Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.Aminopeptidases (APs) catalyze the sequential removal of amino acids from the unblocked N-termini of protein or peptide substrates, a process necessary for intracellular metabolism [1] and implicated in several human diseases [2]. Most APs are metalloproteases and are classified based on substrate preference towards an acidic, basic or neutral amino acid at the P1 position of the scissile peptide bond. Very few acidic APs are known to date, the most extensively studied being the membrane-bound glutamyl aminopeptidase (ENPEP, also known as aminopeptidase A; EC 3.4.11.7) [3]. ENPEP, a membrane-bound Ca2+-activated enzyme, is involved in the renin-angiotensin system (RAS) by catalysing the conversion of angiotensin II to angiotensin III, a key regulator of blood pressure [4,5]. A second, cytosolic acidic AP has been reported in yeast, fungi and mammals, and termed aspartyl aminopeptidase (DNPEP, also known as