MUTATION OF AN IQ-MOTIF: EVIDENCE FOR AN EXOSITE IN CALPAIN-2?

These studies aimed to test the hypothesis that calcium regulates calpain activity by a mechanism analogous to that used by Ca2+- calmodulin (CaM) to regulate its target enzymes. Site directed mutagenesis of a putative IQ motif, IQ413xxxR417 generated catalytic subunits modified at both, or each, of the key residues of the motif to yield ISxxxQ, ISxxxR, and IQxxxQ. Each of the mutant catalytic subunits was co-expressed with a truncated (21k) small subunit in E. coli. The heterodimeric enzymes were purified by standard methods suggesting each was properly folded. Electrophoresis under native conditions, in the absence of calcium, showed no differences between wt and enzyme mutated at R417Q. Each of these enzymes was also stable to incubation at 45 ºC. In contrast enzymes containing Q413S were resolved into multiple bands on native gels and were not stable at 45 ºC. Two calcium dependent activities were assessed: casein hydrolysis and autoproteolysis. Each of the mutants retained significant autolytic activity but lost much of their caseinolytic activity. We propose two interpretations of the results based in part on the calcium-free structures determined for calpain-2 (Hosfield et al , 1999 EMBO J. 18, 6880 and Strobl et al 2000, PNAS 97, 588). The ability of the enzymes to autoproteolyze suggests that the conformational change required for alignment of the catalytic residues is unimpaired in the mutants. Impaired casein hydrolysis may result from 1) inability to release product after cleavage or 2) failure to form a required substrate binding exosite. The latter concept is consistent with many previously known calpain attributes. Exosites provide a mechanism for achieving high substrate specificity amongst enzymes that share conserved active sites, such as those of the thrombin family and perhaps the calpain family. Confirmation and definition of such exosites will be important as they could provide targets for design of isoform selective inhibitors

A biochemical approach to define the interactome for calpain2 in endothelial cells

Current repositories for protein-protein interactions and high throughput screening methods focus on individual gene products and do not consider the significance of calcium induced conformational changes. These limitations suggest the need for alternative strategies to better define the calpain2 interactome. Affinity capture coupled with LC-MS/MS and proteomic analysis of the recovered proteins provides a powerful approach to identify protein-protein interactions for the heterodimeric calpain2. CAPN2 (rat) was modified to be catalytically incompetent (C105A) and fused with a C-terminal 15 residue peptide optimized for biotinylation by the biotin protein ligase, BirA. The resulting CAPN2*, heterodimerized with truncated CAPNS1, was purified from E. coli, and biotinylated in vitro. Biotinylated calpain2* served as ‘bait’ for streptavidin affinity capture of calpain2 and its interacting proteins from lysates of bovine aortic (BAEC) and human umbilical vein (HUVEC) endothelial cells (ECs). Protein-calpain2 complexes were formed in the presence of calcium to allow EGTA elution of interacting proteins and LC-MS/MS analysis in the absence of an abundance of bait peptides. Capture of the well characterized calpain inhibitor protein calpastatin (CAST), and a known substrate, vimentin provide proof of concept and validates the conformational integrity of the bait calpain2*. Significant overlap between datasets (two from BAEC and one HUVEC) is also encouraging. Of numerous other proteins including several annexins, ANXA1 was confirmed as a substrate for calpain2. Findings are expected to contribute to continuing efforts in the field to better characterize calpain2’s selection of substrates and may reveal other important clues to calpain’s localization and regulation