Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

Thermitase and proteinase K: a comparison of the refined three-dimensional structures of the native enzymes

We compare the three-dimensional structures of thermitase and of proteinase K determined by X-ray crystallography to a resolution of 1.4 and 1.48 A respectively. Both enzymes are relatively stable towards heat and denaturating agents and are representative of a subgroup of subtilisins characterized by a free SH group close to the active site histidine. Even though they have low sequence homology, the overall tertiary structures are highly conserved. The high resolution structures are compared in terms of the overall fold of the molecules, the active sites, the calcium binding sites, disulphide bridge positions, the positions of the charged residues and the solvent structure. Most subtilisins such as thermitase are of prokaryotic origin and proteinase K is up to now the only known eukaryotic structure.     

Use of thermitase in the cultivation of embryonal mouse fibroblasts

Thermitase has been investigated as a means for obtaining single cells from tissue material, for enzymatic detachment of cultivated cells from the substrate and rarification of cells with subsequent passaging of mouse embryonic fibroblasts. The action of this enzyme was compared with that of "trypsin for cell cultivation". Tissue digestion showed that thermitase at 1/50 of the enzyme concentration needed with trypsin, was 1.5 fold more effective in yielding single cells. In cell cultivation thermitase is able to detach cells from the substrate at the same low concentration of 0.05 mg enzyme/ml and to give sufficient rarification, without the need of adding complexing agents. Rate of attachment, cell form and multiplication in subcultures corresponded to those after application of trypsin. The best results in cell detachment and rarification and the most uniform cell morphology were obtained with thermitase at a concentration of 0.025 mg enzyme/ml under addition of 4 mM of complexing agent. At that, thermitase proved 50 fold more effective than trypsin. Another advantage of thermitase is its better storage quality at 4 degrees C in dissolved form.     

Complete primary structure of thermitase from Thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteinases

Thermitase, a thermostable alkaline proteinase, consists of a single polypeptide chain, containing 279 amino acid residues (M_r = 28 369). The enzyme shows remarkable structural features of proteinases of the subtilisin type as shown by pronounced sequential homologies. The amino acid replacements, insertions and deletions observed when the amino acid sequence of the enzyme is compared with the sequences of several subtilisins are discussed with respect to substrate specificity and expected tertiary structure. The existence of a cysteinecontaining subgroup of subtilisin-like proteinases is postulated.     

Crystal structure of thermitase and stability of subtilisins

Crystal structure of thermitase, a serine proteinase from Thermoactinomyces vulgaris, has been determined by X-ray diffraction at 1.4 A resolution. The atomic model of thermitase refined to an R-factor of 0.149 contains 1997 protein atoms, 182 water molecules and 2 Ca2+ ions. The tertiary structure of thermitase is similar to that of subtilisin BPN'. The greatest variations are connected with insertions and deletions in the amino acid sequence, which are located on the surface of the molecule. Higher thermostability of thermitase can be explained in terms of the three-dimensional structure. The Ca2+ ions, bound to the protein molecule, as well as the ionic and hydrophobic interactions are supposed to give the main contribution to the stabilization of the structure.     

Molecular dynamics refinement of a thermitase-eglin-c complex at 1.98 A resolution and comparison of two crystal forms that differ in calcium content

The crystal structure of the complex of thermitase with eglin-c in crystal form II, obtained in the presence of 5 mM-CaCl2, has been determined at 1.98 A resolution. The structure was solved by a molecular replacement method, then molecular dynamics crystallographic refinement was started using the thermitase-eglin-c structure as determined for crystal form I. A ten degrees rigid body misplacement of the core of eglin-c was corrected by the molecular dynamics crystallographic refinement without any need for manual rebuilding on a graphics system. A final crystallographic R-factor of 16.5% was obtained for crystal form II. The comparison of the complexes of thermitase with eglin-c in the two crystal forms shows that the eglin-c cores are differently oriented with respect to the protease. The inhibiting loop of eglin binds in a similar way to thermitase as to subtilisin Carlsberg. A tryptophanyl residue at the S4 site explains the preference of thermitase for aromatic residues of the substrate at the P4 site. The difference in the P1 binding pocket, asparagine in thermitase instead of glycine in subtilisin Carlsberg, does not change the binding of eglin-c. The preference for an arginyl residue at the P1 site of thermitase can be explained by the hydrogen bonding with Asn170 in thermitase. Three ion-binding sites of thermitase have been identified. The strong and weak calcium-binding sites resemble the equivalent sites of subtilisin Carlsberg and subtilisin BPN', though there are important amino acid differences at the calcium-binding sites. The medium-strength calcium-binding site of thermitase is observed in the subtilisin family for the first time. The calcium is bound to residues from the loop 57 to 66. A difference in chelation is observed at this site between the two crystal forms of thermitase, which differ in calcium concentration. Additional electron density is observed near Asp60 in crystal form II, which has more calcium bound than form I. This density is possibly due to a water molecule ligating the calcium ion or the result of Asp60 assuming two significantly different conformations.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.     

Amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins

The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr- Pro-Gln-Asn-Gly-Asn-Gly-64His-Gly-Thr-His-68Cys-Ala- Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile- Ala-Gly-Thr-Ala-Pro-Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.     

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83 degrees C (3.5 min) and temperature optimum for activity (Topt, 76 degrees C) are identical with those of thermitase. The Topt of the evolved enzyme is 17 degrees C higher and its half-life at 65 degrees C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90 degrees C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity.     

应用定点突变与分子模建方法研究蛋白酶抑制剂eglin C突变体对蛋白酶furin和kexin的抑制活力

蛋白质前体加工酶参与许多重要蛋白质闪体的加工成熟过程,哺乳动物来源的furin和酵母中的kexin是该家族的重要成员。首先人工合成了编码枯草杆菌蛋白酶抑制剂eglin C的基因片段,组装后在大肠杆菌中得到表达。以定点突变方法在野生型eglin C抑制活性中心的P1、P2和P4位引入碱性氨基酸残基可以将其改造为很强的furin抑制剂（Ki约10^-9mol/L）,和kexin抑制剂（Ki约10^-11mol/L）。同时根据枯草杆菌蛋白酶和eglin C复合物的晶体结构,计算机同源模建了前体加工酶与eglin C突变体结构之间的相互作用,并结合实验数据得到以下结果：（1）P1位引入的碱性残基是该抑制剂活力的前提;（2）P4位碱性残基的引入可以极大地提高抑制剂活力约两个数量级;（3）P2 的碱性残基将有效提高抑制剂的活力。然而同时可以破坏抑制剂本身的稳定性。（4）野生型P3位的疏水性残基参与抑制剂活性环附近疏水核心的构成。     

Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B.

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.     

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.     

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.     

Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution

Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)