoms of Thr3 Another interesting feature is that the direction of the main chain of the tripeptide is antiparallel to the naturally occurring inhibitors It was first speculated whether the C-terminal domain in an uncleaved state could fold back into its own active site, or alternatively into the active site of a neighboring molecule, something which could explain the antiparallel binding However, this can not be verified here since the C-terminal is cleaved off The tripeptide has a conformation most similar to OMTKY3, CI2 and the second binding region of TI2 (Fig 7) The first binding region of TI2 and eglinC have slightly different orientation from the P2¢ residue, probably resulting from different scaffolds of the inhibitors Structures of PRK with inhibitors or sub- Fig Stereo plot illustrating inhibitor binding in subtilases The binding loop of chymotrypsin inhibitor II (CI2) in complex with subtilisin BPN (cyan, PDB 2sni), the inhibitor fragment of lactoferrin in complex with PRK (green, PDB 1bjr), a hepta-peptide in complex with PRK (yellow, PDB 1p7v) are docked on the ribbon figure of SPRK The tripeptide found in SPRK is illustrated as red ball and sticks 68 FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS R Helland et al strate analogues at the prime side of the binding region exist, e.g hepta- or octapeptide substrate analogues or an inhibiting decapeptide fragment of lactoferrin However, these peptides have a completely different binding mode compared to the naturally occurring inhibitors They bind in the S1–S1¢ region, but not always at the other subsites of the enzymes It is therefore not clear as yet whether there are two distinct S’ binding sites in the proteinase K family or whether the different binding modes can be related, i.e to d-amino acids in the P1¢ position of the peptide inhibitors, thus forcing the amino acids on the prime side of the binding site into a different conformation Conclusion The structure of a proteinase K-like enzyme of a psy˚ chrotroph Serratia species has been solved to 1.8 A The fold of the protein reported here is practically identical to the two other enzymes of this family with known 3D structure, with only minor differences in the surface loops In addition, an Ala-Pro-Thr tripeptide is observed at the prime side of the active site, and the direction of the tripeptide is antiparallel to what would be expected of a naturally occurring proteinaceous inhibitor The protein presented in this paper is more similar to the bacterial species (VPRK) than the fungal species (PRK) with respect to disulfide bridges, calcium binding and surface loops This also coincides with the sequence identity, which is about 64% to VPRK and about 40% to PRK Only one calcium binding site is observed in the structure presented here, in contrast to the other proteinase K structures where two or more calcium binding sites are observed The calcium ion in this structure, similar to Ca1 in VPRK, is coordinated to six protein atoms, indicating a very strong binding, whereas the calcium ions in PRK are coordinated to not more than four protein atoms We therefore suggest that the calcium binding site observed in this structure is more crucial for the stabilization of the enzyme than the calcium binding sites observed in PRK This assumption is supported by the observation that SPRK loses about 40% of its activity when incubated in the presence of 10 mm EDTA whereas PRK remains essentially unaffected [18] SPRK should have the ability to bind one more calcium ion since it has the same amino acid sequence as VPRK in the loop comprising residues 58–62, but there is no obvious structural explanation why this does not occur The two disulfide bridges in SPRK are found in the same positions as VPRK, and they are completely different Structure of proteinase K from a Serratia sp from the positions found in PRK One of the disulfide bridges (including Cys98) in SPRK and VPRK is located next to a residue forming a part of the S2 site of the enzyme The disulfide bridge restricts the flexibility of a tight loop which is part of the S2 site The tight loop is further stabilized by a tight hydrogen bond pattern This could confer more rigidity to the active site of the enzyme and may explain the much lower binding affinity (higher KM value) SPRK has towards the synthetic suc-AAPF-nitroanilide substrate than PRK has [18] The volume of the S4 site in the bacterial enzymes is larger than in PRK It is therefore reasonable to believe that larger residues will be favored at this site in SPRK and VPRK and more kinetic data should therefore be obtained in order to verify this assumption The electrostatic potential of SPRK is significantly more negative than PRK Since the N-terminal protective succinyl group is negatively charged, one cannot rule out that differences in activity towards suc-AAPF-pNA when comparing SPRK and PRK [18], at least partly, are due to electrostatic effects Methods and materials Crystallization and data collection Expression and purification of the peptidase from the Serratia sp (SPRK) is described by Larsen and coworkers [18] Crystals of the catalytic domain of SPRK were obtained from 0.1 m Bis ⁄ Tris and Tris buffer pH 6.5–7.5 and 45–47.5% ammonium sulfate at °C by the hanging drop vapor diffusion method Protein concentration was mgỈmL)1 in 25 mm Tris pH 7.5 and 10 mm CaCl2 X-ray ˚ data to 1.8 A were collected at 100 K at Swiss Norwegian Beamlines (BM01) at the European Synchrotron Radiation Facility (ESRF) using reservoir solution containing 25% glycerol as cryoprotectant Data were integrated using denzo [36], scaling and merging was carried out using Scalepack [36] and scala of the CCP4 program suite [37] Molecular replacement, model building and refinement The structure was solved using MolRep [38] in CCP4i, using proteinase K (PRK; PDB 1ic6 [20]), as search model The structure was built using a combination of auto building in ARP ⁄ wARP [39] and manual refitting of side chains using O [40] based on 2FoFc and FoFc electron density maps Refinement was carried out in Refmac5 [41] of the CCP4 suite Solvent molecules were automatically added to the structure using ARP ⁄ wARP Solvent molecules that did not fit into electron density, had no hydrogen binding FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS 69 Structure of proteinase K from a Serratia sp R Helland et al ˚ partners or where the temperature factor exceeded 50 A2 were excluded from further refinement One calcium and two sulfate ions were added based on the electron density and hydrogen binding partners Numbering of SPRK follows that of PRK, and inserted residues get a letter extension after the number (i.e 60B, 60C, etc.) Structure analysis and comparison Software for structure analysis and comparison was chosen from the CCP4 suite and O Numbering in the comparisons refers to SPRK unless stated otherwise The alignment figure is generated using alscript [42] Figures represented as cartoons are generated using molscript [43] and bobscript [44] Electrostatic surfaces are generated using ICM [28] Acknowledgement The authors thank the organizers at the Swiss-Norwegian Beamlines (SNBL) at the European Synchrotron Radiation Facilities (ESRF) for provision of beam time The present study was supported by the national Functional Genomics Programme (FUGE) in The Research Council of Norway and Biotec Pharmacon ASA References Rao MB, Tanksale AM, Ghatge MS & Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases Microbiol Mol Biol Rev 62, 597–635 Rheinnecker M, Baker G, Eder J & Fersht AR (1993) Engineering a novel specificity in subtilisin BPN¢ Biochemistry 32, 1199–1203 Rheinnecker M, Eder J, Pandey PS & Fersht AR (1994) Variants of subtilisin BPN¢ with altered specificity profiles Biochemistry 33, 221–225 Wells JA, Powers DB, Bott RR, Graycar TP & Estell DA (1987) Designing substrate specificity by protein engineering of electrostatic interactions Proc Natl Acad Sci USA 84, 1219–1223 Estell DA, Graycar TP, Miller JV, Powers DB, Burnier JP, Ng PG & Wells JA (1986) Probing steric and hydrophobic effects on enzyme interactions by protein engineering Science 233, 659–663 Cedrone F, Menez A & Quemeneur E (2000) Tailoring new enzyme functions by rational redesign Curr Opin Struct Biol 10, 405–410 Chen RD (2001) Enzyme engineering: rational redesign versus directed evolution Trends Biotechnol 19, 13–14 Hult K & Berglund P (2003) Engineered enzymes for improved organic synthesis Curr Opin Biotechnol 14, 395–400 70 Somero GN (1975) Temperature as a selective factor in protein evolution – adaptational strategy of compromise J Exp Zool 194, 175–188 10 Hochancha PW & Somero GN (1984) Temperature Adaptation in Biochemical Adaptation, pp 377–388 New Jersey, USA, Princeton University Press 11 Sweeney PJ & Walker JM (1993) Proteinase K Methods Mol Biol 16, 305–311 12 Shaked GM, Shaked Y, Kariv-Inbal Z, Halimi M, Avraham I & Gabizon R (2001) A protease-resistant prion protein isoform is present in urine of animals and humans affected with prion diseases J Biol Chem 276, 31479–31482 13 Jackson GS, McKintosh E, Flechsig E, Prodromidou K, Hirsch P, Linehan J, Brandner S, Clarke AR, Weissmann C & Collinge J (2005) An enzyme-detergent method for effective prion decontamination of surgical steel J General Virol 86, 869–878 14 Schechter I & Berger A (1967) On the size of the active site in proteases I Papain Biochem Biophys Res Commun 27, 157–162 15 Siezen RJ & Leunissen JA (1997) Subtilases: the superfamily of subtilisin-like serine proteases Protein Sci 6, 501–523 16 Gron H, Meldal M & Breddam K (1992) Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching Biochemistry 31, 6011–6018 17 Gron H & Breddam K (1992) Interdependency of the binding subsites in subtilisin Biochemistry 31, 8967–8971 18 Larsen AN, Moe E, Helland R, Gjellesvik DR & Willassen NP (2006) Characterization of a recombinant expressed proteinase K-like enzyme from a psychrotrophic Serratia sp FEBS J 273, 47–60 19 Smalas AO, Leiros HK, Os V & Willassen NP (2000) Cold adapted enzymes Biotechnol Annu Rev 6, 1–57 20 Betzel C, Gourinath S, Kumar P, Kaur P, Perbandt M, Eschenburg S & Singh TP (2001) Structure of a serine protease proteinase K from Tritirachium album limber at 0.98 A resolution Biochemistry 40, 3080–3088 21 Arnorsdottir J, Kristjansson MM & Ficner R (2005) Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation FEBS J 272, 832–845 22 Teplyakov AV, Kuranova IP, Harutyunyan EH, Vainshtein BK, Frommel C, Hohne WE & Wilson KS ˚ (1990) Crystal structure of thermitase at 1.4 A resolution J Mol Biol 214, 261–279 23 Wolf WM, Bajorath J, Muller A, Raghunathan S, Singh TP, Hinrichs W & Saenger W (1991) Inhibition of proteinase K by methoxysuccinyl-Ala-Ala-Pro-Ala-chloro˚ methyl ketone: an x-ray study at 2.2-A resolution J Biol Chem 266, 17695–17699 FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS R Helland et al 24 Feller G & Gerday C (1997) Psychrophilic enzymes: molecular basis of cold adaptation Cell Mol Life Sci 53, 830–841 25 Feller G (2003) Molecular adaptations to cold in psychrophilic enzymes Cell Mol Life Sci 60, 648–662 26 Kristjansson MM, Magnusson OT, Gudmundsson HM, Alfredsson GA & Matsuzawa H (1999) Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I Eur J Biochem 260, 752–760 27 Arnorsdottir J, Smaradottir RB, Magnusson OT, Thorbjarnardottir SH, Eggertsson G & Kristjansson MM (2002) Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species Eur J Biochem 269, 5536–5546 28 Abagyan R, Totrov M & Kuznetsov D (1994) icm – a new method for protein modeling and design – applications to docking and structure prediction from the distorted native conformation J Comput Chem 15, 488– 506 29 Radisky ES & Koshland DE Jr (2002) A clogged gutter mechanism for protease inhibitors Proc Natl Acad Sci USA 99, 10316–10321 30 Radisky ES, Kwan G, Karen Lu CJ & Koshland DE Jr (2004) Binding, proteolytic, and crystallographic analyses of mutations at the protease–inhibitor interface of the subtilisin BPN¢ ⁄ chymotrypsin inhibitor complex Biochemistry 43, 13648–13656 31 McPhalen CA & James MN (1988) Structural comparison of two serine proteinase-protein inhibitor complexes: eglin-c-subtilisin Carlsberg and CI-2-subtilisin novo Biochemistry 27, 6582–6598 32 Heinz DW, Priestle JP, Rahuel J, Wilson KS & Grutter MG (1991) Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins: comparison with other serine proteinase inhibitor complexes J Mol Biol 217, 353–371 33 Bode W, Papamokos E & Musil D (1987) The highresolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis: structural Structure of proteinase K from a Serratia sp 34 35 36 37 38 39 40 41 42 43 44 FEBS Journal 273 (2006) 61–71 ª 2005 The Authors Journal compilation ª 2005 FEBS analysis, subtilisin structure and interface geometry Eur J Biochem 166, 673–692 Horn JR, Ramaswamy S & Murphy KP (2003) Structure and energetics of protein–protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases J Mol Biol 331, 497–508 Barrette-Ng IH, Ng KK, Cherney MM, Pearce G, Ryan CA & James MN (2003) Structural basis of inhibition revealed by a : complex of the two-headed tomato inhibitor-II and subtilisin Carlsberg J Biol Chem 278, 24062–24071 Otwinowski Z & Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode Methods Enzymol 276, 307–326 Collaborative Computational Project Number (1994) The CCP4 suite: programs for protein crystallography, Acta Crystallogr Sect D Biol Crystallogr D50, 760–763 Vagin A & Teplyakov A (1997) MOLREP: an automated program for molecular replacement J Appl Crystallogr 30, 1022–1025 Perrakis A, Harkiolaki M, Wilson KS & Lamzin VS (2001) ARP ⁄ wARP and molecular replacement Acta Crystallogr Sect D Biol Crystallogr 57, 1445–1450 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models Acta Crystallogr Sect A Found Crystallogr 47, 110–119 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr Sect D Biol Crystallogr 53, 240–255 Barton GJ (1993) ALSCRIPT: a tool to format multiple sequence alignments Protein Eng 6, 37–40 Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures J Appl Crystallogr 24, 946–950 Esnouf RM (1997) An extensively modified version of MolScript that includes greatly enhanced coloring capabilities J Mol Graph Model 15, 112–113 71 ... the carboxyl oxygen atoms of the side chains of Asp11, Asp14 and Asp21, the amide oxygen atom of Gln15 and the carbonyl oxygen atoms of Asp11 and Asn23 (Fig 3) in an arrangement similar to what... within a few days to thin hexa62 Table Data collection and refinement statistics for SPRK ˚ Resolution (A) Space group Cell parameters ˚ a- axis (A) ˚ b-axis (A) ˚ c-axis (A) b angle (°) Number of. .. bound to the prime side (S¢) of the active site is reported Results and discussion Crystallization, data collection and refinement Crystals of the catalytic domain of the Serratia proteinase K (SPRK)

Tài liệu Báo cáo khoa học: ˚ The 1.8 A crystal structure of a proteinase K-like enzyme from a psychrotroph Serratia species docx

Thông tin tài liệu

Từ khóa liên quan

Tài liệu cùng người dùng

Tài liệu liên quan