The signal peptide of staphylococcal protein A alters the multimeric states of PepV

Signal peptides (SPs) have traditionally been recognized as molecular address tags that direct protein translocation between subcellular compartments. However, emerging evidence suggests that they serve additional functions beyond their canonical role in protein trafficking. In this study, we investigate the signal peptide of staphylococcal protein A (SpA), which contains the YSIRK/GXXS motif and facilitates translocation at bacterial cross-walls. Our findings reveal a previously unrecognized regulatory function of the SpA SP: it modulates the multimerization and enzymatic activity of PepV, a SecA-associated peptidase implicated in surface protein display. While PepV primarily exists as a dimer, it can also form higher-order assemblies, including tetramers, though the functional significance of these multimeric forms remains unclear. We demonstrate that the SpA SP inhibits the formation of PepV tetramers and higher-order oligomers while also suppressing its dipeptidase activity. In contrast, the canonical Sec signal peptide from SasF reduces PepV enzymatic activity without affecting its oligomerization. These findings highlight a novel regulatory dimension of signal peptides, illustrating their ability to modulate enzyme function and protein oligomerization. This expands their functional repertoire beyond protein trafficking, revealing a previously unappreciated layer of cellular regulation.IMPORTANCESignal sequences have traditionally been regarded as address tags that direct protein precursors to their appropriate cellular or extracellular destinations. Emerging evidence indicates that these sequences not only influence targeting and membrane insertion but also can retain functional roles after cleavage from the parent protein. Here, we present evidence for a previously unrecognized regulatory function of an uncommon Gram-positive signal peptide, demonstrating its ability to modulate both protein multimerization and enzymatic activity. These findings suggest that some signal peptides may serve broader regulatory roles beyond their conventional function in protein transport.