NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5

• Main Authors: Wommack, Andrew (Contributor), Robson, Scott A. (Author), Wanniarachchi, Yoshitha A. (Contributor), Wan, Andrea (Contributor), Turner, Christopher J. (Contributor), Wagner, Gerhard (Author), Nolan, Elizabeth Marie (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Chemistry (Contributor), Francis Bitter Magnet Laboratory (Massachusetts Institute of Technology) (Contributor), Nolan, Elizabeth M. (Contributor)
• Format: Article
• Language: English
• Published: American Chemical Society, 2013-12-10T20:08:37Z.
• Subjects: Article
• Online Access: Get fulltext

 Human defensin 5 (HD5) is a 32-residue host-defense peptide expressed in the gastrointestinal, reproductive, and urinary tracts that has antimicrobial activity. It exhibits six cysteine residues that are regiospecifically oxidized to form three disulfide bonds (Cys[superscript 3]-Cys[superscript 31], Cys[superscript 5]-Cys[superscript 20], and Cys[superscript 10]-Cys[superscript 30]) in the oxidized form (HD5[subscript ox]). To probe the solution structure and oligomerization properties of HD5[subscript ox], and select mutant peptides lacking one or more disulfide bonds, NMR solution studies and analytical ultracentrifugation experiments are reported in addition to in vitro peptide stability assays. The NMR solution structure of HD5[subscript ox], solved at pH 4.0 in 90:10 H2O/D2O, is presented (PDB: 2LXZ). Relaxation T[subscript 1]/T[subscript 2] measurements and the rotational correlation time (τc) estimated from a [superscript 15]N-TRACT experiment demonstrate that HD5[subscript ox] is dimeric under these experimental conditions. Exchange broadening of the Hα signals in the NMR spectra suggests that residues 19-21 (Val[superscript 19]-Cys[superscript 20]-Glu[superscript 21]) contribute to the dimer interface in solution. Exchange broadening is also observed for residues 7-14 comprising the loop. Sedimentation velocity and equilibrium studies conducted in buffered aqueous solution reveal that the oligomerization state of HD5[subscript ox] is pH-dependent. Sedimentation coefficients of ca. 1.8 S and a molecular weight of 14 363 Da were determined for HD5[subscript ox] at pH 7.0, supporting a tetrameric form ([HD5ox] ≥ 30 μM). At pH 2.0, a sedimentation coefficient of ca. 1.0 S and a molecular weight of 7079 Da, corresponding to a HD5ox dimer, were obtained. Millimolar concentrations of NaCl, CaCl[subscript 2], and MgCl[subscript 2] have a negligible effect on the HD5[subscript ox] sedimentation coefficients in buffered aqueous solution at neutral pH. Removal of a single disulfide bond results in a loss of peptide fold and quaternary structure. These biophysical investigations highlight the dynamic and environmentally sensitive behavior of HD5[subscript ox] in solution, and provide important insights into HD5[subscript ox] structure/activity relationships and the requirements for antimicrobial action.