Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, beta 1-3 region ((25)SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK(60)) in N-terminal half and alpha 4 beta 9 region ((138)KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved beta 1-3 but not alpha 4 beta 9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted beta 1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored beta 1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the beta 1-3 region is responsible for the inhibition of proteolytic activity of MMP-9. (C) 2018 Elsevier Inc. All rights reserved.