Crystal Structure of a Complex between Pseudomonas aeruginosa Alkaline Protease and Its Cognate Inhibitor

Abstract

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...

Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa

“…The rationale for substitution of Trp at position I2 was that the indole side chain might better occupy the S1Ј pocket, which is non-polar and incompletely filled by the hydroxymethyl side chain of Ser-2I in the complex with the wt inhibitor (9). This pocket contains several aromatic residues that might interact favorably with an aromatic group of the inhibitor.…”

“…In particular, we were interested in Ser-2I, which is conserved among known serralysin inhibitors as well as in the TIMPs. This residue occupies the S1Ј pocket of the target proteinase (9,13,14), and its hydroxyl donates a hydrogen bond to the catalytic base, which is Glu-177P in APR. Our rationale for constructing the particular mutations in APRin position 2 is as follows.…”

“…Crystal structures of the S. marcescens serralysin-Erwinia chrysanthemi inhibitor Inh and APR⅐APRin complex from P. aeruginosa have been determined (9,10). These structures show that both the Erwinia and the Pseudomonas inhibitors fold into disulfide-stabilized, eight-stranded ␤-barrels with an N-terminal trunk of 10 amino acids with the first five residues of the trunk occupying the extended substrate binding site of the enzyme (see Fig.…”

“…They are produced as precursors of about 125 amino acids with an N-terminal signal sequence that is removed to form the mature inhibitor of 103-106 residues (8). The seven serralysin inhibitors known to date share ϳ20% sequence identity and 40 -50% sequence similarity (9). In particular, residue 2 is always Ser, whereas residue 3 is Leu in 6 of 7 known inhibitors and Phe in the seventh.…”

“…Finally, the mechanism of interaction between serralysins and their inhibitors is similar to that of the MMPs and their cognate inhibitors, the TIMPs (9), although the two classes of inhibitor bear no apparent sequence homology. In both the N-terminal residue of the inhibitor chelates the catalytic zinc; in addition, both inhibitors have a hydroxyl group in the penultimate position that hydrogen bonds to the catalytic Glu of the enzyme (9,13,14).…”