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UC Davis UC Davis Previously Published Works UC Davis UC Davis Previously Published Works Title 1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast Permalink https://escholarship.org/uc/item/95t1n2df Journal Biomolecular NMR Assignments, 3(2) ISSN 1874-270X Authors Lim, Sunghyuk Ames, James B. Publication Date 2009-12-01 DOI 10.1007/s12104-009-9191-3 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Biomol NMR Assign (2009) 3:269–271 DOI 10.1007/s12104-009-9191-3 ARTICLE 1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast Sunghyuk Lim • James B. Ames Received: 12 August 2009 / Accepted: 8 October 2009 / Published online: 23 October 2009 Ó The Author(s) 2009. This article is published with open access at Springerlink.com Abstract The neuronal calcium sensor (NCS) proteins 1999)andS. pombe Ncs1p (Hamasaki-Katagiri et al. regulate signal transduction processes and are highly con- 2004). All members of the NCS family have around served from yeast to humans. We report complete NMR 200 amino acid residues, contain N-terminal myris- chemical shift assignments of the NCS homolog from fis- toylation, and possess four EF-hands. sion yeast (Schizosaccharomyces pombe), referred to in Three-dimensional structures are now known for this study as Ncs1p. (BMRB no. 16446). many NCS proteins, including recoverin (Ames et al. 1997; Flaherty et al. 1993), frequenin (Bourne et al. Keywords NCS Á Ncs1p Á Fission yeast Á Calcium Á 2001), Frq1 (Strahl et al. 2007), neurocalcin (Vijay- EF-hand Á NMR Á S. pombe Kumar and Kumar 1999), and GCAPs (Ames et al. 1999; Stephen et al. 2007). The Ca2?-bound NCS proteinsshareacommonfoldwithfourEF-hands Biological context arranged in a tandem array and an exposed N-terminus. The structure of Ca2?-free recoverin contains a cova- Neuronal calcium sensor (NCS) proteins belong to a lently attached myristoyl group buried inside the protein sub-branch of the calmodulin superfamily that regulate hydrophobic core (Tanaka et al. 1995). Binding of Ca2? a variety of physiological target proteins in the brain to recoverin leads to extrusion of its myristoyl group, and retina (Ames et al. 1996;BraunewellandGundel- termed the calcium-myristoyl switch, that enables rec- finger 1999;Burgoyneetal.2004). The best charac- overin to bind to membrane targets only at high Ca2? terized NCS protein is recoverin that serves as a Ca2? levels (Dizhoor et al. 1993; Zozulya and Stryer 1992). sensor in retinal rod and cone cells where it controls the By contrast, frequenin (NCS-1) and yeast Frq1 contain desensitization of rhodopsin (Dizhoor et al. 1991; exposed myristoyl groups in their Ca2?-free state and Erickson et al. 1998;Kawamura1993). The NCS family therefore lack a Ca2? myristoyl switch (Ames et al. also includes neuronal Ca2? sensors such as neurocalcin 2000; O’Callaghan and Burgoyne 2004). Also, the (Hidaka and Okazaki 1993), hippocalcin (Kobayashi recent x-ray structure of Ca2?-bound GCAP1 (Stephen et al. 1993), and frequenin (Pongs et al. 1993), as well et al. 2007) showed the myristoyl group to be seques- as yeast homologs, S. cerevisiae Frq1 (Hendricks et al. teredinauniqueenvironmentflankedbyN-and C-terminal helices, very different from the myristate binding pocket seen in Ca2?-free recoverin. The atomic- resolution structures of other myristoylated NCS pro- teins are needed to better define the range and different types of NCS protein-myristate interactions. We report here NMR resonance assignments of myristoylated & S. Lim Á J. B. Ames ( ) S. pombe Ncs1pintheCa2?-free state as a first step Department of Chemistry, University of California, Davis, CA 95616, USA toward elucidating the protein structure and environ- e-mail: [email protected] ment around the N-terminal myristoyl group. 123 270 S. Lim, J. B. Ames Methods and experiments G59 105 G172 G33 T92 A G133 T20 G188 G162 Expression and purification of Ncs1p G41 I80 G95 S62 110 S40 T165 G2 L183 Q106 S19 T144 F55 Recombinant myristoylated neuronal calcium sensor-1 S184 S134 Y108 F35 I116 F72 D75 15 15 13 N98 V136 I86 Q54 F58 T178 K53 M156 (Ncs1p) was uniformly N- or N/ C-labeled by S90 S181S6 S46 K174 E66 115 K83 D111 F105 Y79 F85 D119 I128 S173 S93 E146 N112 D141 K158 L89 Y186 N154 K76 Q29 E84 D99 co-expressing Ncs1p and yeast N-myristoyl CoA-transfer- Q163 V125 A65 V71 R175 D161 V180 R123 D37 S117 Q28 D11 Q49 M131 W103 E120 K36 E171 V68 D157 F48 L122 M121 Q14 V91 H42 A104 K50 L115 Y31 E167 W30 N) (ppm) C170 K25 L101 D187 ase in E. coli strain, BL21(DE3) grown on M9 medium S9 N150 K160 E26 E168 K147 F169 K45 F56 L8 F22 L107 E96 V149 F64 N44 K130 N70 E142 Y67 15 K3 V17 R18 K151 L164 F69 I51 120 ( 15 13 V132 D126 I102 D15 M135 Q12 F153 R148 C38 A127 L189 I124 δ δ supplemented with N-NH4Cl and/or C6-glucose. D81 Q10 Y52 N159 M155 L27 Q5 D176 V190 Y129 E140 R94 K137 A88 I152 K100 Y118 L43 L166 D109 E47 C87 D73 Recombinant protein expression was induced by exoge- D23 A182 L110 K32 R21 D60 L13 I179 L97 F34 D143 L16 A63 K7 L185 nously adding myristic acid (10 mg/l) and 0.5 mM isopro- A74 125 pyl b-D-1-thiogalactopyranoside (IPTG) to cells grown K24 overnight at 25°C. Typically, a 1-L culture yields about L138 30 mg of myristoylated protein. Detailed procedures for 9.0 8.5 8.0 7.5 7.0 6.5 purifying myristoylated Ncs1p are described elsewhere δ(1H) (ppm) (Hamasaki-Katagiri et al. 2004). I179CD B I86CD NMR spectroscopy I128CD I152CD I124CD I102CD I51CD I116CD Samples of recombinant Ca2?-free myristoylated Ncs1p I80CD 15 (0.5–0.7 mM) were prepared in 90%/10% H2O/D2Oor I116CG2 100% D O with 5 mM Tris-d (pH 7.4), 4 mM DTT-d I124CG2 I179CG2 2 11 11 M155CE M135CE I152CG2 and 0.3 mM EDTA-d12. NMR experiments were conducted M156CE I102CG2 I51CG2 I80CG2 using Bruker Advance 600 or 800 MHz spectrometer M121CE A65CB I128CG2 A74CB M131CE A104CB equipped with a triple resonance cryogenic probe. All A127CB A63CB V136CG2 experiments were performed at 310 K. Backbone and side- A182CB A88CB chain chemical shift assignments were obtained using V190CG 20 I86CG2 15 V17CG2 V68CG2 V190CG V71CG2 N-HSQC, HNCO, HNCACB, CBCACONH, HBHA- V180CG2 C) (ppm) L89CD1 15 V136CG1 13 CONH and N-HSQC-TOCSY (mixing time of 60 ms) T165CG V91CG2 V17CG1 ( δ L101CD2 δ spectra (Ikura et al. 1990). Methyl group side-chain reso- T20CG T92CG V71CG1 V149CG2 13 13 V132CG2 nances were assigned using C-CT-HSQC and C-HCCH- T144CG T178CG L27CD2 V180CG1 L183CD L166CD2 V125CG L189CD L164CD2 TOCSY (Kay et al. 1993). For aromatic side-chain chemical V132CG1 L16CD L138CD V91CG1 L122CD2 L107CD1 L110CD1 L107CD2 shift assignments, HBCBCGCDHD, HBCBCGCDCEHE, L43CD1 L166CD1 25 13 C-CT-HSQC-TOCSY spectra (Yamazaki et al. 1993) V68CG1 13 L97CD2 along with C-HSQC-NOESY, recorded with a mixing L110CD2 L89CD2 L164CD1 time of 120 ms, were used. NMR data were processed using L27CD1 L101CD1 NMRPipe (Delaglio et al. 1995) software package and L43CD2 analyzed using SPARKY. 2.0 1.5 1.0 0.5 0.0 -0.5 δ(1H) (ppm) Assignments and data deposition Fig. 1 Two-dimensional 15N-HSQC a and 13C-CT-HSQC b NMR 2? 2? spectra of Ca -free Ncs1p recorded at 800 MHz proton frequency. Figure 1 presents HSQC spectra of myristoylated Ca -free Amide resonances from residues in the downfield region (N77, G78, Ncs1p at pH 7.4 to illustrate representative backbone and F82, N113, G114, and indole amide protons of W30 and W103) are side chain resonance assignments. NMR assignments were not shown in a. Side chain amide resonances of Asn and Gln are solid lines based on 3D heteronuclear NMR experiments performed on connected with . The assignment of backbone amide and 13 15 side-chain methyl groups are indicated in a and b, respectively. C/ N-labeled Ncs1p (residues 2–190). The protein sample Unresolved methyl groups of valine and leucine are designated as CG in this study consists of 189 native residues with a myristoyl and CD, respectively group covalently attached at the N-terminus (Gly 2). All non- proline residues exhibited strong backbone amide reso- *82% of aliphatic side chain resonances were assigned. The nances with uniform intensities, indicative of a well-defined chemical shift assignments (1H, 15N, 13C) of Ca2?-free three-dimensional protein structure. More than 95% of the Ncs1p have been deposited in the BioMagResBank (http:// backbone resonances (1HN, 15N, 13Ca, 13Cb, and 13CO) and www.bmrb.wisc.edu) under accession number 16446. 123 1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog 271 The chemical shift index of each amino acid residue Dizhoor AM, Chen CK, Olshevskaya E, Sinelnikova VV, Phillipov P, reveals a protein secondary structure in Ncs1p that closely Hurley JB (1993) Role of the acylated amino terminus of recoverin in Ca(2?)-dependent membrane interaction. Science resembles the canonical secondary structure and topology 259:829–832 seen in other NCS proteins. Ncs1p contains 10 a-helices and Erickson MA, Lagnado L, Zozulya S, Neubert TA, Stryer L, Baylor two antiparallel b-sheets (a1: 9–17; a2: 25–35; b1: 42–44; DA (1998) The effect of recombinant recoverin on the photo- a3: 44–55; a4: 61–72; b2: 79–81; a5: 82–90; a6: 101–108; response of truncated rod photoreceptors. Proc Natl Acad Sci USA 95:6474–6479 b3: 115–117; a7: 118–131; a8: 145–154; b4: 163–165; a9: Flaherty KM, Zozulya S, Stryer L, McKay DB (1993) Three- 166–174; a10: 179–186).
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