Serralysins are a family of metalloproteases secreted by Gram-negative bacteria into the medium in the form of inactive zymogens. Usually, all serralysin secretors have on the same operon a gene coding for a periplasmic 10-kDa protein, which is an inhibitor of the secreted protease. The recent characterization of the inhibitor of the alkaline protease from Pseudomonas aeruginosa revealed a surprisingly low dissociation constant of 4 pM, contrary to earlier studies on homologous systems, where inhibition constants in the M range were reported. To approach a more accurate understanding, the crystal structure of the complex between inhibitor and protease from P. aeruginosa was determined at 1.74 Å resolution and refined to R free ‫؍‬ 0.204. The structure reported here shows clearly that the N terminus of the inhibitor forms a coordinative bond to the catalytic Zn 2؉ ion with a nitrogen-zinc distance of 2.17 Å. We conclude that this interaction adds substantially to the complex stability and show also that similar interactions are found in other metzincin-inhibitor complexes.Pseudomonas aeruginosa is a ubiquitous bacterium that is responsible for many nosocomial infections such as pneumonia (1), surgical wound infections (2), and respiratory tract infections in cystic fibrosis patients (3). P. aeruginosa secretes a large variety of proteins (4). One of these proteins is the alkaline protease aeruginolysin (APR), 1 originally characterized by Morihara (5, 6).APR is a member of the serralysin family, a group of 500-kDa bacterial metalloproteases which in turn belong to the metzincin metalloprotease superfamily (7). The metzincins are characterized by the zinc binding motif HEXXHXXGXXH and a conserved methionine, which is located on a turn near the catalytic site, 40 -60 residues toward the C terminus. Although sequence identity 2 between the different subfamilies of the metzincins is very low, their catalytic domains do have similar three-dimensional structures (8 -14). X-ray crystallography of APR (13), of the Serratia marcescens homologue called SMP (Serratia metalloprotease (14)), and of PrtC, one of four serralysins secreted by Erwinia chrysanthemi, 3 shows that serralysins can be divided into an N-terminal catalytic domain and a C-terminal structural domain, each consisting of about 230 to 240 residues. The structural domain consists of the secretion signal located within the last 70 residues and a repetitive glycine-rich nonapeptide (15-17) that folds into a ␤-roll stabilized with calcium ions. These sequence repeats have been found in a variety of toxins, the so-called RTX toxins (18), and appear to be involved also in the secretion process.Like other serralysin secretors, P. aeruginosa can produce a specific protease inhibitory protein of 131 residues, including a signal peptide of 25 residues that is cleaved off during secretion into the periplasm. The inhibitor is entirely located in the periplasm of P. aeruginosa, where its presumed physiological function is to protect the periplasmic proteins against the s...

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The metzincins constitute a subclan of metalloproteases possessing a HEXXHXXGXXH/D zinc-binding consensus sequence where the three histidines are zinc ligands and the glutamic acid is the catalytic base. A completely conserved methionine is located downstream of this motif. Families of the metzincin clan comprise, besides others, astacins, adamalysins proteases, matrix metalloproteases, and serralysins. The latter are extracellular 50 kDa proteases secreted by Gram-negative bacteria via a type I secretion system. While there is a large body of structural and biochemical information available, the function of the conserved methionine has not been convincingly clarified yet. Here, we present the crystal structures of a number of mutants of the serralysin member protease C with the conserved methionine being replaced by Ile, Ala, and His. Together with our former report on the leucine and cysteine mutants, we demonstrate here that replacement of the methionine side chain results in an increasing distortion of the zinc-binding geometry, especially pronounced in the x 2 angles of the first and third histidine of the consensus sequence. This is correlated with an increasing loss of proteolytic activity and a sharp increase of flexibility of large segments of the polypeptide chain.

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