Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.     

Dynamic light scattering study of calmodulin-target peptide complexes

Dynamic light scattering (DLS) has been used to assess the influence of eleven different synthetic peptides, comprising the calmodulin (CaM)-binding domains of various CaM-binding proteins, on the structure of apo-CaM (calcium-free) and Ca(2+)-CaM. Peptides that bind CaM in a 1:1 and 2:1 peptide-to-protein ratio were studied, as were solutions of CaM bound simultaneously to two different peptides. DLS was also used to investigate the effect of Ca(2+) on the N- and C-terminal CaM fragments TR1C and TR2C, and to determine whether the two lobes of CaM interact in solution. The results obtained in this study were comparable to similar solution studies performed for some of these peptides using small-angle x-ray scattering. The addition of Ca(2+) to apo-CaM increased the hydrodynamic radius from 2.5 to 3.0 nm. The peptides studied induced a collapse of the elongated Ca(2+)-CaM structure to a more globular form, decreasing its hydrodynamic radius by an average of 25%. None of the peptides had an effect on the conformation of apo-CaM, indicating that either most of the peptides did not interact with apo-CaM, or if bound, they did not cause a large conformational change. The hydrodynamic radii of TR1C and TR2C CaM fragments were not significantly affected by the addition of Ca(2+). The addition of a target peptide and Ca(2+) to the two fragments of CaM, suggest that a globular complex is forming, as has been seen in nuclear magnetic resonance solution studies. This work demonstrates that dynamic light scattering is an inexpensive and efficient technique for assessing large-scale conformational changes that take place in calmodulin and related proteins upon binding of Ca(2+) ions and peptides, and provides a qualitative picture of how this occurs. This work also illustrates that DLS provides a rapid screening method for identifying new CaM targets.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.     

Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C.

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium-loaded (1H, 15N, and 13C) regulatory N-domain of chicken skeletal troponin-C (1-90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical-shift information, along with detailed NOE analysis and 3JHNH alpha coupling constants, permitted the determination and quantification of the Ca(2+)-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short beta-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761-779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix-B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca(2+)-induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in alpha-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca(2+)-induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.     

Mitochondrial calpain 10 is degraded by Lon protease after oxidant injury

Calpain 10 is ubiquitously expressed and is one of four mitochondrial matrix proteases. We determined that over-expression or knock-down of mitochondrial calpain 10 results in cell death, demonstrating that mitochondrial calpain 10 is required for viability. Thus, we studied calpain 10 degradation in isolated mitochondrial matrix, mitochondria and in renal proximal tubular cells (RPTC) under control and toxic conditions. Using isolated renal cortical mitochondria and mitochondrial matrix, calpain 10 underwent rapid degradation at 37°C that was blocked with Lon inhibitors but not by calpain or proteasome inhibitors. While exogenous Ca(2+) addition, Ca(2+) chelation or exogenous ATP addition had no effect on calpain 10 degradation, the oxidants tert-butyl hydroperoxide (TBHP) or H(2)O(2) increased the rate of degradation. Using RPTC, mitochondrial and cytosolic calpain 10 increased in the presence of MG132 (Lon/proteasome inhibitor) but only cytosolic calpain 10 increased in the presence of epoxomicin (proteasome inhibitor). Furthermore, TBHP and H(2)O(2) oxidized mitochondrial calpain 10, decreased mitochondrial, but not cytosolic calpain 10, and pretreatment with MG132 blocked TBHP-induced degradation of calpain 10. In summary, mitochondrial calpain 10 is selectively degraded by Lon protease under basal conditions and is enhanced under and oxidizing conditions, while cytosolic calpain 10 is degraded by the proteasome.     

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.     

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3-MDa (alphabetagammadelta)(4) hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (alphabetagammadelta)(2) octameric lobes that are oriented back-to-back with overall D(2) symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca(2+) by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca(2+) in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca(2+)-activated PhK. Although the overall dimensions of the complex were not affected by Ca(2+), the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca(2+)-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca(2+)-activated conformers of PhK are consistent with Ca(2+)-induced conformational changes in both the lobes and the interlobal bridges.     

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family

Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.     

Molecular dynamics simulations revealed Ca(2+)-dependent conformational change of Calmodulin

Molecular dynamics simulations were performed to simulate Ca(2+)-dependent conformational change of calmodulin (CaM). Simulations of the fully Ca(2+)-bound form of CaM (Holo-CaM) and the Ca(2+)-free form (Apo-CaM) were performed in solution for 4 ns starting from the X-ray crystal structure of Holo-CaM. A striking difference was observed between the trajectories of Holo-CaM and Apo-CaM: the central helix remained straight in the former but became largely bent in the latter. Also, the flexibility of Apo-CaM was higher than that of Holo-CaM. The results indicated that the bound Ca(2+) ions harden the structure of CaM.     

Solution structure and fluctuation of the Mg(2+)-bound form of calmodulin C-terminal domain

Calmodulin (CaM) is a Ca(2+)-binding protein that functions as a ubiquitous Ca(2+)-signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+)-bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+)-bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+)-induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+)-induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+)-induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+).     

Effect of extremely low frequency magnetic fields on calpain activation

The effects of low intensity, low frequency magnetic fields (MFs) on catalytic activity of the calcium dependent protease calpain was determined following the enzyme activation both in "in vitro" and "in vivo" conditions. We have observed that a 0.3 mT MF induces a significant increase in the requirement of the protease for this metal ion. This change is detectable at low [Ca(2+)] and disappears when the level of Ca(2+) is raised to saturating amounts. The observed effects are not due to transient MF(-) induced conformational changes occurring in calpain, but to direct effects of the MF on Ca(2+) ions, which become less available for the binding sites present in calpain. Altogether, these results indicate that exposure to low intensity, low frequency MFs alters the intracellular Ca(2+) "availability," thereby modifying the related cell response.     

Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.     

Different susceptibility of red cell membrane proteins to calpain degradation.

The presence of low levels of calpastatin activity in erythrocytes of hypertensive rats affects regulation of calpain activity so it is highly susceptible to activation within physiological fluctuations in [Ca2+]. Under identical conditions, in red cells of normotensive rats, calpain activation is efficiently controlled by the high levels of calpastatin activity, and a progressive increase in proteinase activity can only be observed in parallel with a decrease in the level of calpastatin. In intact erythrocytes from hypertensive rats exposed to small variations in [Ca2+], degradation of anion transport protein (band 3) and Ca(2+)-ATPase appears as a primary event indicating that these two transmembrane proteins are probably early recognized as targets of intracellular calpain activity. Furthermore, band 3 protein seems to be structurally modified in erythrocytes from hypertensive rats, as indicated by its increased susceptibility to degradation in the presence of 10-50 microM Ca2+. In addition, when exposed to progressive and limited increases in [Ca2+], erythrocytes from hypertensive rats, but not those from normotensive rats, show a high degree of fragility that can be restored to normal values by inhibition of calpain. These results indicate that, within fluctuations in [Ca2+] close to physiological values, regulation of calpain activity is efficiently accomplished in normal erythrocytes but is completely lost in cells from hypertensive animals. Regulation is of critical importance in maintaining normal structural and functional properties of selective red cell membrane and cytoskeletal proteins, among which band 3 and Ca(2+)-ATPase appear to be the substrates with highest susceptibility to digestion by calpain.     

Conformational dynamics of complementarity-determining region H3 of an anti-dansyl Fv fragment in the presence of its hapten

Antigen-induced structural changes in the Fv fragment of an anti-dansyl immunoglobulin G were studied by X-ray crystallography and stopped-flow fluorescence measurement. The crystal structure of the Fv fragment complexed with dansyl-lysine was determined at a resolution of 1.85 A. The dansyl-lysine molecule bound to a narrow cavity formed by the complementarity-determining regions H3 and H1, the N-terminal region of the VH domain and L2 of the VL domain. The structure of the binding site in the crystal structure explained well the results of the previous nuclear magnetic resonance measurements. The hapten binding caused remarkable conformational changes in H3 and its environmental structures, including the hydration structure from those observed in the unliganded state. The tip of H3 moved about 12 A from its position in the unliganded state. In addition, because of the contacts of H3 with the VL domain at the domain interface, the conformational changes of H3 resulted in the relative rotation of the variable domains by 5 degrees from their association observed in the unliganded state. The hydrophobic interactions at the domain interface seemed to be particularly important for the mutual rotation of the domains. The stopped-flow fluorescence measurement monitoring the interaction of the dansyl group and the binding pocket revealed that H3 was in a conformational equilibrium of three consecutive conformational states in the presence of dansyl-lysine in solution; an unliganded state preventing the access of the hapten, another unliganded state able to bind the hapten and the complex. The conformational dynamics of H3 in recognizing and binding the hapten molecule are discussed on the basis of the structural information from the present and previous studies.     

Effects on the conformation of FVIIa by sTF and Ca²⁺ binding: studies of fluorescence resonance energy transfer and quenching

The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5? for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4? upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. It is known from amidolytic activity measurements that Ca(2+) binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca(2+) binding. Interestingly, this Ca(2+)-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe.     

Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis

Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.     

The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca(2+) in proximity to the two hemes [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. This Ca(2+) did not readily dissociate; however, after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca(2+)-dependent changes in heme spin state and conformation. Addition of H(2)O(2) to the Ca(2+)-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca(2+) to the Ca(2+)-depleted MauG, full TTQ biosynthesis activity and reactivity toward H(2)O(2) were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca(2+) binding to Ca(2+)-depleted MauG indicated a two-step mechanism. Ca(2+) initially reversibly binds to Ca(2+)-depleted MauG (K(d) = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10(-3) s(-1)) but highly favorable (K(eq) = 4.2) conformational change, yielding an equilibrium dissociation constant K(d,eq) value of 5.3 μM. The circular dichroism spectra of native and Ca(2+)-depleted MauG were essentially the same, consistent with Ca(2+)-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases.     

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.