Critical amino acids in the active site of meprin metalloproteinases for substrate and peptide bond specificity

Abstract

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.

Fig. 1. Electrostatic differences in the protease domain of meprins α and ß

Sequence alignment of the amino acids in the meprin (mep) protease domains of mouse (m), rat (r) and human (h) meprins ß (b) and α (a), respectively. The numbering system is based on the nascent mouse meprin α sequence and begins with the N-terminal amino acid of the protease domain. Residues conserved among mouse, rat and human meprin ß are colored blue. Similar amino acids conserved among all six sequences are colored red in meprin α. Basic residues conserved throughout the species in the ß subunits that are nonbasic in α are denoted by * below the sequences. Amino acids that were mutated in mouse α to the corresponding basic ß residues are boxed.

Fig. 2. Critical electrostatic differences within the active site of meprins α and ß

The top panels are homology models in space filling (left) and ribbon (right) representations of the protease domain of mouse meprin α based on the crystal structure of crayfish astacin. For the space filling representations acidic residues, Asp and Glu, are red, basic residues Lys and Arg, are blue; all other residues are white. The zinc located in the center of the active site is green. For the ribbon representation α-helices are red and ß-sheets are cyan. The bottom panels contain the corresponding representations for mouse meprin ß. Active site electrostatic differences are denoted by arrows in space filling models and are numbered in ribbon representation.

Fig. 3. Peptide bonds hydrolyzed in cholecystokinin by meprin mutants

Chromatograms of CCK8s treated with wild-type merpin α, αY199K, αF161R and αP228K mutants separated on a reverse phase HPLC column. The hydrolysis products were identified by MALDI-TOF and correspond to the W-M and D-F hydrolysis sites.

Fig. 4. Protease domains of several proteases of the astacin family

Homology models of the protease domains of rat meprin β(rmepβ), mouse bone morphogenetic protein-1 (mBMP-1; procollagen C-proteinase), flavastacin (FLAV), mmepα (mouse meprin α), crayfish astacin (AST) and high choriolytic enzyme-1 (HCE-1) were produced as described in Experimental Procedures.