Flexibility analysis and structure comparison of two crystal forms of calcium-free human m-calpain

The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papain-related catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and full-length human apo-m-calpain revealed the domain arrangement and explained the inactivity of m-calpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that m-calpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papain-like catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of m-calpain, we have determined and refined the human full-length apo-m-calpain structure of a second crystal form to 3.15 A resolution. Here we present this new structure, compare it with our first structure now re-refined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calcium-induced activation process.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Molecular evolution of intracellular Ca2+-dependent proteases

The structural features and evolutionary interrelationships of the intracellular Ca2+-dependent cysteine enzymes calpains, proteases of the family C2 (EC 3.4.22.17), are considered. A variety of identified sequences of calpains and calpain-like polypeptides found in organisms of different taxons, from the simplest to mammals, are described. Calpains of the major evolutionary groups, typical and atypical, are classified by the analysis of their phylogenetic tree and are differentiated due to the presence of the calmodulin-like Ca2+-binding domain. It is shown that, along with enzymes having "advanced" characteristics (heterodimeric structure, presence of tissue-specific isoforms and splice variants, regulation by the endogenous inhibitor calpastatin, and others), higher organisms contain homologues of calpains of lower eukaryotes. A high degree of homology of the catalytic domain of calpains and the variable structure of other functional domains indicate that calpains are implicated in various physiological processes with the retention of their regulatory role.     

Molecular evolution of intracellular Ca<Superscript>2+</Superscript>-dependent proteases

The structural features and evolutionary interrelationships of the intracellular Ca²⁺-dependent cysteine enzymes calpains, proteases of the family C2 (EC 3.4.22.17), are considered. A variety of identified sequences of calpains and calpain-like polypeptides found in organisms of different taxons, from the protozoa to mammals, are described. Calpains of the major evolutionary groups, typical and atypical, are classified by the analysis of their phylogenetic tree and are differentiated due to the presence of the calmodulin-like Ca²⁺-binding domain. It is shown that, along with enzymes having “advanced” characteristics (heterodimeric structure, presence of tissue-specific isoforms and splice variants, regulation by the endogenous inhibitor calpastatin, and others), higher organisms contain homologues of calpains of lower eukaryotes. A high degree of homology of the catalytic domain of calpains and the variable structure of other functional domains indicate that calpains are implicated in various physiological processes with the retention of their regulatory role.     

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.     

Contribution of distinct structural elements to activation of calpain by Ca2+ ions

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a K(d) of 3400 microm. Phospholipids lower this value to 220 microm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 microm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this "extended transducer" mechanism is presented.     

Crystal structure of the C-terminal three-helix bundle subdomain of C. elegans Hsp70

Hsp70 chaperones are composed of two domains; the 40 kDa N-terminal nucleotide-binding domain (NDB) and the 30 kDa C-terminal substrate-binding domain (SBD). Structures of the SBD from Escherichia coli homologues DnaK and HscA show it can be further divided into an 18 kDa beta-sandwich subdomain, which forms the hydrophobic binding pocket, and a 10 kDa C-terminal three-helix bundle that forms a lid over the binding pocket. Across prokaryotes and eukaryotes, the NBD and beta-sandwich subdomain are well conserved in both sequence and structure. The C-terminal subdomain is, however, more evolutionary variable and the only eukaryotic structure from rat Hsc70 revealed a diverged helix-loop-helix fold. We have solved the crystal structure of the C-terminal 10 kDa subdomain from Caenorhabditis elegans Hsp70 which forms a helical-bundle similar to the prokaryotic homologues. This provides the first confirmation of the structural conservation of this subdomain in eukaryotes. Comparison with the rat structure reveals a domain-swap dimerisation mechanism; however, the C. elegans subdomain exists exclusively as a monomer in solution in agreement with the hypothesis that regions out with the C-terminal subdomain are necessary for Hsp70 self-association.     

Interaction of calpastatin with calpain: a review

Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca2+.     

An extracellular calcium-binding domain in bacteria with a distant relationship to EF-hands

Extracellular Ca(2+)-dependent nuclease YokF from Bacillus subtilis and several other surface-exposed proteins from diverse bacteria are encoded in the genomes in two paralogous forms that differ by a approximately 45 amino acid fragment, which comprises a novel conserved domain. Sequence analysis of this domain revealed a conserved DxDxDGxxCE motif, which is strikingly similar to the Ca(2+)-binding loop of the calmodulin-like EF-hand domains, suggesting an evolutionary relationship between them. Functions of many of the other proteins in which the novel domain, named Excalibur (extracellular calcium-binding region), is found, as well as a structural model of its conserved motif are consistent with the notion that the Excalibur domain binds calcium. This domain is but one more example of the diversity of structural contexts surrounding the EF-hand-like calcium-binding loop in bacteria. This loop is thus more widespread than hitherto recognized and the evolution of EF-hand-like domains is probably more complex than previously appreciated.     

Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.     

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the RIM1alpha C2B domain at 1.7 A resolution

RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.     

Amino-terminal dimerization, NRDP1-rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease 8 (USP8)

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.     

Protein-chemical identification of the major cleavage sites of the Ca2+ proteinase on murine vimentin, the mesenchymal intermediate filament protein

Neutral thiol proteinases (calpains), activated by calcium are involved in the intracellular turnover of intermediate filaments but the precise position of the cleavage points has remained unknown. Here we identify by direct sequence analysis the major cleavage sites found when murine vimentin is digested by limited proteolysis in vitro with calpain purified from porcine kidney. Contrary to some previous suggestions, no absolute sequence specifity could be detected although 10 specific sites have been identified. This result is in line with the cDNA derived amino-acid sequence of a calpain, which pointed to a similarity of the catalytic site with the active sites in papain, cathepsin and actinidin. However, all major cleavage sites are located within regions of the vimentin molecule, which in current models of intermediate filament structure are thought to be non-helical: the amino-terminal headpiece, the carboxy-terminal tailpiece and the spacer separating the two major coiled-coil domains. The sequence information about the cleavage sites was extended to provide the amino-terminal 119 residues of murine vimentin.     

Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.     

Interdomain association in fibronectin: insight into cryptic sites and fibrillogenesis

The process by which fibronectin (FN), a soluble multidomain protein found in tissue fluids, forms insoluble fibrillar networks in the extracellular matrix is poorly understood. Cryptic sites found in FN type III domains have been hypothesized to function as nucleation points, thereby initiating fibrillogenesis. Exposure of these sites could occur upon tension-mediated mechanical rearrangement of type III domains. Here, we present the solution structures of the second type III domain of human FN ((2)FNIII), and that of an interaction complex between the first two type III domains ((1-2)FNIII). The two domains are connected through a long linker, flexible in solution. A weak but specific interdomain interaction maintains (1-2)FNIII in a closed conformation that associates weakly with the FN N-terminal 30 kDa fragment (FN30 kDa). Disruption of the interdomain interaction by amino-acid substitutions dramatically enhances association with FN30 kDa. Truncation analysis of (1-2)FNIII reveals that the interdomain linker is necessary for robust (1-2)FNIII-FN30 kDa interaction. We speculate on the importance of this interaction for FN function and present a possible mechanism by which tension could initiate fibrillogenesis.     

Amino acid sequence of myosin essential light chain from the scallop Aquipecten irradians

The amino acid sequence of the scallop myosin essential light chain (SELC) was determined from analysis of the intact, S-carboxymethylated protein and peptides produced by cleavage at its four methionine residues by cyanogen bromide digestion and at its six arginine residues by citraconylation and tryptic digestion. SELC contains 156 amino acid residues, including three cysteines, four tyrosines, one tryptophan, two histidines, and an unblocked amino-terminal proline. The protein has a calculated Mr of 17,616. SELC is an acidic protein, with a net charge of 18- at physiological pH. Comparative analysis reveals four homologous domains (I-IV), which arose by reduplication of a gene for a small, ancestral calcium binding protein. Each domain has a helix-loop-helix structure, with all the ligands for calcium binding located within a 12-residue segment that spans the loop and the first turn of the following helix. Potential calcium binding sequences were found in the ancestral sites III (residues 94-105) and IV (residues 132-143). Mutations in critical positions in domains I and II seem to preclude the possibility of calcium binding in the amino-terminal half of SELC. An unexpected third potential calcium binding segment (at residues 119-130, predicted to be in helical conformation) was found in domain IV. A reactive thiol group (Cys-78) that is involved in binding of regulatory light chains was tentatively located in an extended "linker region", which connects the two halves of the molecule.