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...

The apr locus of Pseudomonas aeruginosa encodes alkaline proteinase (APR), a member of the metzincin metalloendopeptidase superfamily, and an 11.4-kDa alkaline proteinase inhibitor (APRin). We describe here the expression in Escherichia coli and characterization of full-length and N-terminally truncated APRin proteins. Fluorescence and circular dichroism spectra indicated that the recombinant proteins were folded into native-like structures. Analytical ultracentrifugation showed that APRin was monomeric and formed a Pseudomonas aeruginosa is a ubiquitous microorganism that is usually benign with respect to healthy individuals but pathogenic in a number of clinical settings, including surgical patients, burn victims, and persons with cystic fibrosis (1). Eye infection by P. aeruginosa is relatively common, particularly in wearers of contact lenses (2); if not promptly treated, these infections can result in corneal ulceration and visual impairment. Among the virulence factors of P. aeruginosa are two secreted metalloproteinases (3), elastase (pseudolysin, PL) 1 and alkaline proteinase (aeruginolysin, APR). Substrates for PL include extracellular matrix structural proteins, immunoglobulins, complement components, and ␣ 1 -proteinase inhibitor (3). APR degrades fibrinogen, fibrin (4), laminin (5), and ␥-interferon (6); it also is capable of activating Hageman factor (7) and matrix metalloproteinases (MMPs) (8).APR and PL were initially characterized by Morihora (9, 10). Subsequent structural comparison revealed that PL is related to thermolysin of Bacillus thermolyticus, and APR is homologous to the 50-kDa metalloproteinases secreted by Serratia marcescens and Erwinia chrysanthemi (11,12). These enzymes constitute the serralysin branch of the metzincin superfamily of metalloendopeptidases (13). X-ray crystallography of APR (14) and SMP (15), the Serratia homolog of APR, shows that both consist of an N-terminal catalytic domain of about 230 residues and a C-terminal calcium binding domain of approximately 240 residues that appears to be required for proteinase secretion (16). The catalytic domain contains sequences characteristic of the metzincin superfamily of metalloendopeptidases, namely a zinc-binding motif (HEXXHXXGXXH) and a conserved Met located in a turn near the base of the metal binding pocket. Other metzincins include astacins, MMPs, reprolysins, and the adamalysins (12).Serralysin secretors are also capable of producing specific inhibitory proteins of about 125 residues that are transported to the periplasm where the signal peptide is removed (17, 18). The three known members of this inhibitor family exhibit approximately 20% sequence identity and an additional 20% sequence similarity, including a conserved disulfide bond (19). The mature inhibitors from E. chrysanthemi and P. aeruginosa have N-terminal Ser, whereas the N terminus of the S. marcescens inhibitor is Gly; in addition, 8 of 15 N-terminal residues are identical among the three inhibitors.X-ray crystallography of a complex between SMP and the

The protein networks of cell-wall-biosynthesis assemblies are largely unknown. A key class of enzymes in these assemblies is the lytic transglycosylases (LTs), of which eleven exist in P. aeruginosa. We have undertaken a pulldown strategy in conjunction with mass-spectrometry-based proteomics to identify the putative binding partners for the eleven LTs of P. aeruginosa. A total of 71 putative binding partners were identified for the eleven LTs. A systematic assessment of the binding partners of the rare lipoprotein A (RlpA), one of the pseudomonal LTs, was made. This 37-kDa lipoprotein is involved in bacterial daughter-cell separation by an unknown process. RlpA participates in both the multi-protein and multi-enzyme divisome and elongasome assemblies. We reveal an extensive protein-interaction network for RlpA involving at least 19 proteins. Their kinetic parameters for interaction with RlpA were assessed by microscale thermophoresis, surface-plasmon resonance, and isothermal-titration calorimetry. Notable RlpA binding partners include PBP1b, PBP4, and SltB1. Elucidation of the protein-interaction networks for each of the LTs, and specifically for RlpA, opens opportunities for the study of their roles in the complex protein assemblies intimately involved with the cell wall as a structural edifice critical for bacterial survival.

Angiotensin II-and K ؉ -stimulated aldosterone production in the adrenocortical glomerulosa cells requires induction of the steroidogenic acute regulatory protein (StAR). While both agents activate Ca 2؉ signaling, the mechanisms leading to aldosterone synthesis are distinct, and the angiotensin II response cannot be mimicked by K ؉ . We previously reported that StAR mRNA levels and promoter-reporter gene activity in transiently transfected H295R human adrenocortical cells were stimulated by angiotensin II but not by K ؉ treatment. The current study focused on identifying signaling pathways activated by angiotensin II that contribute to StAR transcriptional activation. We show that the angiotensin II-stimulated transcriptional activation of StAR was dependent upon influx of external calcium and requires protein kinase C activation. Furthermore we describe for the first time that the Janus tyrosine kinase family member, JAK2, was activated by angiotensin II treatment of H295R cells. Treatment of the cells with AG490, a selective inhibitor of JAK2, blocked JAK2 activation and StAR reporter gene activity and inhibited steroid production. Taken together these studies describe a novel pathway controlling StAR expression and steroidogenesis in adrenocortical cells.

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