NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5
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]...
| Main Authors: | , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Chemical Society
2013
|
| Online Access: | http://hdl.handle.net/1721.1/82906 https://orcid.org/0000-0002-6153-8803 |
| _version_ | 1826194418948898816 |
|---|---|
| author | Wommack, Andrew Robson, Scott A. Wanniarachchi, Yoshitha A. Wan, Andrea Turner, Christopher J. Wagner, Gerhard Nolan, Elizabeth Marie |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Wommack, Andrew Robson, Scott A. Wanniarachchi, Yoshitha A. Wan, Andrea Turner, Christopher J. Wagner, Gerhard Nolan, Elizabeth Marie |
| author_sort | Wommack, Andrew |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:55:49Z |
| format | Article |
| id | mit-1721.1/82906 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:55:49Z |
| publishDate | 2013 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | mit-1721.1/829062022-09-26T14:37:32Z NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 Wommack, Andrew Robson, Scott A. Wanniarachchi, Yoshitha A. Wan, Andrea Turner, Christopher J. Wagner, Gerhard Nolan, Elizabeth Marie Massachusetts Institute of Technology. Department of Chemistry Francis Bitter Magnet Laboratory (Massachusetts Institute of Technology) Wommack, Andrew Wanniarachchi, Yoshitha A. Wan, Andrea Turner, Christopher J. Nolan, Elizabeth M. 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. National Institutes of Health (U.S.) (NIH Grant DP2OD007045) National Institute of General Medical Sciences (U.S.) (NIH Grant P01 GM047467) National Institute for Biomedical Imaging and Bioengineering (U.S.) National Institute for Biomedical Imaging and Bioengineering (U.S.) (NIH Grant EB-002026) Massachusetts Institute of Technology. Dept. of Chemistry 2013-12-10T20:08:37Z 2013-12-10T20:08:37Z 2012-12 2012-11 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/82906 Wommack, Andrew J., Scott A. Robson, Yoshitha A. Wanniarachchi, Andrea Wan, Christopher J. Turner, Gerhard Wagner, and Elizabeth M. Nolan. “NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5.” Biochemistry 51, no. 48 (December 4, 2012): 9624-9637. https://orcid.org/0000-0002-6153-8803 en_US http://dx.doi.org/10.1021/bi301255u Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society PMC |
| spellingShingle | Wommack, Andrew Robson, Scott A. Wanniarachchi, Yoshitha A. Wan, Andrea Turner, Christopher J. Wagner, Gerhard Nolan, Elizabeth Marie NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title | NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title_full | NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title_fullStr | NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title_full_unstemmed | NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title_short | NMR Solution Structure and Condition-Dependent Oligomerization of the Antimicrobial Peptide Human Defensin 5 |
| title_sort | nmr solution structure and condition dependent oligomerization of the antimicrobial peptide human defensin 5 |
| url | http://hdl.handle.net/1721.1/82906 https://orcid.org/0000-0002-6153-8803 |
| work_keys_str_mv | AT wommackandrew nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT robsonscotta nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT wanniarachchiyoshithaa nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT wanandrea nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT turnerchristopherj nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT wagnergerhard nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 AT nolanelizabethmarie nmrsolutionstructureandconditiondependentoligomerizationoftheantimicrobialpeptidehumandefensin5 |