pathogens Article Metamorphic Protein Folding Encodes Multiple Anti-Candida Mechanisms in XCL1 Acacia F. Dishman 1,2,†, Jie He 3,†, Brian F. Volkman 1,* and Anna R. Huppler 3,* 1 Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA; [email protected] 2 Medical Scientist Training Program, Medical College of Wisconsin, Milwaukee, WI 53226, USA 3 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA; [email protected] * Correspondence: [email protected] (B.F.V.); [email protected] (A.R.H.) † These authors contributed equally. Abstract: Candida species cause serious infections requiring prolonged and sometimes toxic therapy. Antimicrobial proteins, such as chemokines, hold great interest as potential additions to the small number of available antifungal drugs. Metamorphic proteins reversibly switch between multiple different folded structures. XCL1 is a metamorphic, antimicrobial chemokine that interconverts between the conserved chemokine fold (an α–β monomer) and an alternate fold (an all-β dimer). Previous work has shown that human XCL1 kills C. albicans but has not assessed whether one or both XCL1 folds perform this activity. Here, we use structurally locked engineered XCL1 variants and Candida killing assays, adenylate kinase release assays, and propidium iodide uptake assays to demonstrate that both XCL1 folds kill Candida, but they do so via different mechanisms. Our results suggest that the alternate fold kills via membrane disruption, consistent with previous work, and the chemokine fold does not. XCL1 fold-switching thus provides a mechanism to regulate Citation: Dishman, A.F.; He, J.; the XCL1 mode of antifungal killing, which could protect surrounding tissue from damage associ- Volkman, B.F.; Huppler, A.R. ated with fungal membrane disruption and could allow XCL1 to overcome candidal resistance by Metamorphic Protein Folding switching folds. This work provides inspiration for the future design of switchable, multifunctional Encodes Multiple Anti-Candida antifungal therapeutics. Mechanisms in XCL1. Pathogens 2021, 10, 762. https://doi.org/10.3390/ Keywords: Candida; C. albicans; XCL1; metamorphic protein; fold-switching protein; antifungal peptide pathogens10060762 Academic Editor: Jonathan Richardson 1. Introduction Candida albicans and other fungal pathogens cause severe and costly infections in Received: 11 May 2021 children and adults [1–3]. High incidence of drug toxicity and the emergence of resistance Accepted: 12 June 2021 Published: 17 June 2021 limit the utility of available antifungal drug classes [4,5]. Components of the innate immune system termed antimicrobial peptides, including certain chemokines, are currently under Publisher’s Note: MDPI stays neutral investigation as novel antifungal agents [6,7]. Detailed understanding of the relationship with regard to jurisdictional claims in between protein structure and function can promote the optimal development of these published maps and institutional affil- peptides as therapeutics. iations. In the field of structural biology, the conventional wisdom has been that each amino acid sequence folds into a single structure to carry out its biological role. However, in recent decades, proteins have been discovered that defy this norm, folding into multiple different structures and reversibly interconverting between them. Recent work has shown that these proteins, called metamorphic proteins, may be more common than initially expected [8,9]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Interest in the biological relevance of metamorphic protein folding is growing, and efforts This article is an open access article to understand and harness protein metamorphosis for therapeutic benefit are beginning distributed under the terms and to mount [10–12]. conditions of the Creative Commons One such metamorphic protein is the antimicrobial human chemokine XCL1. Attribution (CC BY) license (https:// Chemokines are small, secreted immune proteins that orchestrate the migration of white creativecommons.org/licenses/by/ blood cells under homeostatic and inflammatory conditions, some of which have antimi- 4.0/). crobial activity [13,14]. XCL1 is unique amongst chemokines because it switches between Pathogens 2021, 10, 762. https://doi.org/10.3390/pathogens10060762 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, x FOR PEER REVIEW 2 of 8 One such metamorphic protein is the antimicrobial human chemokine XCL1. Chem- okines are small, secreted immune proteins that orchestrate the migration of white blood Pathogens 2021, 10, 762 cells under homeostatic and inflammatory conditions, some of which have antimicrobial2 of 8 activity [13,14]. XCL1 is unique amongst chemokines because it switches between the con- served α–β chemokine fold and an all-β alternate fold that forms a dimer [15] (Figure 1). XCL1the occupies conserved theα– twoβ chemokine folds in equal fold and proportion an all-β alternate under near-physiological fold that forms a dimer conditions [15] and interconverts(Figure1). XCL1between occupies the two the twofolds folds in the in equal absence proportion of a trigger under near-physiological[15]. The chemokine fold bindsconditions and activates and interconverts XCL1’s cognate between G-protei the two foldsn coupled in the absence receptor of a (GPCR), trigger [15 XCR1,]. The on the chemokine fold binds and activates XCL1’s cognate G-protein coupled receptor (GPCR), surfaceXCR1, of ona subset the surface of dendritic of a subset cells of dendritic [15,16]. The cells XCL1 [15,16]. alternate The XCL1 fold alternate binds fold to glycosamino- binds glycansto glycosaminoglycans (GAGs), facilitating (GAGs), the formation facilitating of the chemotactic formation of concentration chemotactic concentration gradients [15,17]. It hasgradients been shown [15,17]. that It has the been XCL1 shown alternate that the fold XCL1 directly alternate kills fold E. directly coli via kills physicalE. coli viamembrane disruption,physical membranebut the XCL1 disruption, chemokine but the fold XCL1 does chemokine not [18,19]. fold does Recent not [18 work,19]. Recent has demonstrated work thathas wild-type demonstrated (WT) that human wild-type XCL1 (WT) also human kills C. XCL1 albicans also kills [18].C. The albicans aim[18 of]. Thethis aimstudy of was to determinethis study whether was to determine this antifungal whether activity this antifungal is encoded activity by is one encoded or both by one of orthe both XCL1 of struc- the XCL1 structures, and to further elucidate the molecular mechanism by which XCL1 tures,kills andCandida to further. elucidate the molecular mechanism by which XCL1 kills Candida. Figure 1.Figure In vitro 1. In C. vitro albicansC. albicans killingkilling activity activity of of WT WT XCL1 XCL1 and engineeredengineered XCL1 XCL1 variants variants locked locked in the in chemokine the chemokine fold, fold, alternatealternate fold, and fold, unfolded and unfolded state. state. Left:Left: thethe two two XCL1 XCL1 nativenative structures structur (chemokinees (chemokine fold, left;fold, dimeric left; di alternatemeric alternate fold, right, fold, in right, in colorcolor (subunit (subunit A) A)and and light light grey grey (subunit (subunit B)). B)). CC3, CC3, gold, gold, is an is engineered an engineered XCL1 variantXCL1 thatvariant is locked that inis thelocked chemokine in the fold. chemokine fold. CC5,CC5, blue, blue, is is an an engineered engineered XCL1 XCL1 variant variant that that is locked is locked in the in alternate the alternate fold. WT fold. XCL1, WT orange, XCL1,interconverts orange, interconverts between the between the twotwo folds folds and and occupies occupies eacheach fold fold in equalin equal proportion proportion under under near-physiological near-physiological conditions. conditions.Right: C. albicans Right:killing C. activityalbicans killing activity ofof XCL1 XCL1 (orange), (orange), CC3 CC3 (gold), (gold), CC5 (darkCC5 blue),(dark and blue), CC0 and (grey). CC0 CC0 (grey). is an engineered CC0 is an XCL1 engineered variant lacking XCL1 thevariant disulfide lacking the disulfidebond bond that that has has no defined no defined folded folded structure. structure. Here, we used a panel of engineered XCL1 variants [16,17] and Candida killing dose- responseHere, we and used time a course panel assays,of engineered adenylate XCL1 kinase variants (AK) release [16,17] assays, and and Candida propidium killing dose- responseiodide and uptake time (PI) course assays toassays, show thatadenylate the XCL1 kinase chemokine (AK) fold release and alternate assays, foldand both propidium iodidekill C.uptake albicans (PI). However, assays ourto show data suggest that the that XCL1 the two chemokine folds kill by fold different and mechanisms.alternate fold both kill MultipleC. albicans complementary. However, our assays data indicate suggest that that the the alternate two folds fold kills kill C.by albicans differentvia directmechanisms. membrane disruption, in agreement with previous studies [18], but the chemokine fold Multiple complementary assays indicate that the alternate fold kills C. albicans via direct does not. XCL1 can provide unique therapeutic inspiration as a member of a family of membraneantimicrobial disruption, human immune in agreement system proteins,with prev withious the studies added feature [18], but of switching the chemokine folds fold doesto not. encode XCL1 multiple can distinctprovide antifungal unique mechanisms.therapeutic inspiration as a