Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function.
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.
Translational Medicine Center, the First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, China ; Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
Le
L
Zhao
W David
WD
Tolbert
Bryan
B
Ericksen
Changyou
C
Zhan
Xueji
X
Wu
Weirong
W
Yuan
Xu
X
Li
Marzena
M
Pazgier
Wuyuan
W
Lu
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry; D018360:Crystallography, X-Ray; D015699:HIV Envelope Protein gp120 / Q000737:chemistry; D015700:HIV Envelope Protein gp41 / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D023084:alpha-Defensins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D023084:alpha-Defensins; C030325:anthrax toxin; C514793:gp120 protein, Human immunodeficiency virus 1; C514787:gp41 protein, Human immunodeficiency virus 1; C406413:human neutrophil peptide 1