TY - JOUR
T1 - Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function
AU - Zhao, Le
AU - Tolbert, W. David
AU - Ericksen, Bryan
AU - Zhan, Changyou
AU - Wu, Xueji
AU - Yuan, Weirong
AU - Li, Xu
AU - Pazgier, Marzena
AU - Lu, Wuyuan
N1 - Funding Information:
L.Z. was a Guanghua Scholar supported by Xi’an Jiaotong University School of Medicine. We thank the X-ray Crystallography Core Facility of the University of Maryland at Baltimore for providing crystallographic equipment and resources. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program (P41RR001209), and the National Institute of General Medical Sciences.
PY - 2013/11/13
Y1 - 2013/11/13
N2 - HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr16, Ile20, Leu 25, 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.
AB - HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr16, Ile20, Leu 25, 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.
UR - http://www.scopus.com/inward/record.url?scp=84893524560&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0078937
DO - 10.1371/journal.pone.0078937
M3 - Article
C2 - 24236072
AN - SCOPUS:84893524560
SN - 1932-6203
VL - 8
JO - PLoS ONE
JF - PLoS ONE
IS - 11
M1 - e78937
ER -