Calmodulin binds to Drosophila TRP with an unexpected mode

•An elongated region of the Drosophila TRP tail specifically binds to Ca2+-CaM•Two discrete fragments N- and C-lobes Ca2+-CaM•Unexpectedly, CaM binding does not directly modulate channel activity•Mammalian TRPC4 Ca2+-CaM with a similar mode as is calcium-permeable cation essential for fly visual signal transduction. During phototransduction, Ca2+ mediates both positive negative feedback regulation on activity, possibly via calmodulin (CaM). However, molecular mechanism underlying modulated CaM/TRP interaction poorly understood. Here, we discover an unexpected, Ca2+-dependent between TRP. The contains two sites (CBS1 CBS2) separated by ?70-residue linker. CBS1 N-lobe CBS2 recognizes C-lobe. Structural studies reveal lobe-specific CBS1&2. Mutations introduced in eliminated full-length TRP, but surprisingly had no effect response light under physiological conditions, suggesting alternative mechanisms governing Ca2+-mediated activity. Finally, that TRPC4, closest mammalian paralog adopts mode. Transient receptor potential channels (TRP channels) are extensively expressed different species animals involved diverse processes such responding light, pressure, pain, taste, temperature, other stimuli (Clapham, 2003Clapham D.E. cellular sensors.Nature. 2003; 426: 517-524Crossref PubMed Scopus (2022) Google Scholar; Montell, 2005Montell C. superfamily channels.Sci. STKE. 2005; 2005: re3PubMed Scholar). Based their sequence similarities functional properties, mammals can be classified into seven subfamilies: canonical (TRPCs), melastatin (TRPMs), vanilloid (TRPVs), polycystin (TRPPs), ankyrin transmembrane protein 1 (TRPA), mucolipin (TRPML), mechanosensing TRPN (Drosophila NOMPC) (Li, 2017Li H. Classification.Adv. Exp. Med. 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Chyb vivo analysis drosophila channels, TRPL.Neuron. 19: 1249-1259Abstract (158) (Gu 2005Gu Oberwinkler Postma Mechanisms photoreceptors.Curr. 1228-1234Abstract (73) critical dark noise threshold maintenance photoreceptors (Chu 2013Chu C.H. Sengupta Gupta Common quantum bump amplification Neurophysiol. 2013; 109: 2044-2055Crossref (15) Katz 2012Katz Phospholipase C-mediated suppression enables single-photon detection 32: 2722-2733Crossref (22) which regulates activities largely unknown. At least nonexclusive exist. First, may bind regulate activities, case recently demonstrated TRPMs (Wang 2018Wang L. Fu T.M. Zhou Xia Greka Wu Structures gating human TRPM2.Science. 362: eaav4809Crossref (72) Yin 2019Yin Le S.C. Hsu A.L. Borgnia M.J. Yang Lee S.Y. basis cooling agent lipid sensing cold-activated TRPM8 channel.Science. 2019; 363: eaav9334Crossref (93) Second, might indirectly activity modulating (CaM) 2018Sun Zheng Identification novel site C-terminus.Biochem. Biophys. Res. 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Unexpectedly, found containing CBSs one molecule its engaging Lys75 plays role determining specificity CBS CaM. Guided biochemical TRP/CaM uncovered searched TRPCs, mouse Together, our discoveries serve framework future began performing detailed tail. begins residue S717 ends at L1275 (Figure 1A). Searching Target Database (http://calcium.uhnres.utoronto.ca/ctdb/no_flash.html) (Yap 2000Yap K.L. Kim Truong Sherman Yuan Ikura target database.J. Struct. Funct. Genomics. 2000; 1: 8-14Crossref (442) showed N-terminal part 717-940) several CBSs, rest 940-1275) contain signs sequence. Consistently, 940-1275), purified thioredoxin (Trx)-tagged fusion detectable either absence 1B). contrast, bound based analytical gel filtration chromatography coupled static scattering (SLS). Using Trx-tagged tag-removed proteins, calculated 1:2 stoichiometry 1C), indicating within fragment. further divided 717-940 two. Interestingly, aa 783-940 (we chose start N783, marks beginning nonconserved regions among coiled-coil domain; Figure 1F) manner, CBS-B 1A) 1:1 1D). 717-783 could soluble forms alone bacterial cells. therefore used synthetic peptide test whether certain segment earlier corresponding 728-754 Kd 3.75±0.62 ?M (Tang 1E), refer CBS-A residues overlap entire connecting helix closed state structures TRPC3, TRPC5, TRPC6 (Duan 2018Duan Zeng Chen Peng Wang Clapham Structure channel.Nat. 3102Crossref (60) Duan 2019Duan J.J. Ge J.Y. K.C. X.G. Zhong J.N. Y.X. al.Cryo-EM 2.8-angstrom resolution reveals unique conserved elements function.Sci. 5: eaaw7935Crossref (32) 2018Tang Q. Guo J.X. receptor-activated TRPC3 channels.Cell 28: 746-755Crossref (76) 1F). predicted form single completely buried TRPCs 1G). Therefore, it unlikely accessible channel. Indeed, conditions (see data 4). cannot exclude possibility when gets exposed. preceding structure-based TRPC focus encompassing study. Detailed sequences insects, though 2A). These defined T802-K862) M899-D940), experiments, unless specified otherwise. Trx-fused indeed Isothermal titration calorimetry (ITC)-based assays 0.35±0.09 ?M, 2B1). 0.25±0.03 Neither nor apo-CaM (titration curves red 2B). 2B manner quite strong affinities. SLS 1D), ultracentrifugation (AUC) sedimentation velocity 2C) N783-D940) formed stable stoichiometry. fact, TRP(783-940)/Ca2+-CaM obtained only coexpressing bacteria Removal addition excess amount EDTA led dissociation TRP(783-940) Moreover, precipitated due low solubility (data shown). There possible explanations observations. compete each so engages complex. Alternatively, simultaneously interact thus forming To differentiate these possibilities, mixed CBS1, CBS2, 1:1:1 molar ratio subjected mixture analysis. CBS1/CBS2/CaM eluted peak volume smaller elution CBS1/CaM CBS2/CaM 2D, top). SDS-PAGE fractions Trx-CBS1 Trx-CBS2 bottom). Taken together, suggested triple were (i.e., same moiety). understand CBS12 resorted NMR spectroscopic studies. compared 1H-15N HSQC spectrum coexpressed underwent significant binding-induced chemical shift changes (Figures 2E, S1A, S1B, whole spectra). indicate lobes CBS12. dissect interaction, overlaid 15N-Ca2+-CaM/CBS12 15N-Ca2+-CaM/14N-CBS1 15N-Ca2+-CaM/14N-CBS2 Ca2+-CaM/CBS12 roughly overlapped summed spectra 2E S1B), distinct Ca2+-CaM. Most signals 15N-labeled remain unstructured very sharp peaks suggests CaM-bound S1A although definitive answer will require additional NMR-based measuring residual dipolar coupling constants and/or relaxation small angle X-ray experiments unlabeled S1A). Ca2+-CaM, caused relatively C-lobe Conversely, signature Gly EF-hand zoomed respectively, confirm conclusion derived generated derivatives mutated Ca2+-binding substituting last Glu EF-hands 1&2 Gln E31/67Q) 3&4 E104/140Q). E31/67Q-CaM lost retained CBS2. E104/140Q-CaM S2). formulate model 2F). model, selectively It noteworthy complete spans total ?140 asked able higher affinity synergistic actions Because obtain isolated ITC-based assay, equilibrium measure constant 2G). value (0.10 ± 0.01 ?M) AUC comparable Ca2+-CaM/CBS1 Ca2+-CaM/CBS2 2B), little conformational (or synergism) attempted uncover crystal CBS12/Ca2+-CaM Despite extensive trials, crystallize complex, likely flexibility CBS12-bound 2F Figures S1). independently decided CBS1/N-lobe_CaM CBS2/C-lobe_CaM separately. determine resolutions 1.78Å 2.15Å, respectively 3A S2 Table 1). easy viewing, connected dotted line representing central ?D ?E Ca2+-CaM.Table 1Statistics crystallographic collection refinementData collectionDatasetTRP CBS1/N-lobeTRP CBS2/C-lobeTRPC4 CBS1/N-lobeSpace groupH32I41P6122Wavelength0.978900.978900.97890Unit cell (a,b,c,Å)98.449, 98.449, 134.2658.344, 58.344, 92.2164.46, 46.46, 120.75Unit (?,?,?,?)90, 90, 12090, 9090, 120Resolution range (Å)49.27–1.78 (1.81–1.78)49.31–2.15 (2.19–2.15)50.00-1.90 (1.97–1.90)No. reflections24,105 (1,192)8342 (369)12,754 (1,169)Redundancy19.6 (19.5)12.6 (9.1)7.4 (7.5)I/sigma55.8 (7.27)50.18 (2.71)19.11 (2.29)Completeness (%)100.0 (100.0)99.1 (88.5)99.7 (99.5)RmergeaRmerge = ? |Ii - |/? Ii, where Ii intensity measured reflection mean symmetry-related reflections. (%)6.1 (35.6)5.0 (36.8)10.2 (100)Structure refinementResolution (1.85–1.78)49.31–2.15 (2.09–1.90)RcrystbRcryst ?||Fcalc| – |Fobs||/?Fobs, Fobs Fcalc observed factors./RfreecRfree ?T||Fcalc| T dataset approximately 5% reflections randomly chosen set aside prior refinement. (%)20.48/23.3921.40/25.3221.94/24.98Rmsd bonds (Å)/angles (°)0.007/0.9630.0107/1.450.006/0.909Average factors (Å2)30.8868.5827.54No. atoms Protein atoms1,444664725 Water73030 Other molecules422No. Working set22,889 (2,530)7,910 (513)11,637 (2,800) Test set1,202 (127)415 (34)607 (140)Ramachandran plot Favored (%)98.4098.8598.90 Allowed (%)0.530.001.10 Outliers (%)0.531.150.00Numbers parentheses represent highest-resolution shell.a Rmerge reflections.b Rcryst factors.c Rfree Open table new tab Numbers shell. Both adopt open conformation occupying (Zhang 1995Zhang Tanaka Calcium-induced transition solution apo calmodulin.Nat. 758-767Crossref (634) “helix-turn-heli