Abstract
Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.