Bryan Ericksen - Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function

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      Publication Details (including relevant citation   information):

    Zhao L, Tolbert WD, Ericksen B, Zhan C, Wu X, Yuan W, Li X, Pazgier M, Lu W. Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function. PLoS One. 2013 Nov 13;8(11):e78937. doi: 10.1371/journal.pone.0078937. eCollection 2013. PubMed PMID: 24236072; PubMed Central PMCID: PMC3827289.

      Abstract:

        HNP1 is a human alpha defensin that forms dimers and multimers   governed by hydrophobic residues, including Tyr¹⁶, Ile²⁰, Leu²⁵,   and Phe²⁸. Previously, alanine scanning mutagenesis identified   each of these residues and other hydrophobic residues as   important for function. Here we report further structural and   functional studies of residues shown to interact with one another   across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the   double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the   quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested   binding to HIV-1 gp120 and HNP1 by surface plasmon resonance,   binding to HIV-1 gp41 and HNP1 by fluorescence polarization,   inhibition of anthrax lethal factor, and antibacterial activity   using the virtual colony count assay. Similar to the previously   described single mutant W26A-HNP1, the quadruple mutant displayed   the least activity in all functional assays, followed by the   double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A   single mutations were milder than the double mutant   I20A/L25A-HNP1. Crystallographic studies confirmed the correct   folding and disulfide pairing, and depicted an array of dimeric   and tetrameric structures. These results indicate that side chain   hydrophobicity is the critical factor that determines activity at   these positions.

      Address (URL): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827289/