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.
Highlights
Defensins are a family of antimicrobial peptides of innate immunity broadly active against bacteria, viruses and toxins [1,2,3,4,5,6]
Analogs Crude HNP1 mutants, after HF cleavage and ether precipitation, gave rise to molecular masses in agreement with the expected values calculated on the basis of the average isotopic compositions of reduced defensins
Using the efficient protocol established for folding of HNP1, the reduced analogs Y16A/F28A-HNP1, I20A/ L25A-HNP1, I20A-HNP1, and L25A-HNP1 productively folded in the presence of 2 M urea and 25% DMF, giving the three disulfide bridges of Cys2–Cys30, Cys4–Cys19 and Cys9–Cys29
Summary
Defensins are a family of antimicrobial peptides of innate immunity broadly active against bacteria, viruses and toxins [1,2,3,4,5,6]. For HNP1, the canonical dimer interface is formed by the antiparallel extensions of the b2 strands stabilized by the reciprocal mainchain hydrogen bonds contributed by Thr and Ile, and the hydrophobic packing of the side chains of Tyr, Tyr, Phe and the Cys2–Cys disulfide [27,29]. Underlying these hydrophobic contacts are the aromatic rings of Trp26 [28], constituting the bulk of the hydrophobic core of the monomer
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