Journal of Bacteriology, July 2003, p . 4195-4203, Vol . 185, No . 14

The serralysins are secreted as inactive zymogens into the medium via the hemolysin pathway, involving an ABC transporter, which recognizes a C-terminal secretion signal . Activation occurs in the medium autocatalytically in the presence of divalent ions, especially Zn²⁺ and Ca²⁺ (12-14) .

The implication of zinc metalloproteases in many diseases, e.g., cancer or arthrosis (15), makes structural studies of these enzymes highly interesting .

Here we report the results from X-ray crystallographic studies of PAP, an alkaline protease produced by the psychrophilic Pseudomonas sp . strain TAC II 18, which originated in Antarctica . This strain, identified as a gram-negative bacterium (18), is one of numerous bacteria living at temperatures close to 0°C . To survive and grow under such conditions, these organisms have evolved several adaptations of their cellular components, especially of their enzymes (16-18, 30) . Thus, PAP is three times more active at 20°C than a mesophilic counterpart from Pseudomonas aeruginosa but rapidly inactivated at 45°C . The cold-adapted enzyme also displays a greater sensitivity to moderate EDTA concentrations, and fluorescence studies have indicated enzyme unfolding after 2 h of incubation in 2 mM EDTA (11) .

The PAP molecule is made up of 463 amino acid residues distributed in two domains, and its tertiary structure is comparable to mesophilic homologues from P . aeruginosa alkaline protease (AP) and Serratia marcescens protease (SMP) (1-3) . Four -helices and a mixed five-stranded ß-sheet form the catalytic N-terminal domain to which the active-site Zn²⁺ ion and a Ca²⁺ ion (Ca 700, PAP numbering) are bound . This Ca²⁺ ion is not present in its mesophilic counterparts, AP and SMP, but it should be noticed that a calcium ion has been observed in the 3D structure of the metzincin adamalysin II in a position similar to that of Ca 700 (20) .

The C-terminal domain which may play a role in the folding of the molecule after transmembrane translocation forms an extended ß roll with 20 successive ß-strands wound in a right-handed spiral . The number of Ca²⁺ ions bound to this C-terminal domain varies from six to seven in the different organisms and crystal forms .

When we compared the 3D structure with those of the mesophilic counterparts, AP and SMP, the most remarkable differences were found in the proteolytic domain . First, the active site is more accessible due, in part, to deletions in surrounding loops and to a region in the catalytic domain of PAP (residues 107 to 116) that undergoes a conformational change with a loop movement as large as 13 Å induced by the binding of the extra calcium ion, Ca 700 . This change does not seem to be related to crystal packing effects, since it was observed in crystal forms obtained under very different crystallization conditions . The role of this extra calcium ion in the activity or folding of the PAP molecule still needs to be elucidated . Its weak chelation in the proteolytic domain and its close proximity to the solvent could partly explain the sensitivity of PAP to EDTA, which is also supported by a higher temperature factor for this calcium ion compared to other calcium ions in the 3D structure . This sensitivity could also be related to the lower affinity, compared to AP and SMP, for some calcium ions located in the C-terminal domain .

In order to address these questions, we solved the structures of PAP (form 2) in the presence of various concentrations of EDTA, as well as a new native form (form 3) by X-ray crystallography .

Cocrystallization experiments of PAP have been performed under conditions derived from form 2 [1.6 M (NH₄)₂SO₄ and 0.1 M HEPES (pH 7) at 16°C] . EDTA was added to a final concentration of 1 mM (experiment A), 5 mM (experiment B), and 10 mM (experiment C) in the drops . Crystals grew within 1 week to a final size of about 300 by 100 by 100 µm and were left another 2 months in the drops before data collection in experiments A and C . In experiment B the crystals were left an additional 5 days (B1) or 2 months (B2) in the droplets, respectively, before data collection .

Soaking was achieved by directly adding a solution of EDTA to a final concentration of 85 mM into a drop containing form 2 crystals and leaving it 4 h at 16°C (experiment D) . Higher concentrations of EDTA have been tested, but the crystals cracked at concentrations of EDTA greater than 85 mM .

Cocrystallization or soaking with EDTA did not modify the space group, but small variations in the unit cell dimensions were observed (Table 2); these were certainly due in part to flash freezing of the crystals . The large difference in unit cell dimensions between B1 and the remaining data is a consequence of the B1 data being collected at 288 K and the rest being collected at 100 K .

 
Data collection, structure solution, and refinement . (i) Native form. Diffraction data on form 3 crystals (Table 1) were collected with a charge-coupled device camera by using synchrotron radiation at the beam-line D2AM at the European Synchrotron Radiation Facility (ESRF; Grenoble, France) under cryogenic conditions (100 K) . Initial data reduction and space group determination were carried out with the program XDS (23), and further reduction of the data was performed by using programs from the CCP4 package (10) .

 
The known structure of the AP (3) served as model in a molecular replacement search performed with the program AMoRe (26) . Refinement of the structure was done by using the program CNS (9), applying an overall anisotropic temperature factor correction algorithm, followed by manual fitting . A summary of the refinement statistics can be found in Table 1 .

Due to poor or missing electron densities, as for form 2 (1), residues 1 to 2 and residues 184 to 188 were not inserted into the electron density map .

(ii) Experiments A, B, C, and D. X-ray diffraction data of crystals from experiments A, B2, and C were collected at the BM30A beamline at the ESRF at cryogenic temperatures (100 K) at a wavelength of 0.98 Å on a MarCCD . X-ray data on crystals from experiments B1 and D were collected in house at 288 K and at cryogenic temperatures, respectively, on a MARresearch 345 image plate system associated with a Nonius FR591 rotating anode (CuK radiation) operating at 44 kV and 100 mA and coupled to Osmic confocal mirrors .

Diffracted intensities in experiments A, B2, C, and D were integrated with the program MOSFLM (25), as implemented in the CCP4 software package (10) and scaled with SCALA (10), whereas experiment B1 reflection data were processed and merged with DENZO/SCALEPACK (27) .

Collection and processing statistics of experiments A, B, C, and D are summarized in Table 2, and structures resulting from these experiments will henceforward be referred to as structure A, B1, B2, C, and D, respectively . Structures B1 and B2 were solved by the molecular replacement method by using the AMoRe software as implemented in the CCP4 suite (10), and the native form 2 was resolved to a 1.96-Å resolution (1) as a search model . Withdrawing diffraction data in a resolution range of 15 to 4 Å, a unique solution was obtained with a correlation coefficient of 58.8% and an R factor of 37.5% for B1 and a correlation coefficient of 54.0% and an R factor of 37.5% for B2 .

Since the crystals from experiments A, C, and D were isomorphous with B2 crystals, their 3D structures were determined by direct phasing with the 2.0-Å resolution B2 structure, for which ions as well as water molecules in the active site had been removed in order to avoid bias . The refinement of all models was done by using the simulated annealing protocol as implemented in the software package CNS (9), alternately with visual examination of electron density maps and manual building by using the graphic software TURBO-FRODO (29) .

Water molecules present at similar positions in the respective structures have the same numbering .

In order to confirm the presence or absence of calcium or zinc ions, anomalous difference Fourier maps were calculated by using the program CNS, in which calculated phases are derived from a refined model depleted of metal ions .

For all data set, free and conventional R factors were monitored (8) to avoid overrefinement .

Compared to the native structure of form 2 (PDB entry 1G9K), residues not inserted in the electron density map due to poor or missing electron densities were residues 50 to 51 in structures A, B2, C, and D and Tyr-183 in all five structures . Moreover side chain residues for Asn-52, Asp-53, and Asn-184 were not observed in electron density maps of structures A, B2, C, and D . Double conformations for Ser-15, Ser-138, and Ser-332 were found in structure B2 . Model qualities were examined with PROCHECK (24) and WHATCHECK (22) . The details of the refinement statistics and model quality are given in Table 2 .

Relative B-factors were calculated by dividing the mean B-factor of every residue by the mean B-factor of the whole protein . Due to the rather poor quality of structure D data, B-factors where initially set to 20 Ų and left out of the refinement .

Superimposition of structures was done by using the "rigid" option in TURBO-FRODO . The figures shown here were rendered by using TURBO-FRODO and VIEWERLITE 5.0 (freeware from Accelrys, Inc., San Diego, Calif.) .

Coordinates. The coordinates and structure factors are deposited in the the protein data bank as 1O0Q (A), 1O0T (B1), 1OM6 (B2), 1OM8 (C), 1OM7 (D), and 1OMJ (form 3) .

Effect of EDTA on ion binding . (i) Overall structures. Comparative studies of native PAP (Fig . 1) with structures A, B1, B2, C, and D show that the overall 3D structures are very similar . The rmsd values between C’s in these five structures and the native form 2 of PAP were 0.42, 0.17 . 0.43, 0.41, and 0.37 Å, respectively . No density corresponding to a molecule of EDTA can be seen in any of the calculated electron density maps . Significant differences, however, were observed in the number of metal ions ligated in the structures (Table 2) . In fact, B1 reveals the structure of the apo-form of PAP, since the electron density map calculated on the basis of the 2.5-Å data clearly showed that the active-site zinc ion had disappeared but that all of the calcium ions were present relative to the native structure, as confirmed by an anomalous difference Fourier map .

 
The electron density maps calculated for structures A, B2, C, and D clearly showed that the catalytic zinc ion and the calcium ion located in the catalytic domain (Ca 700) were lacking . However, in the C-terminal domain all calcium ions could be located and were inserted in the electron density of structures A, B2 and C . In structure D, one calcium ion, Ca 702, is replaced by a water molecule .

(ii) Changes in the Zn(II)-binding pocket. In the active site of native PAP, the Zn²⁺ ion is coordinated in a trigonal-bipyramidal geometry to four or five ligands depending on the position of the Tyr-209 . The Zn²⁺, when penta-coordinated, is bound to the side chains of three histidines (His-169, His-173, and His-179), Tyr-209, and a water molecule (1) .

Electron density maps calculated for experiments A, B1, B2, C, and D, clearly show that no metal is present in the active-site region and that no additional water molecule replaces the zinc ion in the binding site (Fig . 2) . As a consequence, displacements in the side chains of the zinc-ion ligands are observed, including His-173 and His-179 . In the apo-form, the N2 from His-173 undergoes a 4.1-Å shift and the imidazole ring of His-179 rotates 40° around its Cß-C, leading to a 1.0-Å displacement of its N2 . Similar changes for these histidines are observed in the other structures . Upon removal of the catalytic zinc ion, the phenol ring of Tyr-209 rotates ca . 60° around the Cß-C bond . The side chain of His-169 does not undergo significant conformational changes . Interestingly, a major part of the water molecules constituting an organized network in native PAP have disappeared, including Wat 1225, which ligates the zinc ion in the holo form (Fig . 2) .

 
Excepting the network of water molecules, similar significant structural changes (rmsd > 0.2 Å) have been noticed in structures A, B2, C, and D . In structure A, Wat 1225, 1102, and 1104 are present, whereas Wat 1102, 1104, and 1304 are found in the catalytic site of structure B2, and Wat 1102 and 1134 are found in structures C and D .

(iii) Calcium-binding sites. Of the seven calcium ions that are bound in the native form 2, Ca 700 is found in the proteolytic domain where it is easily accessible to the solvent, and the others are found in the C-terminal domain (Fig . 1) .

Next to the proteolytic domain, Ca 701 and 702 are heptacoordinated, and the four remaining calcium ions (Ca 703 to Ca 706) are bound in the core of the parallel ß-roll in a hexacoordinated manner . These are supposed to be essential determinants of the parallel ß-roll structure (1, 3) .

The loss of Ca 700 from the proteolytic domain in structures A, B2, C, and D induces structural changes, including that of Asn-57, which moves 1.5 Å toward the former calcium position . Moreover, 2Fo-Fc electron density was lacking for residues 50 and 51 and for the side chains of residues 52 and 53, all of which are located in the loop surrounding the Ca 700 binding site (Fig . 3) . The predominant change caused by EDTA chelating Ca 700 is a partial disorganization of this loop .

 
From the highest-resolution structures (B2 and C at 2.0 Å), it can be concluded that a water molecule replaces the calcium ion; hence, Wat 1500 has been inserted (Fig . 3) .

Although the Ca 700 binding site is accessible to the solvent due to the disorganization of the loop, no additional water molecules were found in the close vicinity in any of the structures . The 13-Å loop movement (residues 107 to 116) upon binding of Ca 700 in native PAP compared to its mesophilic counterparts is not reversible since no significant changes are observed in the backbone or in the side chain conformations of the loop residues when this Ca²⁺ is lacking . It should be noted that Asp-114 is one of the ligands of Ca 700 . With regard to structure B2, in which Ca 700 has not been chelated, the distances to the ligands have increased, especially for Asn-51-O1 (Table 3), suggesting a weaker chelation . The six calcium ions bound in the C-terminal domain and their respective ligands are invariant in structures A and B1 .

 
However, when higher concentrations of EDTA were used, structural changes, especially around Ca 701 and Ca 702, were observed (Table 4) . In structures B2 and C, it appears that Ca 701 and Ca 702 are weakly bound . The increased distance to Asp-282 suggests that these ions go from a hepta- to a hexa-coordinated state .

 
Particularly in structure D, Ca 702 is replaced by a water molecule (Wat 702) (Table 4) . The electron densities corresponding to five calcium ion sites located in the C-terminal domain were relatively strong and observed in the 2Fo-Fc map up to 3 for Ca 706 and to 4 for Ca 701, 703, 704, and 705 . As for the electron density corresponding to Ca 702, this site was only observed in a 2Fo-Fc map contoured at a 1 level and, when a calcium ion is inserted at this site, a strong negative density appears in the Fo-Fc electron density map . Ca 702 was therefore replaced by a water molecule in structure D . Furthermore, despite a medium resolution of the structure, the distances measured between this water molecule and its neighbors suggest that these interactions have hydrogen bonding character rather than being coordination bonds and thus confirms the substitution of Ca 702 by a water molecule .

As a consequence of the loss of Ca 702, a small reorganization of the Ca 701 ligands has taken place (Table 4) . One should note that Asp-287 ligates both calcium ions .

Moreover, significant structural changes (rmsd > 0.2 Å) have occurred in the Ca 703 and Ca 704 binding sites (Table 5), suggesting that these ions are weakly bound .

 
(iii) Crystallographic temperature factors. Crystallographic B-factors to a certain extent reflect the flexibility of a protein structure but are correlated to the data collection temperatures, data quality, and refinement procedure . In order to pinpoint the more flexible parts of the molecule, relative B-factors were calculated for each residue . We have compared the temperature factors of the native form 2 with those of A, B1 (apo-form), B2, and C, and differences were observed in four regions (Fig . 1 and 4) . The first stretch which displays high relative B-factors compared to the native structure comprises residues 47 to 57 in structures A, B2, and C . The loss of Ca 700 might therefore be partly responsible for the higher flexibility of this loop, which is corroborated by the lack of electron density for residues 50 and 51 . The second region concerns residues 169 to 191 in the vicinity of the active site, with relatively high B-factors observed for residues 173 and 179 in structures A, B1, B2, and C, a finding consistent with side chain conformational changes induced by the loss of the zinc ion . It is likely that the removal of the zinc ion by the chelator has conferred a higher flexibility to the active-site region . Considering the C-terminal domain, Fig . 4 shows that residues 282 to 287 display high relative B-factors, thus confirming the structural changes of residues 282 and 287, which bind to Ca 701 and Ca 702 .

 
Finally, mobile regions in the neighborhood of Ca 703 and Ca 704 include residues 341 to 344, 346 to 353, and 366 to 373 .

The relative B-factors for the calcium ions are given in Table 6 . It appears that relative B-factors of Ca 702, followed by Ca 703 and Ca 704, increase with the concentration of EDTA . Despite the absence of structural changes observed for Ca 706 and its ligands, this ion displays high relative B-factors in structures B2 and C, which may be due to increased exposure to the solvent, as emphasized by increased temperature factors and Ca 706 having a water molecule as a ligand .

 
The new crystal form (form 3) confirmed that the differences between the psychrophilic enzyme and its mesophilic counterparts, and especially the binding of the extra calcium ion in the proteolytic domain, are independent of crystallization conditions or crystal packing .

Experiments A to D contribute to elucidate the role of divalent metal ions in PAP. In light of the conditions of experiments B1 and B2 (contact times with EDTA of 12 days and 2 months, respectively), it clearly appears that although the zinc ion was more strongly coordinated than Ca 700 (1), it was the first to be chelated by EDTA . Moreover, the complexation constant of Zn²⁺ by EDTA is weaker than for Ca²⁺ (10^10.7 and 10^16.3, respectively, at 25°C) .

Notwithstanding chelation of Ca 700 by EDTA appears favorable from a thermodynamic point of view, our experiments suggest that the action of EDTA on PAP predominantly is controlled by kinetics in agreement with studies on the ß-roll performed by Rose et al . (28) . Thus, in order to explain the increased sensitivity of PAP compared to mesophilic counterparts, kinetic parameters such as steric hindrance should be considered with the increased accessibility to the active site in PAP as the key factor .

Our studies of all calcium-binding sites in structures A, B1, B2, C, and D and in the native form 2 of PAP have led us to analyze their different roles in the global stability of the molecule and their potential implication in the folding of PAP .

Structures B1 and B2 indicate that Ca 700 is present during the crystallization process and hereby when crystals are formed and is only chelated by EDTA after crystal formation . One can speculate that if this site is not occupied, the molecule is too flexible for crystal formation . It should also be stressed that the major part of the intermolecular interactions to the proteolytic domain is performed with residues from the region surrounding this calcium ion . These findings, taken together with the fact that the 13-Å loop movement upon binding of Ca 700 is nonreversible upon chelation in structures A, B2, C, and D, are indeed in agreement with an increased accessibility to the active site, caused by this same loop movement, being a key factor of the adaptation of this enzyme to cold environments . Thus, Ca 700 does not appear to improve the global stability of PAP, but rather the stability of the proteolytic domain as strengthened by the partial disorganization of the loop surrounding the depleted calcium site .

On the basis of these findings, we have proposed a classification of calcium ions from the least to the most tightly bound . After 2 months, a 1 mM concentration of EDTA appears to be sufficient for chelating the calcium ion located in the N-terminal proteolytic domain (Ca 700) . When the concentration of EDTA was increased to 5 mM, ligands of Ca 702 move away from the calcium-binding site . This weakly bound calcium ion also displays a high B-factor and is thought to be partially occupied, especially in structure C . This effect increases with the concentration of EDTA and, at 85 mM, Ca 702 seems to be replaced by a water molecule . One ligand (Asp-287) is shared by Ca 702 and Ca 701 . When the Ca 702 binding site is modified, structural changes in the Ca 701 binding site are concomitant . After 2 months in 5 mM EDTA, the relative B-factor of Ca 706 increases by 60%, indicating a partial occupation of this site .

Ca 703 and Ca 704 in turn seem to be affected when the concentration of EDTA was increased to 10 mM (structure C), as indicated by their high relative B-factors . Structure D, for which conformational movements among the ligands of Ca 703 and Ca 704 have been observed, confirms this observation .

As for the C-terminal domain, it can be concluded that Ca 702 and hereafter Ca 701 are the less tightly bound, followed by Ca 706, Ca 703, and Ca 704 . Finally, Ca 705 emerges as the most tightly bound ion since its binding site undergoes no significant changes in any of the experiments .

This classification is coherent with coordination states and spatial positions in the structures . Ca 701 and Ca 702, which are located in one extremity of the C-terminal domain and next to the catalytic domain, are relatively accessible to the solvent . Although Ca 706 is bound more internally in the central region of the ß-roll, this ion had one water molecule as ligand and then was more exposed to the solvent . Finally, the last three calcium ions (Ca 703 to 705) seem to hold the structure together, thus confirming their putative implication in the folding of the molecule after transmembrane translocation .

Our studies confirm that PAP is sensitive to low concentrations of EDTA, as described previously (11), since 1 mM EDTA in experiment A and 5 mM EDTA in experiment B1 appear to be sufficient for chelating the zinc ion in the N-terminal proteolytic domain and hence to inactivate the protein .

However, our results do not explain previous fluorescence studies that indicated enzyme unfolding after 2 h of incubation in 2 mM EDTA (11) but, because the experiments described here take place within a crystal, direct comparisons are not possible .

From an overall structural point of view, relative exposure of some calcium ions located in the C-terminal domain to the solvent seems to allow a higher conformational flexibility due to the lack of protein-protein interactions by the bias of crystal contacts .

The rigid nature of the C-terminal domain, which probably plays an important role in the folding of the molecule, and the fact that Ca 703 to Ca 705 are essential determinants of the parallel ß-roll structure (3), a finding supported by the present study, promote restrictions in conformational changes within this domain . This can probably explain why the 3D structure of PAP remains folded in high concentrations of EDTA (up to 85 mM as found in the soaking experiments) . Possibly, the chelation of Ca 705 would result in the unfolding of the protein, which in turn may explain why form 2 crystals cracked in the soaking experiments when the EDTA concentrations were beyond 85 mM .

We thank C . Gerday for providing the enzyme for the crystallographic studies and R . Kahn for assistance with data collection at the ESRF .

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