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.     

A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction

The initial contractile event in muscle is the binding of Ca2+ ions to troponin C of the troponin complex, leading to a series of conformational changes in the members of the thin and thick filaments. Knowledge of the crystal structure of turkey skeletal muscle troponin C has provided a structural basis for the modeling of the first stage of this process in atomic detail. This crystal structure probably represents the molecule in the relaxed state of muscle, with two of the maximum of 4 Ca2+ ions bound. The basis for the model presented here is that upon binding of the additional two Ca2+ ions, the regulatory domain of the molecule undergoes a conformational transition to become closely similar in structure to the domain which always binds Ca2+ or Mg2+ under physiological conditions. The root mean square discrepancy in atomic coordinates between the apo and the modeled Ca2+-bound states of the regulatory domain is 4.8 A, with some shifts as large as 10-15 A in the region near the linker between the two Ca2+ binding sites. It is demonstrated that this Ca2+-bound conformation of the regulatory domain conforms to accepted protein structure rules and that the change in conformation can be accomplished without encountering any barriers too high to be surmounted on the physiological time scale.     

ATP-AMP phosphotransferase from Paracoccus denitrificans

1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80 s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.     

The effect of Ca2+ ions on DNA compaction in the complex with non-histone chromosomal protein HMGB1

The analysis of absorption and circular dichroism spectra in UV and IR regions showed that Ca2+ ions interact both with the phosphate groups of DNA and with the HMGB1 protein. Not only negatively charged C-terminal part of the protein molecule participates in interaction with metal ions but also its DNA-binding domains. The latter fact leads to the change of the mode of protein-DNA interaction. The presence of Ca2+ ions prevents formation of ordered supramolecular structures, specific for the HMGB1-DNA complexes, though promotes intermolecular aggregation. The structure of the complexes between DNA and the protein HMGB1 lacking C-terminal tail appears to be the most sensitive to the presence of Ca2+ ions. The data obtained allow to conclude that Ca2+ ions do not play a structural role in the HMGB1/DNA complexes and the presence of these ions is not necessary to DNA compaction in such systems.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.     

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.     

Structural change of the troponin C molecule upon Ca2+ binding measured in solution by the X-ray scattering technique

The small-angle X-ray scattering technique was used to characterize the overall structural change as well as the state of aggregation of troponin C upon binding various amount of Ca2+ ions: in the Ca2+-free state and at pCa 6.5 and 4.0. Under these conditions, the forward scattering intensities of troponin C are not much different from each other: i.e., they coincide within 4%. From these intensities, the Ca2+-facilitated dimerization of troponin C was not verified, and no appreciable aggregation of troponin C molecules was detected below pCa 4.0. Thus, the small-angle X-ray scattering profiles from troponin C solutions were analyzed assuming a monomeric molecule. The radii of gyration of troponin C were 27.8 +/- 0.3 A, 23.8 +/- 0.2 A, and 22.6 +/- 0.1 A for the Ca2+-free state and at pCa 6.5 and 4.0, respectively. The maximum dimension of the molecule decreases from 111 to 98 A with increasing Ca2+ concentration. These results indicate that the troponin C molecule shrinks remarkably as Ca2+ ions bind to the high affinity sites of the molecule. Ca2+ binding to the low affinity sites, on the other hand, leads to a less pronounced change. Following the interpretation of scattering from the dumbbell-shaped structure (Fujisawa, T., Ueki, T., Inoko, Y., & Kataoka, M. [1987] J. Appl. Cryst. 20, 349-355), the two domains of the molecule move closer to each other. The distance between the centers of the two domains decreases from 46 to 35 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium dialysis measurements of the Ca2+-binding properties of recombinant radish vacuolar Ca2+-binding protein expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity.     

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins

BACKGROUND: The Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. CONCLUSIONS: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.     

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.     

Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351.

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.     

Specific interactions of divalent metal ions with a DNA duplex containing the d(CA)n/(GT)n tandem repeat

Divalent metal ions are essential for maintaining functional states of the DNA molecule. Their participation in DNA structure is modulated by the base sequence and varies depending on the nature of the ion. The present investigation addresses the interaction of Ca2+ ions with a tandem repeat of two CA dinucleotides, (CA)2/(TG)2. The binding of Ca2+ to the repeat is monitored by nuclear magnetic resonance (NMR) spectroscopy using chemical shift mapping. Parallel experiments monitor binding of Mg2+ ions to the repeat as well as binding of each ion to a DNA duplex in which the (CA)2/(TG)2 repeat is eliminated. The results reveal that the direction and the magnitude of chemical shift changes induced by Ca2+ ions in the NMR spectra of the repeat are different from those induced by Mg2+ ions. The differences between the two cations are significantly diminished by the elimination of the (CA)2/(TG)2 repeat. These findings suggest a specific interaction of Ca2+ ions with the (CA)2/(TG)2 motif. The specificity of the interaction resides in the two A-T base pairs of the repeat, and it involves the major groove of the first A-T base pair and both grooves of the second A-T base pair.     

Interactions of calmodulin with metal ions and with its target proteins revealed by conformation-sensitive monoclonal antibodies

Two monoclonal antibodies (mAbs) raised against bovine calmodulin (CaM), CAM1 and CAM4, enable one to monitor conformational changes that occur in the molecule. The interaction of CAM1 with CaM depends on the Ca2+ occupancy of its Ca(2+)-binding sites. CAM4, in contrast, interacts with CaM in a Ca(2+)-independent manner, interacting with both holoCaM and EGTA-treated CaM to a similar extent. Their interaction with various CaMs, CaM tryptic fragments and chemically modified CaM, as well as molecular graphics, led to identification of the CAM1 and CAM4 epitopes on the C- and N-terminal lobes of CAM respectively. The two mAbs were used as macromolecular probes to detect conformational changes occurring in the CaM molecule upon binding of metal ions and target proteins and peptides. MAb CAM1 successfully detected changes associated with Al3+ binding even in the presence of Ca2+, indicating that Al3+ and Ca2+ ions may bind to the protein simultaneously, leading to a new conformation of the molecule. MAbs CAM1 and CAM4 were used to follow the interactions of CaM with its target peptides and proteins. Complexes with melittin, mastoparan, calcineurin and phosphodiesterase showed different immunological properties on an immuno-enzyme electrode, indicating unique structural properties for each complex.     

Fluorescence studies of the calcium binding to whiting (Gadus merlangus) parvalbumin

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changs proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms. The dependence of the relative concentration of the three fomrs on Ca2+ concentrations agree well with the two-step binding of Ca2+ to parvalbumin: Protein + Ca in equilibrium K1 protein x Ca; Protein x Ca + Ca in equilibrium K2 Ca x protein x Ca. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 X 10(8) M-1 and 6 X 10(6) M-1. This scheme and the K1 and K2 value are in a good agreement with the independent experimental data resulting from EGTA titration of Ca2+-saturated parvalbumin and pH titratin of parvalbumin in the presence of EGTA and CA2+.     

Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

The presence of cooperative structures in tryptic fragments of troponin C

Scanning microcalorimetry has been used to study the ability of TR-1 and TR-2 tryptic fragments of troponin C to form an ordered compact structure in solution under different conditions. It has been shown that: (1) in the presence, as well as in the absence of bivalent ions both fragments have a structure which can melt with an intensive heat absorption at heating; (2) the structure of fragment TR-1 containing two Ca2+-specific domains (domains I and II) melts as a whole under all conditions studied and therefore the domains form one cooperative block. Binding of Ca2+ or Mg2+ ions stabilizes the block structure, however, significant conformational rearrangements which would lead to a change of denaturational enthalpy do not occur; (3) Ca2+Mg2+-domains of fragment TR-2 (domains III and IV) represent individual cooperative units, blocks. Stability of these cooperative blocks strongly depends on concentration of bivalent ions and in the presence of 2 mM EDTA the melting temperature of one of them is below 10 degrees. Thermodynamic melting temperature of one of them is below 10 degrees. Thermodynamic melting parameters of cooperative blocks within peptides and in the intact molecule of troponic C are compared.     

Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

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.