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J. Biol. Chem., Vol. 282, Issue 47, 34129-34138, November 23, 2007

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Activation of the SspA Serine Protease Zymogen of Staphylococcus aureus Proceeds through Unique Variations of a Trypsinogen-like Mechanism and Is Dependent on Both Autocatalytic and Metalloprotease-specific Processing^*

Nicholas N. Nickerson¹, Lata Prasad, Latha Jacob, Louis T. Delbaere², and Martin J. McGavin³

From the Department of Laboratory Medicine and Pathobiology, University of Toronto Sunnybrook Health Science Centre, Toronto, Ontario M4N 3M5, Canada and the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA²⁹ , releasing a Leu³⁰ isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, ⁴⁰QQTQSSKQQTPKIQ⁵³. The preferred site is Gln⁴³ with secondary processing at Gln⁴⁷ and Gln⁵³. This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu⁵⁸ and then Val⁶⁹ to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val⁶⁹SspA promote rapid intermolecular processing of remaining zSspA at Glu⁶⁵, producing an N-terminal ⁶⁶HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His⁶⁶ of this penultimate isoform blocks the active site by hydrogen bonding to Ser²³⁷ and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr²³² proximal to Ser²³⁷, permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.

Received for publication, July 10, 2007 , and in revised form, September 14, 2007.

^* This work was supported in part by Canadian Institutes of Health Research Operating Grants FRN-12669 (to M. J. M.) and CIHR IG1-10162 (to L. T. J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a University of Toronto Open Fellowship.

² Recipient of a Canada Research Chair in Structural Biochemistry.

³ To whom correspondence should be addressed. Tel.: 416-480-5831; Fax: 416-480-5737; E-mail: martin.mcgavin{at}sri.utoronto.ca.

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