Similar to bacteria, synthesis of mitochondrial DNA-encoded proteins requires an N-formylated methionine to initiate translation. Thus, the N-formylated methionine peptides originating from mitochondria should be recognized as danger signals. To date, only one such peptide, denoted as mitocryptide-2 (MCT-2), originating from the N-terminal of the mitochondrial cytochrome b, has been isolated from mammalian tissues. Human neutrophils express FPR1 and FPR2 that detect formyl peptides, and the precise structural determinants for receptor recognition remain to be elucidated. MCT-2 is known to activate neutrophils through FPR2 but not FPR1. The aim of this study was to elucidate the structural determinants of importance for receptor preference and human neutrophil activation in MCT-2 by generating a series of MCT-2 variants. We show that there is an absolute requirement for the N-formyl group and the side chain of Met¹ at position 1 of MCT-2 but also the C terminus is of importance for MCT-2 activity. We also uncovered individual side chains that positively contribute to MCT-2 activity as well as those suppressed in the response. The MCT-2 peptide and its two polymorphic variants ([Thr⁷]MCT-2 and [Ser⁸]MCT-2) all activated neutrophils, but MCT-2 containing Ile⁷ and Asn⁸ was the most potent. We also show that some peptide variants displayed a biased FPR2-signaling property related to NADPH oxidase activation and β-arrestin recruitment, respectively. In conclusion, we disclose several critical elements in MCT-2 that are required for neutrophil activation and disclose structural insights into how FPR2 recognition of this mitochondrial DNA-derived peptide may increase our understanding of the role of FPR2 in aseptic inflammation.

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