Abstract
Staphylokinase (Sak) is a plasminogen activator protein that is secreted by many Staphylococcus aureus strains. Sak also offers protection by binding and inhibiting specific antimicrobial peptides (AMPs). Here, we evaluate Sak as a more general interaction partner for AMPs. Studies with melittin, mCRAMP, tritrpticin and bovine lactoferricin indicate that the truncation of the first ten residues of Sak (SakΔN10), which occurs in vivo and uncovers important residues in a bulge region, improves its affinity for AMPs. Melittin and mCRAMP have a lower affinity for SakΔN10, and in docking studies, they bind to the N-terminal segment and bulge region of SakΔN10. By comparison, lactoferricin and tritrpticin form moderately high affinity 1:1 complexes with SakΔN10 and their cationic residues form several electrostatic interactions with the protein’s α-helix. Overall, our work identifies two distinct AMP binding surfaces on SakΔN10 whose occupation would lead to either inhibition or promotion of its plasminogen activating properties.
Highlights
Sample +titrant Sak +LfcinB Sak +Tritrp Sak +mCRAMP Sak +melittin SakΔN10 +LfcinB SakΔN10 +Tritrp SakΔN10 +mCRAMP SakΔN10 +melittin
When Sak was purified from a culture of an S. aureus strain that was selected for its high plasminogen activating activity, the purified protein showed a single observable band on SDS-PAGE, and this form was identified as SakΔN1042
We evaluate the specificity of two forms of staphylokinase for their capacity to bind different AMPs using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy
Summary
Sample +titrant Sak +LfcinB Sak +Tritrp Sak +mCRAMP Sak +melittin SakΔN10 +LfcinB SakΔN10 +Tritrp SakΔN10 +mCRAMP SakΔN10 +melittin. Tritrpticin is a cathelicidin derived from porcine neutrophils that belongs to the Trp- and Arg-rich family of AMPs33 It adopts a unique two-turn structure in the presence of membranes[34]. Bovine lactoferricin (LfcinB) is another Trp- and Arg-rich peptide that is derived through intestinal proteolysis of the milk protein lactoferrin and it adopts an amphipathic β-hairpin structure in aqueous solution[35,36]. Together, these four AMPs represent amphipathic α-helical, β-hairpin and turn-like structures. The two distinct protein-peptide complexes may explain the different effects seen on the plasminogen activating properties of staphylokinase
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