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Discovery and Characterization of a DisulfideLocked C2-Symmetric Defensin Peptide Andrew J. Wommack, Joshua J. Ziarek, Jill Tomaras, Haritha R. Chileveru, Yunfei Zhang, Gerhard Wagner, and Elizabeth M. Nolan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja505957w • Publication Date (Web): 02 Sep 2014 Downloaded from http://pubs.acs.org on September 3, 2014
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Journal of the American Chemical Society
Discovery and Characterization of a Disulfide-Locked C 2 -Symmetric Defensin Peptide Andrew J. Wommack,1 Joshua J. Ziarek,2 Jill Tomaras,1 Haritha R. Chileveru,1 Yunfei Zhang,1 Gerhard Wagner,2 and Elizabeth M. Nolan1* 1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
Supporting Information Placeholder ABSTRACT: We report the discovery of HD5-CD, an
unprecedented C2-symmetric β-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical αdefensin CysII—CysIV (Cys5—Cys20) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii.
Disulfide bonds provide structures and enable the biological functions of various naturally occurring and synthetic peptides.1 Defensins are prominent examples of disulfide-linked peptides found in Nature.2 These cysteine-rich host-defense peptides (2-5 kDa) are ribosomally synthesized by lower and higher eukaryotes, and participate in the innate immune response.2 In humans, oxidized defensins exhibit three regiospecific disulfide bonds with the patterns of CysI—CysVI/CysII— CysIV/CysIII—CysV (α-defensins) or CysI—CysV/CysII— CysIV/CysIII—CysVI (β-defensins) that maintain a threestranded β-sheet fold. Defensins self-associate to afford non-covalent oligomers.3 Despite many structure/activity relationship studies, our understanding of what defensin structures (i.e., oxidized or reduced, various oligomeric states) are physiologically relevant in various biological microenvironments, and precisely how these structures contribute to innate immunity or other biological phenomena, remains unclear. To address such questions in the context of the human gut, our laboratory is investigating human α-defensin 5 (HD5), which is
abundantly produced by small intestinal Paneth cells and is an important contributor to mucosal immunity.2b,4 This peptide exhibits broad-spectrum antimicrobial activity in vitro and may help control the composition of the resident gut microbiota.5,6 Here, we describe an unappreciated facet of the molecular gymnastics displayed by defensins and report that this human α-defensin has the propensity to assemble into a novel disulfide-locked C2-symmetric covalent dimer. During an exploratory set of oxidative folding assays, 32-residue HD5 (Figure 1A,B) in its reduced (HD5red) or oxidized (HD5ox) form, was incubated at pH 7.0 (75 mM HEPES) under anaerobic conditions in buffers of varying redox potentials (-275 to -235 mV) defined by the GSH/GSSG redox couple. The presence of a new peak with a HPLC retention time of 13.1 min (HD5red, 20.0 min; HD5ox, 15.0 min) was observed in the chromatogram of every sample (Figure 1C). LC-MS of the new peak (denaturing conditions) provided a deconvoluted m/z value of 7164.1, which is twice the m/z value of HD5ox (m/z calc 3582.2) (Figure 1D), and thiol quantification supported no (0.55±0.39) free Cys residues. Moreover, two ions corresponding to [M+5H]+5 and [M+7H]+7 identified in the mass spectrum of the new species were not observed for HD5ox (Supporting Information). The paucity of other new HPLC peaks observed with peptide folding, which would be suggestive of other nonnative disulfide-linked species or glutathione adducts, was striking. In total, this work suggested that anaerobic buffers containing the GSH/GSSG redox couple enable an unexpected HD5 congener to predominate. We reasoned that the new species is a disulfide-linked covalent dimer (CD) of HD5 monomers with six disulfide bonds. We named this peptide HD5-CD.
ACS Paragon Plus Environment
Journal of the American Chemical Society
A
B β3
β2
HD5ox ATCYCRTGRCATRESLSGVCEISGRLYRLCCR 10
β1
Coil
15
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D -235 mV
1024.39333 [M+7H]+7
-248 mV -257 mV -266 mV
7164.1 Da
1194.95543 [M+6H]+6
1433.85562 [M+5H]+5
1792.07113 [M+4H]+4
-275 mV 10
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HD5-CD HD5ox
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HD5ox (PDB ID 2LXZ)
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Figure 1. (A) Previously determined NMR structure of the HD5ox monomer (PDB 2LXZ)3e with disulfide bonds shown. (B) Primary sequence of HD5. The solid lines indicate the regiospecific disulfide bonds of HD5ox. The Cys5—Cys20 disulfide bond is highlighted in green. The secondary structure elements are taken from the HD5ox NMR structure (PDB 2LXZ).3e C) HPLC traces showing HD5-CD formation from 25 µM HD5ox in redox buffers containing GSH/GSSG (75 mM HEPES, pH 7.0). The minor peak at 20.0 min is HD5red. (D) ESI-TOF ionization pattern and deconvolution for HD5-CD. (E) Example sedimentation equilibrium data, fits, and residuals for 62 µM HD5-CD (Supporting Information).
Under the anaerobic redox buffer conditions, HD5CD formed irrespective of initial peptide concentration (5–100 µM) and in the presence of millimolar concentrations of NaCl (100 mM), MgCl2 (10 mM), or CaCl2 (10 mM). Using the same GSH/GSSG-containing buffer and aerobic conditions, HD5-CD also formed, albeit to a lesser degree. In total, the conditions and redox potentials that afford HD5-CD suggest that this species has the potential to exist in vivo. HD5-CD formation was attenuated when GuHCl (≥250 mM) was added to the buffer, which increased the amount of HD5red in the mixture. A standard folding protocol performed aerobically that affords HD5ox in high yield employs a 10:1 GSH/GSSG ratio as well as ~2 M GuHCl.7 Evaluation of HPLC traces from prior HD5ox preparations revealed that a peak with the HD5-CD retention time is often observed as a minor (
