Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and

Nannette Y. Younta,b, Deborah Kupferwassera,b, Alberto Spisnic, Stephen M. Dutzd, Zachary H. Ramjand, Shantanu Sharmad, Alan J. Waringe, and Michael R. Yeamana,b,e,1

aDivision of Infectious Diseases, LAC-Harbor University of California, Los Angeles Medical Center, Torrance, CA 90509; bSt. John’s Cardiovascular Research Center, Los Angeles Biomedical Research Institute at Harbor–University of California, Los Angeles, Torrance, CA 90502; cDepartment of Experimental Medicine, Section of Chemistry and Structural Biology, University of Parma, 43100 Parma, Italy; dDepartment of Chemistry and Center for Macromolecular Modeling and Materials Design, California State Polytechnic University, Pomona, CA 91768; and eDepartment of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA 90024

Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved June 24, 2009 (received for review April 23, 2009) Recent discoveries suggest -stabilized and antimi- fensin and crotamine-like families, but conserved cysteine- crobial have structure–activity parallels derived by com- array and ␥-core motifs (4). In the present study, evolutionary, mon ancestry. Here, human antimicrobial hBD-2 and rat- structural, and mechanistic investigations were performed to ad- tlesnake -toxin crotamine were compared in phylogeny, 3D dress the hypothesis that reciprocal relationships exist between structure, target cell specificity, and mechanisms of action. Results antimicrobial and cytotoxic host defense peptides. indicate a striking degree of structural and phylogenetic congru- ence. Importantly, these polypeptides also exhibited functional Results reciprocity: (i) they exerted highly similar antimicrobial pH optima Phylogenetic, structural, mechanistic, and target interaction and spectra; (ii) both altered membrane potential consistent with comparisons between hBD-2 and crotamine were investigated ion channel-perturbing activities; and (iii) both peptides induced through complementary methods. phosphatidylserine accessibility in eukaryotic cells. However, the Nav channel-inhibitor tetrodotoxin antagonized hBD-2 mecha- Phylogenetic Analysis. Crotamine and related serpentine toxins

nisms, but not those of crotamine. As crotamine targets eukaryotic formed a monophyletic group with robust statistical confidence IMMUNOLOGY ion channels, computational docking was used to compare hBD-2 (Fig. 1). A single subclade in this group was represented by crotasin, versus crotamine interactions with prototypic bacterial, fungal, or a ␤- from the nonvenomous somatic tissues of Crotalus mammalian Kv channels. Models support direct interactions of each durissus terrificus (7). A sister group was comprised of 2 crotamine- peptide with Kv channels. However, while crotamine localized to like (CLP) from the venom of the bearded dragon Pogona occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 barbata (6). The next most closely related sequences are ␤- interacted with prokaryotic and eukaryotic Kv channels but did not from various avian species. Notably, the serpentine and avian occlude either. Together, these results support the hypothesis that sequences form a unified clade within the Sauropsida. These antimicrobial and cytotoxic polypeptides have ancestral structure- findings suggest a divergence of peptides optimized for antimicro- function homology, but evolved to preferentially target respective bial versus cytotoxic functions appeared concomitant with the microbial versus mammalian ion channels via residue-specific in- divergence of synapsids (mammals) from sauropsids (aves/reptiles) teractions. These insights may accelerate development of anti- (SI Text and Figs. S1 and S2). infective or therapeutic peptides that selectively target microbial or abnormal host cells. Structural and Biophysical Comparison. Three-dimensional (3D) alignment between hBD-2 and crotamine revealed a striking degree ͉ ͉ ͉ channel defensin host defense toxin of identity (Fig. 2A). The greatest degree of 3D alignment (RMSD Յ2) occurs between respective ␣-helical and ␥-core regions. Thus, ysteine-stabilized antimicrobial polypeptides are thought to evolutionary selective pressures have favored conservation of 3D Cfunction as a first line of host defense. One of the most structure in the face of limited sequence identities (28%) of the 2 well-characterized groups of such molecules is the ␤-defensins peptides (2). Despite striking conformational homology, biophys- from myeloid and epithelial tissues of mammalian and avian ical analyses revealed significant physicochemical differences be- species. The ␤-defensins contain a highly conserved cysteine- tween hBD-2 and crotamine that likely relate to differences in array that affords structural rigidity and promotes hypervari- target preference. For example, the solvent accessible surface area ability for accelerated evolutionary adaptation. of hBD-2 (Ϸ70% lacking charged residues; Fig. 2B) is markedly Several parallels suggest structural, functional, and evolutionary more hydrophobic than that of crotamine. commonalities between defensins and other host defense and/or offense molecules, such as toxins. Like antimicrobial peptides, many Antimicrobial Activity. Crotamine antimicrobial activities paralleled toxins are small, cysteine-stabilized, and cationic (1, 2), and share those of hBD-2 at both experimental pH values against most a striking degree of conservation with host defense peptides that contain a ␥-core motif (3). One group of toxins with particularly close homology to ␤-defensins is the crotamine- family Author contributions: N.Y.Y. and M.R.Y. designed research; N.Y.Y., D.K., S.M.D., Z.H.R., S.S., from South American (4, 5). As in ␤-defensins, toxin A.J.W., and M.R.Y. performed research; N.Y.Y., A.S., S.S., and M.R.Y. contributed new expression is often targeted for mucosal or extracorporeal secretion reagents/analytic tools; N.Y.Y., S.M.D., Z.H.R., S.S., A.J.W., and M.R.Y. analyzed data; and N.Y.Y., A.S., S.S., A.J.W., and M.R.Y. wrote the paper. (6). Such toxins typically induce rapid paralysis resulting in local Conflict of interest statement: M.R.Y. is a shareholder of NovaDigm Therapeutics, Inc., and myonecrosis in skeletal muscle. Crotamine-like toxins are thought has received research funding from Pfizer, Inc., Amgen, Inc., Cubist Pharmaceuticals, and to induce such effects through targeting of ion channels and altering Novozymes Pharmaceuticals. None of these entities provided support for the current membrane transport or conductivity (5). studies. Understanding structural and mechanistic reciprocity is of direct This article is a PNAS Direct Submission. relevance to immunologic roles and potential therapeutic develop- 1To whom correspondence should be addressed. E-mail: [email protected]. ment of antimicrobial and cytotoxic peptide-based therapies. Prior This article contains supporting information online at www.pnas.org/cgi/content/full/ studies have noted overall diversity in primary structures of ␤-de- 0904465106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904465106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 BNBD12_BOSTA BNBD8_BOSTA A HBD-2 CRO BNBD3_BOSTA TAP_BOSTA LAP_BOSTA BD402_BOSTA Artiodactyl SBD2_OVIAR

SBD1_OVIAR β-defensins Synapsida BD1_CAPHI BD2_CAPHI EBD_BOSTA NBD4_BUBBU BNBD5_BOSTA BNBD4_BOSTA MBD8_MUSMU RBD3_RATNO Rodent RBD5_RATNO β-defensins MBD4_MUSMU BD2_MACMU Primate BD2_PANTR β-defensins BD2_HOMSA BD1_SUSSC BD1_EQUCA Mammalian BD1_CHILA β-defensins BD103_CANFA BD3_SUSSC BD7_GALGA BD_ANAPL BD2_GALGA GAL2_GALGA Avian BD5_GALGA β-defensins

GAL9_GALGA Sauropsida GAL1_GALGA THP1_MELGA CLPPOGL2_POGBA Reptilian CLPPOGL3_POGBA Toxins CROTASIN_CRODU MYO3_CRODU CROT1_CRODU CROT3_CRODU MYO_CRODU Serpentine MYOA_CROVIV Toxins MYO2_CROVIC MYO3_CROVIV HBD-2 Crotamine MYO1_CROVIC B BD4_GALGA Avian GAL12_GALGA A BD8_GALGA β-defensins

B Fig. 1. Phylogenetic parallels between crotamine and hBD-2. Neighbor-joining tree visualization (36) of the hBD-2/crotamine polypeptide family. Branch signif- icance was validated by bootstrap analysis. 180o

C organisms (P Ͼ 0.05; Fig. 3). The only exception to this observation was for the prokaryote Staphylococcus aureus at pH 7.5, where hBD-2 had significantly greater efficacy (P Ͻ 0.05). Notably, both crotamine and hBD-2 had marked activity against the eukaryotic D pathogen Candida albicans at pH 7.5 or 5.5. Generally, hBD-2 had greater efficacy at pH 7.5, except against C. albicans, where pH had no discernable impact. E Cytotoxic Properties. To assess relative cytotoxicities of crotamine 90o versus hBD-2, flow cytometry was used to compare membrane electrophysiology (⌬⌿), permeabilization, and phosphatidylserine accessibility in bacteria and 2 eukaryotic cell systems. Fig. 2. Structure and biophysical comparison of crotamine and hBD-2. (A)3D Membrane Energetics. Membrane potential (⌬⌿) was evaluated alignment of crotamine/hBD-2 was performed by combinatorial extension (34). using 3,3-dipentyloxacarbocyanine (DiOC5), a charged lipophilic Coloration is per secondary structure schema: ␤-Sheet (blue); turn (gray); ␣-helix dye that emits a fluorescent signal proportional to ⌬⌿. The validity (red), molecular visualization MOLMOL (42). (B) Biophysical parallels in crotamine of this method to assess channel activity approaches the reliability and hBD-2. (A) ␥-Core domain, in red, hBD-2 and crotamine; (B) partial transpar- of classical patch clamp methods, as borne out by a compelling ency of the ␥-core domain (red); (C) Kyte-Doolittle hydrophathy plot overlay: body of evidence (8–10). Overall, hBD-2 and crotamine altered Brown, most hydrophobic; green, intermediate; blue, most hydrophilic; (D) ⌬⌿ charge overlay: Blue, basic (Arg, Lys); red, acidic (Asp, Glu); (E) visualization of in several target organisms versus untreated controls (Fig. 4 cationic (blue) and hydrophobic (Kyte-Doolittle coloration, as above) residues A–D, Figs. S3 and S4, and SI Text). In Escherichia coli, hBD-2 within the ␤1-␤2 loop for hBD-2 and ␤2-␤3 loop for crotamine. Data are for caused cell membrane hyperpolarization, as evidenced by in- crotamine (1H5O) and hBD-2 (1FD3) as visualized using University of California, creases in the overall percentage of cells polarized, as well as San Francisco (UCSF) Chimera (43).

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904465106 Yount et al. Downloaded by guest on September 26, 2021 caused marked increases in membrane potential (respective 2.2- and 2.8-fold increases in mean channel fluorescence; P Ͻ 0.01; Fig. 4C). Importantly, addition of the Nav channel inhibitor tetrodotoxin (TTX) mitigated hBD-2-induced hyperpolarization of E. coli, but had little impact on activities of hBD-2 on membrane potential in other cells.

Membrane Permeabilization. Peptide permeabilization of cells was measured using the intercalating dye propidium iodide (PI). This fluorescent probe enters permeabilized cells and binds to double- stranded nucleic acids, but is excluded from cells with normal membrane integrity. Defensin hBD-2 caused marked permeabili- zation of E. coli and S. aureus (P Ͻ 0.01; Fig. 4 A and B). Interestingly, crotamine did not increase E. coli permeability, but significantly increased the ratio of permeated S. aureus cells (P Ͻ 0.05; Fig. 4 A and B). Moreover, in the eukaryote C. albicans, hBD-2 or crotamine caused significant permeabilization (respective Fig. 3. Comparative antimicrobial efficacies and pH optima of crotamine and 99- or 95-fold increases in percent of cells permeabilized; P Ͻ 0.01; hBD-2. Antimicrobial activity of crotamine and hBD-2 were determined using Fig. 4C). In human umbilical vein endothelial cells (HUVECs), radial diffusion against a panel of Gram-positive and Gram-negative bacteria and neither hBD-2 nor crotamine caused significant increases in cell fungi. Antimicrobial activity was measured at pH 5.5 and 7.5. Data are displayed permeability versus controls (Fig. 4D). As with membrane ener- as the zone of inhibition. Coloration: Complete clearing (blue); partial clearing getics, the Na channel blocker TTX revealed peptide-specific (red). †, P Յ 0.05 vs. same peptide at alternate pH; *, P Յ 0.05 vs. alternate peptide v at same pH. consequences on target cell permeability. In bacteria, TTX caused a complete abrogation of cell permeabilization by hBD-2 (no significant difference from control). Conversely, TTX enhanced their mean channel fluorescence (P Ͻ 0.05 versus control). permeabilization of S. aureus by crotamine (P Ͻ 0.05), but did not Neither peptide had a detectable affect on membrane ⌬⌿ of S. do so for E. coli.InC. albicans, TTX only modestly increased IMMUNOLOGY aureus (Fig. 4B). In C. albicans, both hBD-2 and crotamine permeabilization by hBD-2 or crotamine.

Fig. 4. Reciprocal activities of crotamine and hBD-2 on distinct prokaryotic and eukaryotic cells. Comparative flow cytometric analysis of membrane energetics (DiOC5), membrane permeabilization (PI), and phosphatidylserine accessibility (annexin V)inA E. coli,(B)S. aureus,(C)C. albicans, and (D) HUVECs in response to peptide exposure for 1 h. Lower left quadrant depicts basal fluorescence. Cells above the quadrant threshold are considered positive for permeability and phosphatidylserine access. For cellular energetics, movement relative to the control cell population indicates either hyper- or hypopolarization. Percent of cells gating positive are indicated on each panel, with mean channel fluorescence provided in parentheses. Data were generated using FCS Express V3 software (De Novo Software).

Yount et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 Phosphatidylserine (PS) Accessibility. One of the earliest markers of A 1Y B E cellular transition to an apoptotic state is the translocation of PS 2K

from the inner to the outer leaflet of the plasma membrane (11). 375D There, PS is accessible to staining by fluorescent-labeled annexin V, 373Y a phospholipid-binding with specificity for PS. In bacteria, C D 10H neither hBD-2 nor crotamine caused significant PS accessibility 31R Kv1.2

(Fig. 4 A and B). Likewise, hBD-2 did not lead to increased PS Crotamine 375D accessibility in C. albicans in the presence or absence of TTX. 374G However, crotamine did induce a substantial increase in PS acces- F G J 350R sibility in C. albicans, which was not affected by TTX (Fig. 4C). 22R Interestingly, both study peptides influenced PS accessibility in 375D 4D HUVECs. In these cells, hBD-2 or crotamine alone caused signif- icant increases in PS accessibility (30-fold and 24-fold, respectively; H I

hBD-2 351D P Ͻ 0.01; Fig. 4D). However, consistent with other mechanistic 19F results, TTX inhibited this effect of hBD-2, but not of crotamine. 374G 23R Predicted Interactions with Kv Channels. Docking studies were per- formed to compare crotamine versus hBD-2 interactions with A B E prokaryotic and eukaryotic Kv channels, including this model of the 7K 14K C. albicans Kv channel. 452D 452D Crotamine. Crotamine preferentially targeted eukaryotic over pro- C D Ϫ1 karyotic Kv channels [interaction energies (E; kcal mol ): Kv1.2, 27K

Crotamine 35K Ϫ1119; CaKv, Ϫ618; KcsA, Ϫ355]. Moreover, crotamine interacted KcsA CAK 452D with the eukaryotic Kv1.2 and CAKv channels so as to completely 452D occlude their apertures (Fig. 5). For Kv1.2, crotamine residues Arg31 F 22R G J and Tyr1 participated in electrostatic and hydrogen-bonding inter- actions with Asp375 and Tyr373 of the channel pore. A similar 25K

interaction was observed for crotamine and the fungal CAKv 452D 452D channel, although the opposite facet of the peptide was involved in H I hBD-2 this interaction. In this association, peptide residues Lys27 and Trp32 19F 36K occupied the channel via electrostatic and hydrogen bonding interac- 452D tions with aspartic acid residue Asp452 of the channel pore. In contrast, 452D the predicted interaction between crotamine and the prokaryotic KcsA channel did not occlude the channel pore (Fig. 5). A 31R B E

78Y 31R hBD-2. While its binding energies were similar, the orientation and localization of hBD-2 were distinguishable from those of crotamine 79G and eukaryotic Kv channels at the molecular level. Notably, the C D hBD-2 backbone did not occlude either eukaryotic channel pore. 1Y

Rather, hBD-2 targeted the outer region of these channels through Crotamine 32W 80D 84V residues of its ␤1-␤2 loop (Arg22 and Phe19; Figs. 2 and 5). In the prokaryotic KcsA model, hBD-2 did not interpose the channel F G J aperture, but localized to partially occlude this pore. Furthermore, 82Y 41P 22R the interaction between hBD-2 and KcsA was mediated by Arg22 and Arg23 from the ␤1-␤2 loop of hBD-2 (Figs. 2 and 5). 80D H I Discussion hBD-2 22R 23R Prior reports have noted structural homology among certain 79G antimicrobial peptide and toxin classes (4, 12). However, struc- 80D tural, mechanistic, and evolutionary relatedness among these molecules is less clear. If such reciprocal relationships exist, they Fig. 5. Computational modeling of predicted interactions between crotamine could provide vital insights into molecular origins of innate or hBD-2 with Kv1.2, CaKv, and KcsA Kv channels. Residue-specific interactions immunity, overcome previous barriers to development of non- between crotamine (green; A–D) or hBD-2 (orange; I–L) and respective tetrameric toxic peptide therapeutics, and reveal agents for targeting ab- Kv channels (gray) backbones are visualized. Predicted ligand/receptor complexes normal host cells. are shown as ribbon models in top view orientations (E and J). Side chain Structure-function reciprocity of hBD-2 and crotamine was coloration is per backbone of the specific peptide and atomic elements thereof. Molecular interactions were visualized with Visual Molecular Dynamics (VMD) predicted based upon congruent conformational homology, pro- software (44). pensity for membrane disruption, proclivity for cationic charge at neutral pH, and affinities for electronegative microbial surface targets. Supporting these predictions, the current data demonstrate hBD-2 and crotamine to have parallel yet distinct structure-activity leading to programmed cell death; (v) a preferential trophism of relationships: (i) Charge and hydrophobic topologies consistent crotamine for eukaryotic (CAKv or Kv1.2) and hBD-2 for prokary- with conserved antimicrobial and cytotoxic functions; (ii) similar otic (KcsA) channel targets, respectively; and (vi) antagonism of antimicrobial spectra and pH optima; (iii) prokaryotic (hBD-2) or hBD-2 for TTX-sensitive ion channels. Together, these findings eukaryotic (crotamine) preference with respect to membrane dis- substantiate the hypothesis that overall 3D structural homology ruptive activities; (iv) an ability to evoke asymmetric membrane provides a framework for reciprocal antimicrobial and cytotoxic phosphatidylserine expression consistent with cytotoxic injuries propensities of hBD-2 and crotamine, respectively. Moreover, these

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904465106 Yount et al. Downloaded by guest on September 26, 2021 results point to residue-specific interactions as potentially critical to be attributable in-part to structure-mechanism correlates parallel- differentiating antimicrobial versus cytotoxic proclivities of these ing those of crotamine-like toxins. Crotamine causes rapid paralysis peptides. and myonecrosis following prey envenomation. Early studies sug- Kv, NaKv, and Nav channels represent an evolutionary continuum gested an interaction with voltage-dependent Nav channels pre- of ion-selective channels bridging prokaryotes to eukaryotes (13– dominant in fast-twitch muscles of mammals (28). However, recent 17). TTX, a specific inhibitor of Nav channel function (17), was used studies by Rizzi et al. (29), using expressed Nav1.1–1.6 ␣-subunits, to probe potential differential effects of crotamine and hBD-2 on did not find an interaction between crotamine and Nav channels. target cell Nav-related functions. Generally, TTX mitigated effects Further support for the interaction of defensin-like toxins with of hBD-2, but failed to inhibit crotamine functions, and in some Kv ion channels derives from recent structural mapping of the Kv cases enhanced crotamine action. This pattern of results suggests channel surface and, in some cases, toxin/channel complexes hBD-2 and crotamine preferentially target Nav, NaKv, or other (23–25). Together, these facts support the concept that hBD-2 TTX-sensitive targets in eukaryotes, and orthologous targets in and crotamine may preferentially but not exclusively target prokaryotes (13, 14). Whether such mechanisms may be direct or respective Nav, NaKv, or other TTX-sensitive ion channels— indirect or exclusively involve Nav or NaKv channels is not yet clear. versus Kv channels—contributing to their net cytotoxic effects. It is conceivable that hBD-2 antagonizes cell functions distinguish- Ongoing studies are designed to assess the potential direct and/or able from or in addition to TTX-sensitive Nav or NaKv channels. indirect interactions through which such peptides may differen- For example, hBD-2 and crotamine may perturb lipid membrane tially target and antagonize these or other channels structurally integrity in relation to ion-channel function in certain target cells. or mechanistically. Investigations to determine how hBD-2 or other host defense The present findings may have significant implications for de- peptides may antagonize voltage-sensitive ion channels or related velopment of polypeptide anti-infectives or other therapeutics. In targets are ongoing. The observation that defensin-like peptides the past decade, a sizeable investment has been made in exploiting may interact with Na channels is not entirely unprecedented. v microbicidal activities of CS-stabilized peptides such as defensins to Rogachevskii et al. suggested an interaction between human de- address the mounting resistance of many important human patho- fensin NP-1 and the slow Na channels of rat ganglial neurons (18). v gens to conventional antibiotics. However, for systemic use, toxicity Likewise, plant ␥-thionin defensins inhibit the sodium current in cultured GH3 cells (19). If so, such interactions may begin to explain not unlike that induced by cytotoxins has been a significant barrier toxicity in animal models wherein bolus systemic administration of to such advances (20–22). For example, the current data suggest

defensin-like peptides leads to neurotoxic- and cytotoxic-like ef- that crotamine and hBD-2 initiate apoptotic pathways in eukaryotic IMMUNOLOGY fects (20–22). target cells (Candida, HUVECS). Such events have been linked to Mechanistic findings above suggested biologically distinct inter- peptide perturbation of ion channel functions in fungal as well as mammalian cells (11). Identification of molecular determinants actions of hBD-2 or crotamine with Nav versus Kv channels. Therefore, as a complement to these studies, quantitative modeling that differentiate cytotoxicity from antimicrobial activity may en- was used to compare hBD-2 versus crotamine interactions with able optimization of molecules for therapeutic efficacy without prototypic Kv channels of prokaryote, fungal, and mammalian concomitant host toxicity. Alternatively, engineering peptides to organisms. Potential interactions between peptides and Nav chan- have selective host cell toxicity may offer approaches to prevent or nels were not studied, as no NMR- or X-ray-validated structure treat cancer, autoimmune, or other diseases. Thus, a clearer un- models for these channels were available. Relative docking affini- derstanding of the ancient molecular features that both unite and ties may reflect the relative electrostatic attractions of cationic distinguish host defense, toxin, and venom peptides may aid in crotamine and hBD-2 peptides to anionic Kv channel surfaces development of anti-infectives or other therapeutics to address 21st (most electronegative to least: Kv1.2 Ͼ CAKv Ͼ KcsA) or larger century medical challenges. molecular areas of eukaryotic versus prokaryotic channels. How- ever, the finding that the peptides were distinct in TTX antagonism Materials and Methods suggests that specific stereogeometric interactions influence rela- Microorganisms. Microorganisms representing Gram-positive (S. aureus; ATCC tive targeting of hBD-2 and crotamine for specific ion channels. 27217; and Bacillus subtilis; ATCC 6633); Gram-negative (E. coli; ML-35); and Computational docking analyses suggested a preferential reci- fungal (C. albicans; ATCC 36082) human pathogens were studied. Microorgan- procity of hBD-2 and crotamine for interactions with prokaryotic isms were cultured overnight in brain heart infusion (BHI) broth (Difco) at 37 °C (bacteria) or 30 °C (fungi). Cells were sonicated and adjusted to 106 CFU/mL. versus eukaryotic Kv channels, respectively. In these analyses, crotamine localizes to the inner-pore domain through a classical Endothelial Cells. Studies using HUVECs were conducted in accordance with cationic-aromatic (e.g., Lys27 and Trp32) functional dyad. Consis- National Institutes of Health (NIH) and institutional guidelines for human sub- tent with this theme is the fact that Lys27 occurs at the ‘‘X’’ position ␥ jects. HUVECs were harvested as described previously (30). Cells were cultured to of the GXnC element of the -core domain in many defensin-like confluency (M-199 medium; Invitrogen; 10% FBS; Gemini Bio-Products; 2 mM toxins and is present within crotamine (GKMDC). However, a Lys L-glutamine, penicillin, and streptomycin; Irvine Scientific), detached with 0.1% residue is absent from the GXnC element of the hBD-2 ␥-core motif trypsin EDTA, washed, and enumerated (30). (GTC). The current results also indicate that crotamine perturbs eukaryotic Kv channels as do the charybdotoxin (23), kaliotoxin Molecules. TTX (Sigma). hBD-2 (Peptides International). Crotamine was enriched (24), and cobatoxin (25) family of scorpion toxins (26). Basic from yellow venom of Crotalus durissus terrificus as described previously (5). residues in such toxins form a cationic facet integrating Lys or Arg Crotamine purification was achieved by RP-HPLC on a C18 column (Vydac) equil- ibrated with 0.01% triflouroacetic acid and eluted with a 0–40% gradient of residues of their ␥-core GXnC motif (2, 3). In turn, this cationic water:acetonitrile. Crotamine identity and purity were authenticated using facet binds to 4 highly conserved Asp452 residues symmetrically MALDI-TOF spectrometry. distributed on the extracellular P-loops of Kv tetramers (27). In addition, a highly-conserved aromatic residue, typically Phe or Tyr, also participates in this pore-occluding complex, forming the clas- Radial Diffusion Antimicrobial Assay. Antimicrobial assays were performed using a radial diffusion method (3). As pH can influence peptide antimicrobial efficacy, sical cationic-aromatic residue dyad (25). Collectively, the current the assays were conducted at pH 5.5 or 7.5 (31, 32). These conditions reflect the data support the hypothesis that residue-specific differences in relevant contexts in which antimicrobial peptides often function (e.g., intracel- peptides that have conserved overall 3D homology may contribute lular phagolysosome, pH 5.5, or extracellular milieu, pH 7.5) and which can to target ion-channel preferences. influence peptide activities. Organisms were inoculated (106 CFU/mL) into buff- Many defensins exert considerable cytotoxicity in cell and animal ered agarose (10 mM Pipes, pH 7.5, or 10 mM MES, pH 5.5); peptides (10 ␮g/well) models (21, 22). The present data suggest that such toxicities may were aliquoted into wells in the seeded matrix and incubated for3hat37°C.

Yount et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 Zones of inhibition were measured 24 h later. Independent experiments were P-loop domains used for docking studies. The monomeric C. albicans Kv channel repeated a minimum of 2 times. was then assembled using MUSTANG (38) implemented in YASARA (39). The resulting tetramer (CAK) was energetically minimized with conjugant gradients Flow Cytometry. Multicolor flow cytometry was used to assess the comparative for 5,000 steps using NAMD (40). The structural coordinates for mammalian mechanisms of hBD-2 and crotamine versus prokaryotic (E. coli, S. aureus)or Kv1.2–2.1 (2R9R), S. lividans KcsA (1BL8), crotamine (1H5O), and HBD-2 (1FD3) eukaryotic (C. albicans, HUVECs) target cells. The fluorophores used were as were obtained from the Protein Data Bank (www.pdb.org). follows: Membrane permeabilization, PI (Ex535nm/Em620nm; Sigma); transmem- Computational models for Kv channel-peptide complexes were generated brane potential, DiOC5 (Ex484nm/Em500nm; Invitrogen); phosphatidylserine acces- using RosettaDock (www.rosettacommons.org) implemented in CAPRI (41). In sibility, annexin V (allophycocyanin conjugate; Ex650nm/Em660nm; Invitrogen). For brief, the docking method used a 2-step process: (i) rigid-body Monte Carlo experiments, 105 cells were incubated with peptide (20 ␮g/mL) or peptide with searches and (ii) parallel optimization of backbone displacement and side-chain ␮ TTX (50 nM; subinhibitory concentration) in 100 L 10 mM Pipes, pH 7.5, for 15 conformations using Monte Carlo minimization. The Kv channel domains avail- min or 1 h with shaking at 30 °C (C. albicans) or 37 °C (bacteria, HUVECs). Pipes is able for docking were restricted to extracellular regions. The initial search yielded a zwitterionic organic-based buffer that is not absorbed through cell membranes Ϸ2 ϫ 104 decoys for each ligand (crotamine or hBD-2). For each of the top 50 and is nontoxic to study cells as assayed (31–33). Cells were stained for 10 min at ranked conformers, the ␣-carbon RMSD of the decoy was compared against each ␮ ␮ ␮ room temperature by adding 900 L stain buffer (PI, 5.0 g/mL; DiOC5, 0.5 M; member of the conformational set in the second search. Iterative refinement ␮ ϩ annexin V, 2.5 L/mL in 50 mM K MEM). Control cells were exposed to SDS (0.5%; resulted in 8 top-scoring conformations per ligand. Interaction sites were ranked ␮ ϩ Sigma), CCCP (100 M; Sigma), or K MEM buffer (Sigma) alone. Flow cytometry by binding energy, and the energy contributions per residue (5 Å radius) tabu- ϩ was performed using a FACSCalibur instrument (Becton Dickinson) in 10 mM K lated. Ligand-protein residue pairs were then ranked based on total energy ϫ 3 MEM, pH 7.2. Fluorescence of a minimum of 5 10 cells was acquired for contribution and orientation-dependent hydrogen bonding. The 8 most favor- statistical analysis. able docking complexes were evaluated as potential binding sites.

Bioinformatics. Structural superimpositions and root mean squared deviation Statistical Analyses. Experiments were performed a minimum of 2 independent (RMSD) calculations were carried out using combinatorial extension [http:// times on different days. Unpaired Student’s t test was used to compare differ- cl.sdsc.edu./ce; (34)]. Sequences for phylogenetic analyses were identified in ences in data exhibiting normal distributions; data exhibiting discontinuous iterative BLASTp searches using ␤-defensin and toxin sequences. Sequences were distributions were analyzed using the standard nonparametric Kolmogorov- aligned with CLUSTALW (35), and phylogenetic trees were constructed using the Smirnoff methodology. P values Յ 0.05 were considered significant. neighbor-joining method (36).

ACKNOWLEDGMENTS. We thank Trang Phan and Scott G. Filler (Division of Computational Modeling and Protein Docking. AK channel model from C. v Infectious Diseases, Harbor–UCLA Medical Center, and the General Clinical Re- albicans was generated using homology modeling [Phyre; 3D-PSSM folding search Center at Harbor–UCLA Medical Center) for providing HUVECs for these server; (37)]. The highest scoring template was the mammalian shaker-family studies and H. Ronald Kaback, Terry J. Smith, Robert I. Lehrer, Eric P. Brass, and ϫ Ϫ5 Kv1.2 channel (95% estimated precision; E-value, 4.5 10 ; PDB code, 2A79). John E. Edwards, Jr., for helpful discussions. This work was supported by National Channel regions spanning S2 and S4–S6 were derived from C. albicans [residues Institutes of Health, National Institute of Allergy and Infectious Diseases Grants 271–293 and 365–494, respectively (gi:68486701)], which includes all of the 5R01AI39001 and 5R01AI48031 to M.R.Y.

1. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resis- 23. Yu L, et al. (2005) Nuclear magnetic resonance structural studies of a potassium tance. Pharmacol Rev 55:27–55. channel-charybdotoxin complex. Biochemistry 44:15834–15841. 2. Yeaman MR, Yount NY (2007) Unifying themes in host defence effector polypeptides. 24. Zachariae U, et al. (2008) The molecular mechanism of toxin-induced conformational Nat Rev 5:727–740. changes in a potassium channel: Relation to C-type inactivation. Structure 16:747–754. 3. Yount NY, Yeaman MR (2004) Multidimensional signatures in antimicrobial peptides. 25. Jouirou B, et al. (2004) Cobatoxin 1 from Centruroides noxius scorpion venom: Chem- Proc Natl Acad Sci USA 101:7363–7368. ical synthesis, three-dimensional structure in solution, pharmacology and docking on ϩ 4. Nicastro G, et al. (2003) Solution structure of crotamine, a Na channel affecting toxin Kϩ channels. Biochem J 377:37–49. from Crotalus durissus terrificus venom. Eur J Biochem 270:1969–1979. 26. Zhu S, Bosmans F, Tytgat J (2004) Adaptive evolution of scorpion 5. Mancin AC, et al. (1998) The activity of crotamine, a neurotoxin from Crotalus toxins. J Mol Evol 58:145–153. durissus terrificus (South American ) venom: A biochemical and pharma- 27. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001) Chemistry of ion coordi- cological study. Toxicon 36:1927–1937. nation and hydration revealed by a Kϩ channel-Fab complex at 2.0 A resolution. 6. Fry BG, et al. (2006) Early evolution of the venom system in and . Nature Nature 414:43–48. 439:584–588. 28. Chang CC, Tseng KH (1978) Effect of crotamine, a toxin of South American rattlesnake 7. Radis-Baptista G, et al. (2004) Identification of crotasin, a crotamine-related of Crotalus durissus terrificus. Toxicon 43:751–759. venom, on the sodium channel of murine skeletal muscle. Br J Pharmacol 63:551–559. 8. Baxter DF, et al. (2002) A novel membrane potential-sensitive fluorescent dye improves 29. Rizzi CT, et al. (2007) Crotamine inhibits preferentially fast-twitching muscles but is cell-based assays for ion channels. J Biomol Screen 7:79–85. inactive on sodium channels. Toxicon 50:553–562. 9. Dorn A, et al. (2005) Evaluation of a high-throughput fluorescence assay method for 30. Filler SG, Swerdloff JN, Hobbs C, Luckett PM (1995) Penetration and damage of HERG potassium channel inhibition. J Biomol Screen 10:339–347. endothelial cells by Candida albicans. Infect Immun 63:976–983. 10. Slack M, Kirchhoff C, Moller C, Winkler D, Netzer R (2006) Identification of novel Kv1.3 31. Tang YQ, Yeaman MR, Selsted ME (2002) Antimicrobial peptides from human platelets. blockers using a fluorescent cell-based ion channel assay. J Biomol Screen 11:57–64. Infect Immun 70:6524–6533. 11. Andres MT, Viejo-Diaz M, Fierro JF (2008) Human lactoferrin induces apoptosis-like cell 32. Tang YQ, et al. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by death in Candida albicans: Critical role of Kϩ channel-mediated Kϩ efflux. Antimicrob the ligation of two truncated alpha-defensins. Science 286:498–502. Agents Chemother 52:4081–4088. 33. Good NE, et al. (1966) Hydrogen ion buffers for biological research. Biochemistry 12. Froy O, Gurevitz M (2004) Arthropod defensins illuminate the divergence of scorpion 5:467–477. neurotoxins. J Pept Sci 10:714–718. 34. Shindyalov IN, Bourne PE (1998) Protein structure alignment by incremental combi- 13. Ren D, et al. (2001) A prokaryotic voltage-gated sodium channel. Science 294:2372– natorial extension (CE) of the optimal path. Protein Eng 11:739–747. 2375. 35. Higgins DG, Sharp PM (1988) CLUSTAL: A package for performing multiple sequence 14. Alam A, Jiang Y (2009) High-resolution structure of the open NaK channel. Nat Struct alignment on a microcomputer. Gene 73:237–244. Mol Biol 16:30–34. 36. Saitou N, Nei M (1987) The neighbor-joining method: A new method for reconstructing 15. Gordienko DV, Tsukahara H (1994) Tetrodotoxin-blockable depolarization-activated phylogenetic trees. Mol Biol Evol 4:406–425. ϩ Na currents in a cultured endothelial cell line derived from rat interlobar arter and 37. Kelley LA, MacCallum RM, Sternberg MJ (2000) Enhanced genome annotation using human umbilical vein. Pflugers Arch 428:91–93. structural profiles in the program 3D-PSSM. J Mol Biol 299:499–520. 16. Walsh KB, Wolf MB, Fan J (1998) Voltage-gated sodium channels in cardiac microvas- 38. Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM (2006) MUSTANG: A multiple cular endothelial cells. Am J Physiol 274:H506–H512. structural alignment algorithm. Proteins 64:559–574. 17. Lewis RJ, Garcia ML (2003) Therapeutic potential of venom peptides. Nat Rev Drug Dis 39. Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative 2:790–802. models with YASARA NOVA—a self-parameterizing force field. Proteins 47:393–402. 18. Rogachevskii IV, et al. (2000) The defensin receptor: A possible mechanism responsible for reduced excitability of the neuronal sensory membrane. Dokl Biol Sci 375:595–598. 40. Phillips JC, et al. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 19. Kushmerick C, de Souza Castro M, Santos Cruz J, Bloch C Jr, Beirao PS (1998) Functional 26:1781–1802. and structural features of gamma-zeathionins, a new class of sodium channel blockers. 41. Gray JJ, et al. (2003) Protein-protein docking predictions for the CAPRI experiment. FEBS Lett 440:302–306. Proteins 52:118–122. 20. Gordon YJ, Romanowski EG, McDermott AM (2005) A review of antimicrobial peptides 42. Koradi R, Billeter M, Wuthrich K (1996) MOLMOL: A program for display and analysis and their therapeutic potential as anti-infective drugs. Curr Eye Res 30:505–515. of macromolecular structures. J Mol Graph 14:51–55, 29–32. 21. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389– 43. Pettersen EF, et al. (2004) UCSF Chimera—a visualization system for exploratory 395. research and analysis. J Comput Chem 25:1605–1612. 22. Ganz T, Oren A, Lehrer RI (1992) Defensins: Microbicidal and cytotoxic peptides of 44. Humphrey W, Dalke A, Schulten K (1996) VMD: Visual molecular dynamics. J Mol Graph mammalian host defense cells. Med Microbiol Immunol 181:99–105. 14:33–38, 27–28.

6of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904465106 Yount et al. Downloaded by guest on September 26, 2021