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

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.