Native Thrombocidin-1 and Unfolded Thrombocidin-1 Exert Antimicrobial Activity via Distinct Structural Elements

Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 286(50):43506-14. DOI: 10.1074/jbc.M111.248641
Source: PubMed


Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

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    • "Indeed, lysine substitution of negative or neutral residues bordering the positive patch enhanced the activity. Interestingly, reduced TC-1 had equal antimicrobial activity as the folded protein, even though the positive patch was disrupted (Kwakman et al. 2011). The structural elements of unfolded TC-1 were likely localized in the N-terminal region of the protein, since linear 15-mer synthetic peptides corresponding to the N-terminal part of TC-1 had very potent antimicrobial activity. "
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    • "The disulfide reduced form of TC-1 is almost equally effective as an antimicrobial as native TC-1 and it adopts a similar membrane-induced structure despite the loss of secondary and tertiary structure in aqueous solution (Kwakman et al., 2011). Although disulfide bonds are usually essential for retaining chemokine structure in aqueous solution and for maintaining their cell signaling functions (Rajagopalan and Rajarathnam, 2006), linearized analogs of chemokines such as CXCL9/MIG (monokine-induced by γ-interferon) and CCL28 also retain antimicrobial activity (Egesten et al., 2009; Liu and Wilson, 2010). "
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