IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr tag fluorescence tag visualisation by fluorescence tag fluorescence Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. ANS (8-Anilino-1-napthalenesulphonate) fluorescence emission spectra (400 to 600 nm) were used to monitor the exposed hydrophobic surfaces of CML42. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. Clontech Matchmaker Yeast Two Hybrid System J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. 27-Oct-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation The calmodulin-related calcium sensor CML42 plays a role in trichome branching. The calmodulin-related calcium sensor CML42 plays a role in trichome branching. J. Biol. Chem. (0021-9258) 2009 imex curation Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca 27-Oct-2007: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Dobney S., Chiasson D., Lam P., Smith SP., Snedden WA. wayne.snedden@queensu.ca Accepted 2009-OCT-8 by JYOTI. J. Biol. Chem. (0021-9258) 2009 jyoti Only protein-protein interactions imex curation cml42_arath Calcium-binding protein CML42 CML42 Calmodulin-like protein 42 F21C20.130 At4g20780 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MESNNNEKKKVARQSSSFRLRSPSLNALRLQRIFDLFDKNGDGFITVEELSQALTRLGLNADLSDLKSTVESYIQPGNTGLNFDDFSSLHKTLDDSFFGGACGGGENEDDPSSAAENESDLAEAFKVFDENGDGFISARELQTVLKKLGLPEGGEMERVEKMIVSVDRNQDGRVDFFEFKNMMRTVVIPSS 3B4FDEEC6D4BC037 calcium(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) kic_arath Calcium-binding protein KIC KIC At2g46600 KCBP-interacting calcium-binding protein F13A10.13 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEPTEKSMLLETTSTTKMETKYEDMLPVMAEKMDVEEFVSELCKGFSLLADPERHLITAESLRRNSGILGIEGMSKEDAQGMVREGDLDGDGALNQTEFCVLMVRLSPEMMEDAETWLEKALTQELCNHNLSSMP DIP protein Q9ZPX9 original sequence version: 96 2D7A9E3971F3697B cml42-ca 7 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Two dimensional 1H-15N HSQC NMR spectra of uniformly 15N-labeled CML42 in the absence (A) and presence of Ca2+ (B). The overlay of the two spectra (C) indicates the extent of the Ca2+-induced conformational changes. Figure 2 cml42-ca-1 5 9 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) direct interaction direct interaction Two dimensional 1H-15N HSQC NMR spectra of uniformly 15N-labeled CML42 in the absence (A) and presence of Ca2+ (B). The overlay of the two spectra (C) indicates the extent of the Ca2+-induced conformational changes. Figure 3 cml42-ca-2 1 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Changes in exposed hydrophobicity of CML42 in the presence of Ca2 Figure 4 cml42-ca-3 4 9 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction The latter model is also consistent with the experimental stoichiometry (N) of 3, and overall these observations are consistent with the sequence-based prediction that NtermCML42 comprises only one Ca2+-binding site (Fig. 1) Figure 5 Table 1 Magnesium ions Mg2+ 0.34+/- 0.02 Kd without 5mM magnesium ions 07.14 +/- 0.66 Kd with 5mM magnesium ions -7.31+/- 0.04 Delta H without 5mM magnesium ions -6.49 +/- 0.15 with 5mM magnesium ions cml42-kic 3 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association In order to identify possible effectors of CML42, we conducted a yeast two-hybrid screen using CML42 as bait (Fig. 6). Three positive interactors were identified and all represented a Ca2+-binding, caltractin/centrin-like protein (At2g46600) termed KIC cml42-kic-1 6 8 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association Interaction between CML42 and KIC in the yeast 2-hybrid system. Figure 6 cml42-kic-2 6 10 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Figure 6 cml42-kic-3 2 10 unspecified role unspecified role bait bait n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 7 Pulldown assays show CML42 and KIC interact in vitro in a Ca2+- dependent manner. Interaction observed only in the presence of Ca++ ions.