Protein Cell 2013, 4(12): 942–950 DOI 10.1007/s13238-013-3091-0 Protein & Cell RESEARCH ARTICLE Crystal structure of the N-terminal ankyrin repeat domain of TRPV3 reveals unique conformation of fi nger 3 loop critical for channel function Di-Jing Shi1*, Sheng Ye2*, Xu Cao1, Rongguang Zhang2, KeWei Wang1,3,4 1 Department of Neurobiology, Neuroscience Research Institute, Peking University Health Science Center, Beijing 100191, China 2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China Cell 3 Department of Molecular and Cellular Pharmacology, State Key Laboratory of Natural and Biomimetic Drugs, Peking University & School of Pharmaceutical Sciences, Beijing 100191, China 4 PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China Correspondence: [email protected] (R. Zhang), [email protected] (K. W. Wang) Received August 23, 2013 Accepted October 9, 2013 Protein ABSTRACT of cation channels that function as cellular sensors for numer- ous stimuli (Clapham, 2003). According to their sequence In all six members of TRPV channel subfamily, there is homology, TRP channels are divided into seven subfamilies: an ankyrin repeat domain (ARD) in their intracellular N- TRPV, TRPA, TRPN, TRPM, TRPML, TRPP, and TRPC. termini. Ankyrin (ANK) repeat, a common motif with typi- Among the TRPV subfamily, all six members can be further cally 33 residues in each repeat, is primarily involved divided into two groups: the nonselective and heat-activated in protein-protein interactions. Despite the sequence TRPV1–4 channels primarily expressed in sensory neurons similarity among the ARDs of TRPV channels, the struc- and keratinocytes, and highly Ca2+ selective TRPV5–6 chan- ture of TRPV3-ARD, however, remains unknown. Here, nels primarily expressed in epithelial tissues (Montell et al., we report the crystal structure of TRPV3-ARD solved at 2002; Venkatachalam and Montell, 2007). TRPV1–4 channels 1.95 Å resolution, which reveals six-ankyrin repeats. While are activated by temperature, voltage, protons, and chemicals, overall structure of TRPV3-ARD is similar to ARDs from and are responsible for maintaining homeostasis and sensing other members of TRPV subfamily; it, however, features a environmental changes (Guler et al., 2002; Voets et al., 2004; noticeable fi nger 3 loop that bends over and is stabilized Caterina, 2007; Dhaka et al., 2009). by a network of hydrogen bonds and hydrophobic pack- TRPV3 can be activated by warm temperature with the ing, instead of being fl exible as seen in known TRPV-ARD threshold about 33°C. TRPV3-knockout mice have deficits structures. Electrophysiological recordings demonstrated in response to innocuous and noxious heat, suggesting the that mutating key residues R225, R226, Q255, and F249 of participation of TRPV3 in thermosensation and nociception fi nger 3 loop altered the channel activities and pharmacol- (Moqrich et al., 2005). Like other TRPV channels, TRPV3 is ogy. Taken all together, our findings show that TRPV3- a multimodal cation channel, and can be activated by natural ARD with characteristic fi nger 3 loop likely plays an im- compounds such as camphor, thymol (Moqrich et al., 2005; portant role in channel function and pharmacology. Xu et al., 2006), endogenou s ligands farnesyl pyrophos- phate (FPP), NO (Yoshida et al., 2006; Bang et al., 2010), and KEYWORDS TRPV3, ARD, keratinocyte, 2-APB, skin a synthetic small molecules 2-APB (Chung et al., 2004a; Hu et al., 2004, 2009). TRPV3 has two distinct features. First, both in native and expression systems, TRPV3 does not desensitize INTRODUCTION in response to repetitive stimulations (Chung et al., 2004a, Transient receptor potential (TRP) channels are a large family 2004b). This property is independent of the stimuli (Moqrich *These authors contributed equally to the work. 942 | December 2013 | Volume 4 | Issue 12 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 Crystal structure of TRPV3-ARD RESEARCH ARTICLE et al., 2005) and is thought to result from sequential reduction structure of TRPV3-ARD with molecular replacement by using of calcium inhibition on TRPV3 from both sides of cells, likely the structure of rTRPV1-ARD (PDB entry: 2PNN) as a search- because of conformational changes that occur during succes- ing model. In the fi nal model of TRPV3-ARD, there are two sive activation of channel (Xiao et al., 2008a). Second, TRPV3 protein molecules in one asymmetric unit (ASU), which do not is abundantly expressed in the keratinocytes of skin contain- dimerize due to the lack of suffi cient interactions. The overall ing nerve endings that are physiologically required to respond crystal structure of TRPV3-ARD reveals a common feature of to physical and chemical stimuli. Consistent with its specific six ankyrin (ANK) repeats with each repeat consisting of a pair localization, TRPV3 plays critical roles in skin physiology and of anti-parallel α-helices and a loop linking the adjacent repeats pathology. TRPV3 knockout mice exhibit wavy hair and curled (Fig. 1B). Since the inner helices pack more tightly than the whiskers (Cheng et al., 2010), likely resulting from misaligned outer helices, the six repeats stack with a slight counterclock- and curved follicles with deranged angles. wise twist of about 2–3° along the stacking axis. The overall TRP channels are assumed to function as tetramers with structure forms the shape of a palm up with outward projecting each subunit containing six transmembrane helices and in- loops as fi ngers 1–5 (Fig. 1B). A groove is formed by concave tracellular N- and C-termini. In the TRPV subfamily, all the six surface of the palm, which can function as binding sites of in- members share conserved ankyrin repeat domains (ARDs) in teracting proteins (Fig. 1B). their N-termini. These ankyrin repeats are involved in protein- TRPV3-ARD shares overall folds with other TRPV-ARD protein interactions (Stokes et al., 2005; Kim et al., 2006), structures, consistent with their high sequence homology channel assembly (Chang et al., 2004; Erler et al., 2004), and among the ARDs of this subfamily. However, several distinct traffi cking (Arniges et al., 2006). Visualizing the ARDs of TRPV structural features are noted. The most striking variance occurs Cell channels is critical for understanding their molecular basis of at fi nger 3 that bends over towards fi nger 2 and is stabilized by channel function and regulatory mechanisms. The ARD struc- interactions with inner helix of repeat 3 (Fig. 1C). Other struc- & tures, with or without ATP binding, of several TRPV channels tural differences reside in fi nger 4 and 5 near the outer helices, have been reported, including TRPV1, TRPV2, TRPV4, and where these two fingers are two- and four-residue shorter, TRPV6 (Jin et al., 2006; McCleverty et al., 2006; Lishko et al., respectively, in the sequence of TRPV3-ARD, as compared 2007; Phelps et al., 2008; Auer-Grumbach et al., 2010; Inada to TRPV1-ARD. Also, a sequence insertion in TRPV3-ARD Protein et al., 2012). All these structures show that TRPV-ARD con- causes the loop between outer helix 1 and inner helix 2 to be tains six ankyrin repeats with each repeat having an anti-paral- much longer than that of TRPV1-ARD (Fig. 1C). Because the lel helix-turn-helix fold followed by a β-hairpin loop connecting fi ngers may function as binding sites in ankyrin groove, these adjacent repeats. The overall fold forms a hand-up shape with differences suggest the divergence of interacting proteins of the helices as a curved palm and outward projecting loops as different TRPV-ARDs. fi ngers. Compared with classical ankyrin repeats, TRPV-ARDs have obvious twists between repeats 4 and 5. In spite of high The conformation of TRPV3-ARD fi nger 3 similarities in overall folds, TRPV-ARDs have apparent differ- ences in hydrophobic surfaces that mediate interactions with To understand the characteristic conformation of finger 3 in regulatory factors (Phelps et al., 2008; Inada et al., 2012). Until TRPV3-ARD, we made a comparison with previously reported now, however, there is no structural information for TRPV3- TRPV-ARD structures in which three of them are in com- ARD or other domains available for structural insights into mo- plex with ATP (rTRPV1-ARD, cTRPV4-ARD, and hTRPV4- lecular basis of TRPV3 channel function. ARD) and four are ligand-free (rTRPV2-ARD, hTRPV2-ARD, In this study, we solved the crystal structure of TRPV3-ARD hTRPV4-ARD, and mTRPV6-ARD). Since all TRPV-ARD at 1.95 Å resolution. The structural analysis of TRPV3-ARD re- structures share common ANK repeats, the r.m.s.d. among veals six-ankyrin repeats with a long fi nger 3 loop that differen- them is in the range of 1 .4–1.8 Å for backbone Cαs. However, tiates itself from known ARDs of TRPV channels. Patch clamp signifi cant variation in fi nger 3 conformations suggests that the recordings of TRPV3 mutated in key residues of fi nger 3 loop loop is the most fl exible region of TRPV-ARDs (Fig. 2A). Most confi rmed the role of TRPV3-ARD with characteristic fi nger 3 fi nger 3 of TRPV-ARDs bear higher B-factors than their adja- in channel function and pharmacology. cent helical regions (Fig. 2B). In fact, the fi nger 3 of TRPV2- ARD even adopts three different conformations in three dif- RESULTS ferent crystal forms, showing its remarkable fl exibility (Jin et al., 2006). With or without ATP binding, most fi nger 3 have no Overall structure of TRPV3-ARD direct interactions with the helices of ANK repeats. The mTRPV3-ARD proteins containing residues 118 to 367 Unlike other TRPV-ARDs, the fi nger 3 of both TRPV3-ARD were expressed and purifi ed from Rosetta (DE3) with affi nity molecules in one ASU bends over and forms a unique con- an d size exclusion chromatography (SEC).