The 3D Crystal Structure of BSAP - a Unique Double-Zinc Aminopeptidase from Bacillus subtilis

Aminopeptidases (APs) are enzymes that catalyze the cleavage of single amino acids from the amino terminus of peptides and proteins. These enzymes are involved in a wide range of biological and medically-related processes, such as protein digestion, protein degradation and cell-cycle control. One such enzyme is the extracellular aminopeptidase from Bacillus subtilis (BSAP), a protein monomer consisting of 455 amino acid residues (MW of 46kD) and a double-zinc catalytic center. The goal of the current project was to determine the three-dimensional structure and the structure-function relationships of BSAP, in order to characterize this unique sub-family of high-MW APs within the larger family of M28 metallo-enzymes. The crystal structure of BSAP has been recently determined in its double-zinc form to 2.5 Å resolution. The density shows three monomers in the crystallographic asymmetric unit. The crystallographically-observed part of the protein consists of 417 residues, of which 265 show high resemblance to Streptomyces griseus AP (SGAP) and the rest consists of a PA domain and an unstructured C-terminal domain. The PA domain seems to serve as a lid that is positioned above the substrate binding site, yet far enough to allow access to the site through a funnel. The C-terminal folds into this funnel and blocks access to the binding site completely, interacting with the catalytic zinc ions. We propose a model in which activation of BSAP is initiated by removal of the C-terminal. Such an activation mechanism presents a novel strategy of regulation for peptidases that appears to be prevalent in other bacteria, some of which with pathogenic potential.