Subscriber access provided by Caltech Library Article Switchable membrane remodeling and antifungal defense by metamorphic chemokine XCL1 Acacia F Dishman, Michelle W. Lee, Jaime de Anda, Ernest Y. Lee, Jie He, Anna Huppler, Gerard Wong, and Brian F. Volkman ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00011 • Publication Date (Web): 03 Apr 2020 Downloaded from pubs.acs.org on April 3, 2020
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Figure 1.Machine learning based analysis indicates that XCL1’s α-helix has drastically reduced levels of 29 membrane remodeling activity compared to those of antimicrobial chemokines CCL20 and CXCL4. At left, 30 the mean σ-score for a window size of 30 are projected on the 3D structures of CCL20, CXCL4, and XCL1. 31 Sequences of the helices are shown below each cartoon. At right, each chemokine’s full sequence is 32 visualized on a 2D probability map, with helices highlighted by a white box. X axis, amino acid sequence. Y 33 axis, window size. Color, mean σ-score. 34 35 83x56mm (300 x 300 DPI) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment ACS Infectious Diseases Page 2 of 45
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 2.XCL1 induces Negative Gaussian Curvature in model bacterial membranes, but not model 32 mammalian membranes. (A) SAXS spectra for XCL1 incubated with SUVs that have bacteria-like membrane 33 compositions (DOPG/DOPE 20/80) at a P/L ratio of 1/1 (light blue) and 1/2 (dark blue). To facilitate 34 visualization, spectra have been manually offset in the vertical direction by scaling each trace by a 35 multiplicative factor. Spectra show characteristic Q peaks of NGC-rich Pn3m cubic phases (dashed grey 36 boxes), which are indexed in (B). (C) SAXS spectra for XCL1 incubated with model mammalian SUVs 37 (DOPS/DOPE/DOPC 20/40/40) in a P/L ratio of 1/2 show no detectable Q peaks (dashed grey box). 38 167x129mm (300 x 300 DPI) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 3 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Figure 3. Structural and functional features of XCL1 variants tested for membrane remodeling activity. (A) 46 Panel of seven XCL1 variants tested in this study. WT XCL1 can access a β-sheet structure and chemokine- 47 like α-β structure and is thought to interconvert between the two via complete unfolding. W55D can access the β-sheet and unfolded state, but not the chemokine structure. CC5 can access only the β-sheet structure. 48 XCL1 1-72 is a truncated version of WT XCL1 lacking the extended C-terminal tail (residues 73-93). XCL1 49 R23A and R43A are variants of XCL1 with reduced affinity for glycosaminoglycans which preferentially 50 occupy the chemokine structure but can access the unfolded and all-β structures. CC0 can access only the 51 unfolded state. CC3 can access only the chemokine structure. (B) Published values for WT XCL1 and select 52 XCL1 structural variants’ minimum effective concentrations (MEC, μM) against E. Coli BL21 cells.12 CC3, an 53 XCL1 structural variant locked in the chemokine structure, is notably less active. 54 79x156mm (300 x 300 DPI) 55 56 57 58 59 60 ACS Paragon Plus Environment ACS Infectious Diseases Page 4 of 45
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Figure 4. XCL1 requires access to its unfolded state or β-sheet structure, but not its C-terminal tail, for 33 membrane remodeling. (A) SAXS spectra for model bacterial membranes (DOPG/DOPE 20/80) incubated 34 with XCL1 structural variants. To facilitate visualization, spectra have been manually offset in the vertical 35 direction by scaling each trace by a multiplicative factor. All variants except CC3, which is locked into the 36 chemokine fold, induce NGC in model bacterial membranes. (B) Indexation plots for all variants shown in (A) that induce NGC. Inset: isolated indexation plot for CC0 to provide additional clarity of the coexisting Pn3m 37 and Im3m cubic phases. (C) Summary of NGC induction by each variant in model bacterial and mammalian 38 membranes. Check marks indicate induction of NGC. 39 40 174x138mm (300 x 300 DPI) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 5 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 5.XCL1 induces NGC in model fungal membranes in a structurally dependent manner and can kill 41 Candida in vitro. (A, B) SAXS spectra for model fungal membranes (DOPS/DOPE 20/80) incubated with XCL1 and the panel of XCL1 structural variants described in Figure 3. To facilitate visualization, spectra have been 42 manually offset in the vertical direction by scaling each trace by a multiplicative factor. (C, D) Indexation 43 plots for the spectra in A and B. (E) Summary of NGC induction by each XCL1 variant in model fungal 44 membranes. (F) In vitro Candida killing assay data for XCL1 (blue) and CCL28 (positive control, grey). 45 46 173x180mm (300 x 300 DPI) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment ACS Infectious Diseases Page 6 of 45
1 2 3 4 Switchable membrane remodeling and antifungal defense by 5 6 7 metamorphic chemokine XCL1 8 9 1 † 2 † 2 10 Acacia F. Dishman and Michelle W. Lee , Jaime de Anda , Ernest 11 12 Y. Lee2, 3, Jie He4, Anna R. Huppler4, Gerard C. L. Wong2 and Brian 13 14 F. Volkman1 * 15 16 17 1Department of Biochemistry, Medical College of Wisconsin, 18 19 20 Milwaukee, WI 53226 USA 21 22 2 23 Department of Bioengineering, University of Los Angeles, Los 24 25 Angeles, CA 90095, USA 26 27 28 3UCLA-Caltech Medical Scientist Training Program, David Geffen 29 30 School of Medicine at UCLA, Los Angeles, CA 90095, USA 31 32 33 4 34 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, 35 36 WI 53226 USA 37 38 39 † 40 These authors contributed equally to this work 41 42 43 *Corresponding Author: Brian F. Volkman, [email protected] 44 45 46 47 48 49 50 KEYWORDS: metamorphic proteins, XCL1, chemokines, small-angle X- 51 52 53 ray scattering, antimicrobial, antifungal 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 7 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ABSTRACT 16 17 18 Antimicrobial peptides (AMPs) are a class of molecules which 19 20 21 generally kill pathogens via preferential cell membrane 22 23 disruption. Chemokines are a family of signaling proteins that 24 25 direct immune cell migration and share a conserved tertiary 26 27 28 structure. Recently, it was found that a subset of chemokines can 29 30 also function as AMPs, including CCL20, CXCL4, and XCL1. It is 31 32 therefore surprising that machine learning based analysis predicts 33 34 35 that CCL20 and CXCL4’s -helices are membrane disruptive, while 36 37 XCL1’s helix is not. XCL1, however, is the only chemokine known to 38 39 be a metamorphic protein which can interconvert reversibly between 40 41 42 two distinct native structures (a -sheet dimer and the 43 44 chemokine structure). Here, we investigate XCL1’s antimicrobial 45 46 mechanism of action with a focus on the role of metamorphic 47 48 49 folding. We demonstrate that XCL1 is a molecular ‘Swiss army knife’ 50 51 that can refold into different structures for distinct context- 52 53 dependent functions: whereas the chemokine structure controls 54 55 56 cell migration by binding to G-Protein Coupled Receptors (GPCRs), 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 8 of 45
1 2 3 we find using Small Angle X-ray Scattering (SAXS) that only the - 4 5 6 sheet and unfolded XCL1 structures can induce negative Gaussian 7 8 curvature in membranes, the type of curvature topologically 9 10 required for membrane permeation. Moreover, the membrane 11 12 13 remodeling activity of XCL1’s -sheet structure is strongly 14 15 dependent on membrane composition: XCL1 selectively remodels 16 17 bacterial model membranes but not mammalian model membranes. 18 19 20 Interestingly, XCL1 also permeates fungal model membranes and 21 22 exhibits anti-Candida activity in vitro, in contrast to the usual 23 24 mode of antifungal defense which requires Th17 mediated cell-based 25 26 responses. These observations suggest that metamorphic XCL1 is 27 28 29 capable of a versatile multi-modal form of antimicrobial defense. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 9 of 45 ACS Infectious Diseases
1 2 3 Antimicrobial peptides (AMPs) are innate immune molecules 4 5 6 that often exhibit a common antimicrobial mechanism of disrupting 7 8 pathogen membranes, which leads to depolarization, leakage, and 9 10 eventual cell death.1-2 Examples of AMPs include cathelicidins, 11 12 defensins, bacteriocins, histatins, protegrins, and more recently, 13 14 15 an increasing number of chemokines, which are more commonly thought 16 17 of as signaling molecules.2-8 Among the human chemokines that 18 19 exhibit antibacterial, antifungal, or antiviral properties are 20 21 CCL209, CXCL410, CCL2811, CXCL1712, and XCL113. Chemokines are a 22 23 24 family of ~50 small, secreted proteins that direct cellular 25 26 migration by binding to glycosaminoglycans (GAGs) to form 27 28 concentration gradients, and by binding to G-Protein Coupled 29 30 Receptors (GPCRs) to induce pro-migratory cellular changes. All 31 32 33 members of the chemokine family share a conserved tertiary 34 35 structure consisting of a three-stranded antiparallel -sheet and 36 37 38 C-terminal -helix. 39 40 41 42 XCL1 provides a unique opportunity to demonstrate in an 43 44 unambiguous manner the relationship between sequence, secondary 45 46 and tertiary structure, and distinct forms of immune activity in 47 48 a chemokine. XCL1 is unique among chemokines because it is a 49 50 51 metamorphic protein, meaning that it reversibly switches between 52 53 two or more native structures.14-15 Thus, using XCL1 as a model 54 55 system, it becomes possible to interrogate what happens to 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 10 of 45
1 2 3 signaling activity or membrane remodeling activity when the 4 5 6 organization of secondary structure changes without changing the 7 8 amino acid content. While most chemokines bind to GAGs and GPCRs 9 10 using a single structure, XCL1 partitions these roles between its 11 12 native states, which are (1) the highly conserved chemokine fold 13 14 15 that binds its target receptor XCR1, and (2) a novel four-stranded 16 17 -sheet dimer that binds to GAGs.16-17 Of XCL1’s two native 18 19 20 conformations, the alternative -sheet structure displays strong 21 22 antibacterial activity.13 23 24 Here, we investigated XCL1’s ability to remodel membranes 25 26 like AMPs as a function of its available conformational states, 27 28 29 since previous work has attributed the bactericidal activity of 30 31 XCL1 to its ability to permeabilize bacterial membranes.13 We 32 33 applied a recently-developed machine learning classifier capable 34 35 36 of reliably predicting membrane-active -helical peptide sequences 37 38 to several prototypical chemokines.18 The classifier identified 39 40 the -helices of CCL20 and CXCL4 as motifs that impart membrane 41 42 43 activity to these chemokines. Surprisingly, however, the cognate 44 45 helix of XCL1 is not predicted to induce negative Gaussian 46 47 curvature (NGC) in membranes. We used synchrotron small angle X- 48 49 50 ray scattering (SAXS) to measure membrane curvature deformations 51 52 induced by XCL1 and a panel of XCL1 structural variants in 53 54 membranes modeling Escherichia coli, Candida albicans, and 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 11 of 45 ACS Infectious Diseases
1 2 3 mammalian cells. We found that XCL1 generates NGC in E. coli-like 4 5 6 model membranes in a manner that depends on its metamorphic 7 8 structural state: The -sheet structure exhibits strong remodeling 9 10 11 activity whereas the chemokine structure does not. Whereas 12 13 antifungal defense usually requires clearance by Th17 mediated 14 15 cell-based immune responses, we find that XCL1 induces NGC in 16 17 Candida-like membranes via a structural motif distinct from 18 19 20 classical chemokines, and exhibits anti-Candida activity in 21 22 vitro. This suggests the possibility of a versatile multi-modal 23 24 antifungal defense associated with this metamorphic chemokine. 25 26 27 28 29 RESULTS AND DISCUSSION 30 31 The presence or absence of membrane activity in different 32 33 34 folded states of XCL1 is central to the manner in which different 35 36 immune functions are programmed into this metamorphic chemokine. 37 38 A large body of work has found that specific physicochemical 39 40 properties, including positive net charge and amphipathicity, of 41 42 43 AMPs contribute to their bactericidal activity by facilitating 44 45 electrostatic and hydrophobic interactions between the peptide and 46 47 cell membranes.19 These interactions result in membrane 48 49 destabilization, which can manifest in pore formation20-23, 50 51 24-25 26 52 blebbing, and vesicle budding. A topological requirement 53 54 shared by many membrane-permeation processes is the induction of 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 12 of 45
1 2 3 negative Gaussian curvature (NGC) in target membranes.27 NGC, also 4 5 6 known as saddle-splay curvature, can be seen in the hole of a 7 8 transmembrane pore or the neck of a fusion pore: for a flat 9 10 horizontal surface labeled by cardinal directions (north (N), east 11 12 (E), south (S), west (S)), NGC can be generated by deforming the 13 14 15 surface upward in the N and S directions and downward in the E and 16 17 W directions. A broad range of studies identified a strong 18 19 correlation between the ability of AMPs, AMP mutants, and synthetic 20 21 AMP analogues to disrupt bacterial membranes and their capacity to 22 23 19, 28-34 24 generate NGC in membranes . 25 26 27 28 XCL1 lacks a membrane active -helix 29 30 31 Many antimicrobial peptides are thought to destabilize 32 33 phospholipid bilayers by inducing NGC, a topological feature of 34 35 membrane pores and blebs19, 28-34. Previously, we developed a machine- 36 37 38 learning support vector machine (SVM) classifier trained to 39 40 identify α-helical peptide sequences with the ability to remodel 41 42 membranes by generating negative Gaussian curvature18. In recent 43 44 work, we have shown that even though the SVM was trained on short 45 46 47 -helical peptide sequences, the classifier is able to identify 48 49 the existence of membrane active segments within larger membrane 50 51 active proteins35-36. This machine learning approach constitutes a 52 53 54 new kind of bioinformatic analysis that is based on function rather 55 56 than structural or sequence homology. We aimed to apply this 57 58 59 60 ACS Paragon Plus Environment 21 Page 13 of 45 ACS Infectious Diseases
1 2 3 approach to chemokines, by determining whether the ability to 4 5 6 generate NGC can be found in the α-helical domain of the 7 8 characteristic α-β chemokine fold. We assessed differences between 9 10 the sequences of XCL1, and two other antimicrobial chemokines, 11 12 CXCL4 and CCL20, using the SVM classifier to predict membrane 13 14 18 15 active peptide subdomains . This sequence-based approach allows 16 17 us to score individual amino acid segments and identify α-helical 18 19 (or disordered) regions that are potentially involved in membrane 20 21 remodeling. 22 23 24 We screened the chemokine sequences using a ‘moving window’ 25 26 scan to isolate individual segments of n amino acids. Each 27 28 subsequence was scored by the SVM classifier with a -score 29 30 31 corresponding to its probability to be membrane disruptive, P(+1). 32 33 The mean -score for each amino acid position is an average of the 34 35 36 scores for all the sequences in which that amino acid appears for 37 38 a given window size n. A large positive -score ( > 0.89) 39 40 corresponds to a high probability of membrane activity (P(+1) > 41 42 43 0.95), while a negative -score ( < 0) indicates a small 44 45 probability of membrane activity (P(+1) < 0.50). The scores for 46 47 each chemokine’s full sequence are visualized on a 2D probability 48 49 50 map for different window sizes (Figure 1). For CXCL4 and CCL20, 51 52 only the -helical C-terminal regions were predicted to have 53 54 membrane remodeling ability. In contrast, the entire XCL1 sequence 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 14 of 45
1 2 3 scored poorly for membrane activity (Figure 1) even though XCL1 4 5 13 6 exhibits antimicrobial activity . The individual -helical 7 8 sequence for CCL20 scored slightly higher than the CXCL4 -helix 9 10 11 (TWVKYIVRLLSK, = 1.0, P=0.97; PLYKKIIKKLLE, = 0.72, P=0.92, 12 13 respectively), whereas the XCL1 -helix scored significantly lower 14 15 16 (TWVRDVVRSMDRK, = 0.13, P=0.61). Although the tertiary α-β 17 18 structure is similar between the three chemokines (Figure 1), the 19 20 machine-learning classifier approach was able to identify subtle 21 22 23 differences in these sequences using its optimized physicochemical 24 25 descriptors. The presence of a membrane active -helix in CXCL4 26 27 28 and CCL20 is consistent with the antimicrobial character of these 29 30 chemokines in their native - form and provides contrast to 31 32 understand the minimal antimicrobial activity of XCL1 in its - 33 34 35 conformation, with a membrane inactive -helix. Given that the SVM 36 37 38 classifier is optimized to predict membrane active -helical 39 40 sequences and no membrane activity is identified in the XCL1 - 41 42 43 helix cognate to the helices in the other chemokines examined, 44 45 we hypothesize that membrane remodeling activity in XCL1 arises 46 47 from its ability to switch into a β-sheet structure, since such 48 49 50 membrane activity is not detectable by an -helical classifier. 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 15 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 1.Machine learning based analysis indicates that XCL1’s 26 27 -helix has drastically reduced levels of membrane remodeling 28 29 activity compared to those of antimicrobial chemokines CCL20 and 30 31 32 CXCL4. At left, the mean -score for a window size of 30 are 33 34 projected on the 3D structures of CCL20, CXCL4, and XCL1. Sequences 35 36 of the helices are shown below each cartoon. At right, each 37 38 39 chemokine’s full sequence is visualized on a 2D probability map, 40 41 with helices highlighted by a white box. X axis, amino acid 42 43 sequence. Y axis, window size. Color, mean -score. 44 45 46 47 48 XCL1 induces negative Gaussian curvature necessary for membrane 49 50 permeation in membranes modeling bacterial, but not mammalian 51 52 53 cells 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 16 of 45
1 2 3 To understand the specific biophysical mechanisms driving 4 5 6 XCL1’s ability to selectively kill bacteria, we employed 7 8 synchrotron SAXS experiments to quantitatively characterize XCL1- 9 10 induced curvature deformations in model cell membranes. Although 11 12 AMPs can kill microbes via a range of mechanisms, it has been 13 14 15 recently shown that what AMPs have in common is their ability to 16 17 generate NGC in microbial membranes,18 which is related to the 18 19 ability to kill microbes through membrane disruptive modes such as 20 21 pore formation and blebbing27, 30, 37-38. 22 23 24 Since selectivity of AMP-induced killing is thought to arise 25 26 in part from the ability to recognize differences in membrane lipid 27 28 composition between microbes and host cells, we compared the 29 30 curvature deformations induced by XCL1 in two different membranes: 31 32 33 one modeling E. coli membranes and one modeling mammalian cell 34 35 membranes. Small unilamellar vesicles (SUVs) with lipid 36 37 compositions mimicking that of E. coli membranes and mammalian 38 39 40 cell membranes were incubated with XCL1, and the resulting 41 42 crystalline structures were characterized using SAXS. Bacterial 43 44 membranes were modeled using a lipid composition of 1,2-dioleoyl- 45 46 sn-glycero-3-[phospho-rac-(1-glycerol)]/1,2-dioleoyl-sn-glycero- 47 48 49 3-phosphoethanolamine at a molar ratio of 20/80 (DOPG/DOPE 20/80). 50 51 Mammalian cell membranes were modeled using a lipid composition of 52 53 1,2-dioleoyl-sn-glycero-3-phospho-L-serine/1,2-dioleoyl-sn- 54 55 glycero-3-phosphoethanolamine/1,2-dioleoyl-sn-glycero-3- 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 17 of 45 ACS Infectious Diseases
1 2 3 phosphocholine at a molar ratio of 20/40/40 (DOPS/DOPE/DOPC 4 5 6 20/40/40). XCL1 was incubated with the SUVs at protein/lipid charge 7 8 ratios (P/L) of 1/2 and 1/1 (i.e. molar ratios of 1/85 and 2/85, 9 10 respectively). 11 12 XCL1 restructured E. coli-like membranes into phases rich in 13 14 15 NGC (Figure 2A), while control samples of the SUVs alone displayed 16 17 a broad characteristic feature consistent with the form factor 18 19 expected for unilamellar vesicles (Supplemental Figure 1). SAXS 20 21 spectra for SUVs with the E. coli-like membrane composition that 22 23 24 were exposed to XCL1 exhibited correlation peaks with Q-ratios of 25 26 √2:√3:√6:√8:√9 (for P/L = 1/2) and √2:√3:√4 (for P/L = 1/1), which index to Pn3m 27 28 cubic phases with lattice parameters of 32.17 nm and 19.68 nm, 29 30 31 respectively (Figure 2A-B). Bicontinuous lipidic cubic phases 32 33 (QII), such as the Pn3m “double diamond” cubic lattice seen here, 34 35 are also commonly formed by prototypical AMPs with model bacterial 36 37 19, 28-32 38 membranes . A bicontinuous cubic phase consists of two 39 40 nonintersecting aqueous regions that are separated by a lipid 41 42 bilayer with NGC at every point on its surface. In a cubic phase, 43 44 the average amount of Gaussian curvature, K, can be calculated 45 46 2 47 using the equation
1 2 3 membrane, XCL1 generated Pn3m cubic phases with
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 2.XCL1 induces Negative Gaussian Curvature in model 24 25 bacterial membranes, but not model mammalian membranes. (A) SAXS 26 27 spectra for XCL1 incubated with SUVs that have bacteria-like 28 29 membrane compositions (DOPG/DOPE 20/80) at a P/L ratio of 1/1 30 31 32 (light blue) and 1/2 (dark blue). To facilitate visualization, 33 34 spectra have been manually offset in the vertical direction by 35 36 scaling each trace by a multiplicative factor. Spectra show 37 38 39 characteristic Q peaks of NGC-rich Pn3m cubic phases (dashed grey 40 41 boxes), which are indexed in (B). (C) SAXS spectra for XCL1 42 43 incubated with model mammalian SUVs (DOPS/DOPE/DOPC 20/40/40) in 44 45 a P/L ratio of 1/2 show no detectable Q peaks (dashed grey box). 46 47 48 49 50 A panel of structural XCL1 variants display differing membrane- 51 52 53 disruptive activities in E. coli-like membranes 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 20 of 45
1 2 3 XCL1 has been well studied, and a number of variants have 4 5 6 been engineered to stabilize XCL1 in its chemokine, all-, and 7 8 unfolded states40-41. These variants are named CC3, CC5, and CC0 9 10 respectively. Additionally, an XCL1 point mutant, XCL1 W55D, 11 12 13 destabilizes the hydrophobic core of XCL1’s chemokine structure by 14 15 replacing hydrophobic Trp55 with a negatively charged aspartate, 16 17 and thus allows access to the all- and unfolded states, but not 18 19 17 20 the chemokine structure . Two other point mutants, XCL1 R23A and 21 22 XCL1 R43A, shift XCL1’s conformational equilibrium to favor its 23 24 chemokine structure, in addition to dramatically diminishing 25 26 XCL1’s affinity for GAGs42-43. Finally, a truncated version of XCL1 27 28 29 (XCL1 residues 1-72) is missing its extended C-terminal tail, 30 31 without alteration to the structural equilibrium. The structural 32 33 behaviors of these variants are illustrated in Figure 3A, and the 34 35 36 antimicrobial activities of select variants against E. coli as 37 13 38 measured by a radial diffusion assay are catalogued in Figure 3B. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 21 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Figure 3. Structural and functional features of XCL1 variants tested 36 37 for membrane remodeling activity. (A) Panel of seven XCL1 variants 38 39 40 tested in this study. WT XCL1 can access a -sheet structure and 41 42 chemokine-like structure and is thought to interconvert between 43 44 45 the two via complete unfolding. W55D can access the -sheet and 46 47 unfolded state, but not the chemokine structure. CC5 can access 48 49 50 only the -sheet structure. XCL1 1-72 is a truncated version of WT 51 52 XCL1 lacking the extended C-terminal tail (residues 73-93). XCL1 53 54 R23A and R43A are variants of XCL1 with reduced affinity for 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 22 of 45
1 2 3 glycosaminoglycans which preferentially occupy the chemokine 4 5 6 structure but can access the unfolded and all- structures. CC0 7 8 can access only the unfolded state. CC3 can access only the chemokine 9 10 structure. (B) Published values for WT XCL1 and select XCL1 11 12 13 structural variants’ minimum effective concentrations (MEC, M) 14 15 against E. Coli BL21 cells.13 CC3, an XCL1 structural variant locked 16 17 18 in the chemokine structure, is notably less active. 19 20 21 22 23 To determine which specific structural features are 24 25 influential in XCL1’s antibacterial activity, we used synchrotron 26 27 SAXS to systematically examine each of these seven variants’ 28 29 30 membrane disruption capabilities in SUVs with lipid compositions 31 32 that mimic those of E. coli and mammalian cell membranes. 33 34 We found that CC5 (locked -sheet structure), CC0 (unfolded 35 36 37 variant), and W55D (accesses unfolded state and -sheet structure) 38 39 are all capable of generating NGC in E. coli-like membranes, in a 40 41 42 manner similar to that of WT XCL1. The SAXS spectra of the SUVs 43 44 after exposure to these proteins revealed the formation of NGC- 45 46 rich cubic phases (Figure 4A). For W55D and CC5 at P/L = 1/2, 47 48 correlation peaks indexed to a Pn3m phase with a lattice parameter 49 50 51 of 25.16 nm and 24.57 nm, respectively (Figure 4B). For CC0 at P/L 52 53 = 1/2, we observed a coexistence of Pn3m and Im3m cubic phases, 54 55 39 the latter of which is defined by χ = –4 and A0 = 2.345. The 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 23 of 45 ACS Infectious Diseases
1 2 3 respective lattice parameters of these coexisting cubic phases 4 5 6 were 20.07 nm and 25.45 nm, which had a ratio close to the Bonnet 7 8 ratio of 1.279, indicating that the two cubic phases are near 9 10 equilibrium with the amount of curvature being balanced across 11 12 both. 13 14 15 SAXS spectrum from exposure of CC3 to E. coli-like membranes 16 17 (P/L = 1/2) showed a broad feature that is consistent with the 18 19 form factor of unilamellar vesicles and resembled the spectrum of 20 21 the control SUVs, indicating that NGC was not induced and that the 22 23 24 chemokine conformation does not effectively disrupt membranes at 25 26 this P/L ratio (Figure 4A). In other words, XCL1 requires access 27 28 to its -sheet structure or unfolded state in order to readily 29 30 31 function as an antimicrobial agent with the mechanism of membrane 32 33 permeation. 34 35 XCL1 R23A and XCL1 R43A also restructured E. coli-like SUVs 36 37 38 (P/L = 1/2) into Pn3m cubic phases, as shown in Figure 4A, B. This 39 40 indicates that despite their loss of GAG-binding affinity, these 41 42 variants retain the ability to induce membrane disruption in E. 43 44 coli-like membranes, disentangling XCL1’s GAG-binding activity 45 46 47 from its antimicrobial function. Compared with WT XCL1, the 48 49 structural equilibria of these variants are shifted to favor the 50 51 chemokine fold42, 44. Nonetheless, they are still able to induce NGC 52 53 in model bacterial membranes. In conjunction with the results from 54 55 56 CC3, CC0, W55D, and CC5, this suggests that access to the unfolded 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 24 of 45
1 2 3 and -sheet structures is critical for membrane-disruptive 4 5 6 function, and that proteins can remain highly effective as membrane 7 8 disruptors even when these structures are less highly populated 9 10 than in WT XCL1. 11 12 13 XCL1 1-72 (tail-less variant) was also able to induce NGC in 14 15 E. coli-like membranes, as demonstrated by the generation of a 16 17 Pn3m phase at P/L = 1/2, indicating that XCL1’s extended C-terminal 18 19 tail (residues 73-93) is not required for membrane-disruptive 20 21 22 activity. No XCL1 variants induced NGC in mammalian cell-like 23 24 membranes even when structural and GAG-binding profiles are 25 26 altered (Supplemental Figure 3). 27 28 The results described above indicate that XCL1 functions as 29 30 31 a molecular ‘swiss army knife’ metamorph that partitions distinct, 32 33 context-dependent functions into its different folded structures. 34 35 While the chemokine structure binds and activates its cognate 36 37 38 GPCR XCR1, only the -sheet and unfolded XCL1 structures can induce 39 40 NGC in membranes. It is interesting to compare these structural 41 42 43 tendencies of XCL1 to recently studied metaphilic peptides, which 44 45 have an architecture that allow sidechains to re-organize in 46 47 response to different environments33-34. In the case of XCL1, such 48 49 functional changes are made possible by changes in secondary 50 51 52 structure. 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 25 of 45 ACS Infectious Diseases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Figure 4. XCL1 requires access to its unfolded state or -sheet 32 33 structure, but not its C-terminal tail, for membrane remodeling. 34 35 (A) SAXS spectra for model bacterial membranes (DOPG/DOPE 20/80) 36 37 incubated with XCL1 structural variants. To facilitate 38 39 40 visualization, spectra have been manually offset in the vertical 41 42 direction by scaling each trace by a multiplicative factor. All 43 44 variants except CC3, which is locked into the chemokine fold, 45 46 induce NGC in model bacterial membranes. (B) Indexation plots 47 48 49 for all variants shown in (A) that induce NGC. Inset: isolated 50 51 indexation plot for CC0 to provide additional clarity of the 52 53 coexisting Pn3m and Im3m cubic phases. (C) Summary of NGC 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 26 of 45
1 2 3 induction by each variant in model bacterial and mammalian 4 5 6 membranes. Check marks indicate induction of NGC. 7 8 9 10 11 12 SAXS reveals XCL1’s ability to induce NGC in a conformation- 13 14 dependent dependent manner in Candida-like artificial membrane 15 16 compositions 17 18 19 Since fungi are eukaryotes, fungal membranes are more similar 20 21 to mammalian membranes than bacterial membranes, and 22 23 discrimination is often a challenge. As the structural motif for 24 25 26 generating NGC in XCL1 is different than that of other chemokines 27 28 (-sheet rather than the fold) we investigated whether XCL1 is 29 30 able to disrupt Candida-like membranes. SUVs with a Candida-like 31 32 33 membrane composition, 1,2-dioleoyl-sn-glycero-3-phospho-L- 34 35 serine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine in a molar 36 37 ratio of 20/80 (DOPS/DOPE 20/80), were incubated with XCL1 and the 38 39 resulting membrane structures were characterized using synchrotron 40 41 42 SAXS. As with other lipid-only control samples, the SAXS profile 43 44 from DOPS/DOPE 20/80 SUVs exhibited a form factor that is 45 46 consistent with unilamellar vesicles (Supplemental Figure 1). 47 48 ( 49 However, the lipid vesicles restructured when exposed to XCL1 P/L 50 51 = 1/1), as indicated by correlation peaks with ratios √2:√3:√4 that 52 53 index to a Pn3m cubic phase with a lattice parameter of 21.16 nm and
1 2 3 The seven XCL1 structural variants described earlier (CC3, 4 5 6 CC0, CC5, XCL1 W55D, XCL1 R23A, XCL1 R43A, and XCL1 1-72) were 7 8 also each incubated with Candida-like membranes and characterized. 9 10 We found that the structural dependence observed in bacteria-like 11 12 membranes was also consistent in Candida-like membranes, in which 13 14 15 variants that could access the unfolded or -sheet structure were 16 17 able to generate NGC. SAXS spectra for CC0, CC5, and W55D showed 18 19 correlation peaks with Q-ratios indicative of the presence of Pn3m 20 21 22 and Im3m cubic lattices rich in NGC (Figure 5B,D). As with E. coli- 23 24 like membranes, CC3 was not able to generate NGC in Candida-like 25 26 membranes. Interestingly, XCL1 R23A also did not generate NGC in 27 28 these membranes (Figure 5B,D), deviating from its pattern of 29 30 31 membrane-disruptive activity with bacteria-like membranes. 32 33 However, XCL1 R43A and XCL1 1-72 both generated NGC in Candida- 34 35 like SUVs, consistent with their activity with bacterial membranes 36 37 38 (Figure 5B,D). Collectively, these data suggest that similar 39 40 structure-function relationships hold for XCL1’s activity with 41 42 candidal membranes as for bacterial membranes, with a slight nuance 43 44 at GAG binding residue Arg23. 45 46 47 48 49 XCL1 exhibits antifungal activity against Candida 50 51 Several human mucosal chemokines, including CCL2811, 45, 52 53 CXCL1712, and CCL209, kill Candida but it is not clear whether this 54 55 is a general feature of chemokines. Moreover, given the metamorphic 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 28 of 45
1 2 3 nature of XCL1, it is also not known whether antifungal activity 4 5 6 can exist in a molecule whose structures are transiently occupied. 7 8 Our SAXS data demonstrate that XCL1 induces structural 9 10 rearrangement necessary for membrane disruption in Candida-like 11 12 membranes, suggesting that XCL1 should exhibit antifungal 13 14 15 activity. To investigate this hypothesis, we performed in vitro 16 17 killing assays against C. albicans and found XCL1 to be a potent 18 19 anti-candidal agent, similar to CCL28, which has known anti- 20 21 candidal activity11 (Figure 5F). While CCL28 is a slightly more 22 23 24 potent killer of C. albicans perhaps due to XCL1’s structural 25 26 interconversion, both proteins achieve complete killing at 27 28 concentrations of 1 µM. These findings are in agreement with the 29 30 previously identified correlations between NGC generation and 31 32 28, 30 33 membrane-disruptive AMP activity, including that of mammalian 34 35 α-defensins, which also have β-sheet-rich secondary structures.29 36 37 Moreover, antifungal defense in mammals usually requires clearance 38 39 40 by Th17 mediated neutrophil-based responses: Surprisingly, we find 41 42 that XCL1 induces membrane permeating NGC in Candida-like 43 44 membranes via a structural motif distinct from classical 45 46 47 chemokines. This suggests the possibility of a multi-layered 48 49 antifungal defense associated with this metamorphic chemokine. 50 51 Interestingly, both XCL1 and IL-17A are produced by NKT cells47-48 52 53 and are involved in antifungal defense49, suggesting a possible 54 55 56 functional, synergistic relationship between the two molecules, as 57 58 59 60 ACS Paragon Plus Environment 21 Page 29 of 45 ACS Infectious Diseases
1 2 3 well as a possible mechanism by which NKT cells participate in 4 5 6 host defense against fungi. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Figure 5.XCL1 induces NGC in model fungal membranes in a 34 35 structurally dependent manner and can kill Candida in vitro. (A, 36 37 38 B) SAXS spectra for model fungal membranes (DOPS/DOPE 20/80) 39 40 incubated with XCL1 and the panel of XCL1 structural variants 41 42 described in Figure 3. To facilitate visualization, spectra have 43 44 been manually offset in the vertical direction by scaling each 45 46 47 trace by a multiplicative factor. (C, D) Indexation plots for the 48 49 spectra in A and B. (E) Summary of NGC induction by each XCL1 50 51 variant in model fungal membranes. (F) In vitro Candida killing 52 53 assay data for XCL1 (blue) and CCL28 (positive control, grey). 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 30 of 45
1 2 3 4 5 CONCLUSIONS 6 7 8 Many antimicrobial peptides, such as bacteriocins, defensins, 9 10 histatins, protegrins, and chemokines,2-6, 12 kill microbes via 11 12 membrane disruption, for example by forming pores or blebs. For 13 14 example, the human chemokine CXCL17 has been shown to kill E. coli 15 16 12 17 by inducing membrane disruption . The generation of NGC in the 18 19 target membrane is a topological requirement for the formation of 20 21 these structures27, 30, 37-38. We and others have previously detected 22 23 antimicrobial activity for the human chemokine XCL1, a metamorphic 24 25 13, 46 26 chemokine with two native structures . In this work, we find 27 28 that XCL1 can selectively, physically disrupt bacterial and fungal 29 30 membranes, and that XCL1’s metamorphic folding allows for 31 32 33 restriction of its membrane remodeling function to its -sheet 34 35 structure. 36 37 Our results suggest that XCL1’s dynamic interconversion could 38 39 40 provide the opportunity for switchable, context-dependent 41 42 function. Because XCL1’s chemokine structure cannot induce 43 44 45 membrane disruption and XCL1’s -sheet structure cannot bind to 46 47 XCL1’s cognate GPCR, shifts in equilibrium could tune XCL1 towards 48 49 either membrane disruptive activity or receptor-binding activity 50 51 in different environments, allowing for synergistic, context- 52 53 54 dependent tuning of function. In fact, XCL1’s structural 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 31 of 45 ACS Infectious Diseases
1 2 3 equilibrium is known to shift to favor the -sheet state in the 4 5 17 6 presence of one of its binding partners, GAGs . It is possible 7 8 that the presence of microbial membranes can similarly shift XCL1’s 9 10 11 equilibrium toward the -sheet structure, a hypothesis which will 12 13 be explored in future studies. 14 15 In the present paradigm, proteins typically evolve to 16 17 minimize structural frustration in the thermodynamically favored 18 19 50-52 20 native state . The structural and functional divergence observed 21 22 for XCL1 has fundamental consequences for this classical picture 23 24 of a folded protein: it is possible for the same sequence to encode 25 26 different structures with distinct patterns of frustrations. We 27 28 29 speculate that metamorphosis has evolved in XCL1 because it is 30 31 associated with a gain of function that may enhance XCL1’s 32 33 ‘fitness’53. Such a gain of function is discovered here: XCL1’s 34 35 36 conformation-specific, physical disruption of antibacterial and 37 38 antifungal membranes illustrates how protein metamorphosis can 39 40 allow for adjustability of function via modulation of structural 41 42 equilibria. The principles uncovered in this study can be extended 43 44 45 to the design of tunable, multifunctional antimicrobial protein 46 47 therapeutics. 48 49 50 MATERIALS AND METHODS 51 52 53 Identification of membrane-active sequences using an SVM 54 55 classifier 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 32 of 45
1 2 3 The amino acid sequences of the human chemokines XCL1, CXCL4, 4 5 6 and CCL20 were screened for membrane active domains using a 7 8 previously validated support vector machine (SVM) classifier 9 10 trained to recognize membrane permeable helical sequences.18 We 11 12 implemented a variable moving window along the full sequences of 13 14 15 each protein to give individual segments a -score indicative of 16 17 the degree of likelihood that the segment is a membrane active 18 19 peptide. To visualize the localization of these scores along the 20 21 22 main sequence, we created a probability map displaying the scores 23 24 along the sequence for each window size used. For a given window 25 26 size, the score given to each amino acid position was calculated 27 28 29 by averaging the mean -score of all the segments in which that 30 31 amino acid appears. The resulting probability map was plotted as 32 33 a window size vs amino acid position heat map, in which the 34 35 36 intensity values correspond to the mean -score, i.e. the 37 38 probability that the segment can induce membrane permeability. 39 40 41 42 43 Mutagenesis, Expression, and Purification of Recombinant XCL1 44 45 Proteins 46 47 The QuikChange Site-directed mutagenesis kit (Stratagene) 48 49 was used to perform site-directed mutagenesis on the XCL1 pET28a 50 51 52 expression vector and the XCL1 pQE30 expression vector in order 53 54 to create the following XCL1 variants: CC3 (XCL1 V21C V59C), CC0 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 33 of 45 ACS Infectious Diseases
1 2 3 (XCL1 C11A C48A), XCL1 1-72 (truncated at residue 72), XCL1 R23A, 4 5 6 XCL1 R43A, XCL1 W55D, and CC5 (XCL1 A36C A49C). Each protein was 7 13, 17, 54 8 then expressed and purified as previously described. XCL1 9 10 and CCL28 were also produced and purified using this pipeline. 11 12 In brief, each of these proteins was then expressed recombinantly 13 14 15 in BL21 DE3 E. coli with pET28a or pQE30 expression vectors 16 17 containing the sequence for His6-SUMO-XCL1 or XCL1 variant 18 19 sequence, except CC5 (XCL1 A36C A49C), which contained His8 20 21 affinity tag. Cultures were grown in terrific broth containing 22 23 24 50 lg/mL kanamycin to an optical density of 0.5-0.7 at 37°C, at 25 26 which point 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) was 27 28 used to induce protein expression. After induction, cultures were 29 30 grown for 5h at 37°C and cells were harvested by centrifugation 31 32 33 and stored at -80°C. Following expression and harvesting, 34 35 proteins were purified. Cell pellets were resuspended in 50 mM 36 37 sodium phosphate (pH 8.0), 300 mM sodium chloride, 10 mM 38 39 40 imidazole, 0.1% (v/v) -mercaptoethanol, and 1 mM 41 42 phenylmethylsulfonyl fluoride (PMSF). Cells were then lysed by 43 44 three passages through a French press and lysates were 45 46 47 centrifuged at 12000g for 20 min to collect inclusion bodies. 48 49 Soluble fractions and resuspended inclusion bodies were incubated 50 51 with Ni2+-NTA resin (Qiagen) for 1 hour at room temperature. 52 53 Columns were rinsed and proteins were eluted with 6M guanidinium 54 55 56 chloride, 50 mM sodium phosphate (pH 7.4), 300 mM NaCl, 500 mM 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 34 of 45
1 2 3 imidazole, 0.2% sodium azide, and 0.1% (v/v) β-mercaptoethanol. 4 5 6 Infinite dilution refolding in 20 mM Tris (pH 8.0), 200 mM NaCl, 7 8 10 mM cysteine, and 0.5 mM cystine, was performed for eluted 9 10 proteins, followed by incubation at room temperature overnight 11 12 with gentle stirring. The refolded protein solutions were 13 14 15 concentrated to ~50 mL and the fusion tag was removed by overnight 16 17 cleavage with ULP1 protease (His6-SUMO tag) or TEV protease (His8 18 19 tag). The tag was separated from the protein of interest by either 20 21 cation exchange chromatography on SP Sepharose Fast Flow resin 22 23 24 (GE Healthcare UK Ltd.) or reverse nickel chromatography using 25 26 Ni2+-NTA resin (Qiagen) as above. Lastly, proteins were purified 27 28 by reverse-phase high-performance liquid chromatography with a 29 30 C18 column. Protein samples were then frozen and lyophilized. 31 32 33 Sample identities, purities, and homogeneities were confirmed by 34 35 matrix-assisted laser desorption ionization time-of-flight 36 37 (MALDI-TOF) spectroscopy. Note that because the TEV system was 38 39 40 used for CC5, the N-terminus of this protein was non-native, 41 42 missing the N-terminal Val1. 43 44 45 46 Liposome Preparation for SAXS Experiments 47 48 49 Liposomes were prepared for SAXS experiments as previously 50 51 described19, 34. In brief, lyophilized phospholipids DOPG (1,2- 52 53 dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]), DOPE (1,2- 54 55 dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl- 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 35 of 45 ACS Infectious Diseases
1 2 3 sn-glycero-3-phosphocholine), and DOPS (1,2-dioleoyl-sn-glycero- 4 5 6 3-phospho-L-serine) purchased from Avanti Polar Lipids were 7 8 dissolved in chloroform at 20 mg/mL to produce individual lipid 9 10 stock solutions. Model membrane lipid compositions were prepared 11 12 from the lipid stock solutions as mixtures at specified molar 13 14 15 ratios. Each lipid mixture was subsequently evaporated under 16 17 nitrogen and then desiccated overnight under vacuum to form a dry 18 19 lipid film, which was resuspended in aqueous 140 mM NaCl, 10 mM 20 21 HEPES (pH 7.4) to a concentration of 20 mg/mL. Lipid suspensions 22 23 24 were incubated overnight at 37°C, sonicated until clear, and then 25 26 extruded through a 0.2 m pore size Anopore membrane filter 27 28 (Whatman) to form SUVs. 29 30 31 32 33 SAXS Experiments with Model Membranes 34 35 Methods used for SAXS experiments and data fitting were based 36 37 30, 33-34, 55 38 around those that have been previously described . 39 40 Lyophilized proteins (WT XCL1 and XCL1 variants, i.e. CC3 (XCL1 41 42 V21C V59C), CC5 (XCL1 A36C A49C), CC0 (XCL1 C11A C48A), XCL1 1-72 43 44 (truncated at residue 72), XCL1 R23A, XCL1 R43A, and XCL1 W55D) 45 46 47 were solubilized in aqueous 140 mM NaCl, 10 mM HEPES (pH 7.4) and 48 49 mixed with SUVs at P/L of 1/2 and 1/1. Samples were hermetically 50 51 sealed into quartz capillaries (Hilgenberg GmbH, Mark-tubes) for 52 53 SAXS measurements taken at the Stanford Synchrotron Radiation 54 55 56 Lightsource (SSRL, beamline 4−2) using monochromatic X-rays with 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 36 of 45
1 2 3 an energy of 9 keV. The scattered radiation was collected using a 4 5 6 DECTRIS PILATUS3 X 1M detector (pixel size, 172 m) and the 7 8 resulting 2D SAXS powder patterns were integrated using the Nika 9 10 1.5056 package for Igor Pro 6.31 and FIT2D.57 Using Origin Lab 11 12 13 software, the integrated scattering intensity I(Q) was plotted 14 15 against Q. Ratios of the measured peak positions (Qmeasured) were 16 17 compared with those of permitted reflections for different crystal 18 19 phases to identify the phase(s) present in each sample. A linear 20 21 22 regression through points corresponding to the peaks was used to 23 24 calculate the lattice parameter, a, of each identified cubic phase. 25 26 For a cubic phase, each peak is represented by a point with 27 28 coordinates of the assigned reflection (in terms of Miller indices 29 30 2 2 2 31 h, k, l) and Qmeasured. For a cubic phase, Q = (2/a)√(h + k + l ). 32 33 Therefore, the slope of the regression (m = 2/a) of Q vs. 34 measured 35 2 2 2 36 √(h + k + l ) can be used to calculate a. Similarly, for a lamellar 37 38 phase, each peak is represented by a point with coordinates of the 39 40 order of the reflection, n, and Q with the relation Q = 2n/d. 41 measured 42 43 In this case, the slope (m = 2/d) of the regression of Qmeasured vs. 44 45 n is used to determine the periodic spacing, d. 46 47 48 49 50 Candida Killing Assays 51 52 Candida killing assays were performed in a similar manner to that 53 54 which has been previously described11, 45. In brief, yeast peptone 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 37 of 45 ACS Infectious Diseases
1 2 3 dextrose (YPD) medium was used to culture a single clone of C. 4 5 6 albicans strain CAF2-1 for 16-20 hours at 30 °C and 250 rpm. 7 8 Candida cultures were washed twice with low-salt buffer, 1 mM 9 10 potassium phosphate buffer (PPB). Washed cultures were diluted to 11 12 4 13 10 mL stock at final concentration of ~5x10 cells/mL cell/ L in 1 14 15 mM PPB, pH 7.0, WT XCL1 and positive control chemokine CCL28 in 16 17 lyophilized powder form were resuspended to a final concentration 18 19 20 of 400 M in 1 mM PPB and stored at -20 °C in 50 L aliquots. 21 22 Proteins were serially diluted and mixed 1:1 with the Candida stock 23 24 at ~5x104 cells/mL to a total volume of 100 L in a 96-well plate. 25 26 27 We performed a negative control (no protein) with vehicle 28 29 treatment. After incubation of the 96-well plates at room 30 31 temperature for 2h with gentle shaking, appropriate dilutions of 32 33 34 the suspensions were plated on YPD agar plates and incubated at 30 35 36 °C for 48 hours. Colonies were then counted, and viability was 37 38 calculated as a percentage of the colony number of the no-protein 39 40 control. Each protein condition was tested in triplicate, and 41 42 43 assays were performed at least in duplicate. 44 45 46 47 48 49 AUTHOR INFORMATION 50 51 52 Corresponding Author 53 54 55 *Brian F. Volkman: [email protected] 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 38 of 45
1 2 3 Author Contributions 4 5 6 AFD, MWL, ARH, GCLW, and BFV conceived and planned experiments. AFD produced and 7 8 purified proteins, analyzed data, prepared manuscript figures, and wrote the manuscript. MWL, 9 10 JdA, AFD, and EYL performed SAXS experiments and MWL, JdA, and EYL analyzed data. 11 12 13 JdA screened chemokine sequences using the machine-learning classifier and analyzed results. 14 15 MWL and JdA assisted in manuscript writing, figure preparation, and revision. JH performed in 16 17 vitro Candida assays and both JH and ARH analyzed results and assisted in manuscript revision. 18 19 20 All authors have given approval to the final version of the manuscript. 21 22 23 Competing interests 24 25 26 B.F.V. has ownership interests in Protein Foundry, LLC. 27 28 29 30 31 ACKNOWLEDGMENTS 32 33 34 This work was supported in part by National Institutes of Health Grants R01 AI058072 and R01 35 36 AI120655 (to B.F.V.), K08 DE026189 (to A.R.H.), and F30 CA236182 (to A.F.D). A.R.H. was 37 38 also supported by the Children’s Hospital of Wisconsin Research Institute. A.F.D. is a member 39 40 41 of the NIH supported (T32 GM080202) Medical Scientist Training Program at the Medical 42 43 College of Wisconsin (MCW). J.d.A. is supported by the National Science 44 45 Foundation Graduate Research Fellowship under Grant No. DGE- 46 47 1650604. M.W.L. and G.C.L.W. are supported by NIH R01AI052453, 48 49 50 NSF DMR1808459, and the National Psoriasis Foundation. E.Y.L. 51 52 acknowledges support from the Systems and Integrative Biology 53 54 Training Program (T32GM008185), the Medical Scientist Training 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Page 39 of 45 ACS Infectious Diseases
1 2 3 Program (T32GM008042), and the Dermatology Scientist Training 4 5 6 Program (T32AR071307) at UCLA, and an Early Career Research 7 8 Grant from the National Psoriasis Foundation. 9 10 11 12 13 14 15 ASSOCIATED CONTENT 16 17 18 Supporting Information Available: 19 20 21 Supplemental Figure 1. SAXS spectra for SUV-only control samples. 22 23 24 Supplemental Figure 2. SAXS spectra for protein-only control 25 samples. 26 27 28 29 Supplemental Figure 3. SAXS spectra for XCL1 variant panel 30 31 32 incubated with mammalian cell-like membranes. 33 34 35 36 37 This material is available free of charge via the internet at 38 39 http://pubs.acs.org. 40 41 42 REFERENCES 43 44 1. Shai, Y., Mechanism of the binding, insertion and 45 destabilization of phospholipid bilayer membranes by alpha- 46 47 helical antimicrobial and cell non-selective membrane-lytic 48 peptides. Biochim Biophys Acta 1999, 1462 (1-2), 55-70. 49 2. Zasloff, M., Antimicrobial peptides of multicellular 50 organisms. Nature 2002, 415 (6870), 389-95. 51 3. Wolf, M.; Moser, B., Antimicrobial activities of 52 chemokines: not just a side-effect? Front Immunol 2012, 3, 213. 53 4. Crawford, M. A.; Margulieux, K. R.; Singh, A.; Nakamoto, R. 54 55 K.; Hughes, M. A., Mechanistic insights and therapeutic 56 57 58 59 60 ACS Paragon Plus Environment 21 ACS Infectious Diseases Page 40 of 45
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1 2 3 4 Table of Contents Graphic: 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 21