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/ Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism. Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism. Cell (0092-8674) 2006 imex curation Schumacher MA., Karamooz E., Zikova A., Trantirek L., Lukes J. maschuma@mdanderson.org 25-OCT-2006: Contacted by JYOTI. Only protein-protein interactions 2 in vitro In vitro fps fluorescence polarization spectroscopy Fluorescence anisotropy FPS FPS Fluorescence anisotropy fps predetermined predetermined participant predetermined Schumacher MA., Karamooz E., Zikova A., Trantirek L., Lukes J. maschuma@mdanderson.org Accepted 2006-SEP-26 by JYOTI. 25-OCT-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 2 imex curation Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism. Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism. Cell (0092-8674) 2006 imex curation Schumacher MA., Karamooz E., Zikova A., Trantirek L., Lukes J. maschuma@mdanderson.org 25-OCT-2006: Contacted by JYOTI. Only protein-protein interactions 2 in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Schumacher MA., Karamooz E., Zikova A., Trantirek L., Lukes J. maschuma@mdanderson.org Accepted 2006-SEP-26 by JYOTI. 25-OCT-2006: Contacted by JYOTI. Cell (0092-8674) 2006 Only protein-protein interactions 2 imex curation gnd7-506_trybr_grna Trypanosoma brucei guide RNA gND7-506 (I and II) Stem/Loops I and II rna ribonucleic acid RNA rna 9tryp Trypanosoma brucei AAAUACGAUGUAAAUAACCUGUAGUAUAGUUAGUGUAUA 06D78883CD82EBC7 q952g2_9tryp protein protein 9tryp Trypanosoma brucei MLRRIISQQTTLIRKPLGAALFATSALRLEAASSSDADGKEVGSSGEGNRATGGKWRRPSLAQQRARRAQLPPAFDVVHWNDEDISRGHLLRVLHRDTFVVLDYHRQARMLTEEGNKAERVVSVMLPAVYTARFLAVLEGRSEKVEVHSRYTNATFTPNPAAPYTFTLKCTSTRPAQQKQQVAGEEGDETFEWTVEFDVAESLMLQRFLTQALHYNTGFARTSV A0836ABF429825BC p90629_9tryp gBP21 protein protein 9tryp Trypanosoma brucei MIRLACLRGPAMRSAWSRGASTFSGVQSLPKFEIHDVRDDPALGTMTRVAVDGKLLLISQYPQLGPRKVDPNDLSPQFDADRRISVRLRHVDLAYLVGVCKERVPRHRMETKAYTLDFEKSAQGYHLHGKVHRVASQRMEDWSVKFDNHFAVTLEHFLESALDESFGFRQHYATRAAEGGEKIAATSSAEGGARRKRSVSDTSRYH BE2EB3B9AFC9228F p90629-mrp2 2 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 110 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 125 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 204 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 110 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 125 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 204 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei 5 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 46 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 109 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 139 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei direct interaction direct interaction The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). 1, Table 1 MRP1 and MRP2 combine to form a heterotetramer The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. The model includes 8 acetate molecules and 572 water molecules. The modified amino acids are detailed in the pdb file. p90629-mrp2-1 2 5 unspecified role unspecified role neutral component neutral component 46 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 109 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 139 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 110 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 125 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 204 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 110 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 125 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 204 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei direct interaction direct interaction The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). 1, Table 1 A second, rhombohedral, crystal form of apoMRP1/MRP2 was obtained (Table 1), solved by Molecular Replacement using EPMR (Kissinger et al., 1999), and was minimally refined to an Rfree of 29.9% to 3.35 A � resolution. The model contains two tetramers that are identical to the P21 tetramer structure and consists of residues 28�173 of each of the four MRP1 subunits, 60�175 and 188�221 of one MRP2 subunit and 59�175 and 188�221 of three of the MRP2 subunits and 56 solvent molecules. The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. The modified amino acids are detailed in the pdb file. p90629-mrp2-2 2 5 unspecified role unspecified role neutral component neutral component 46 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 109 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 139 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei 3 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 4 unspecified role unspecified role neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain 110 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 125 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 204 se(s)met L-selenomethionine Delta:S(-1)Se(1) Se(S)Met Selenium replaces sulphur semetm certain certain sequence position certain certain certain sequence position certain 9tryp Trypanosoma brucei direct interaction direct interaction The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. The modified amino acids are detailed in the pdb file. Crystals of this complex were grown under low salt conditions using a gRNA fragment encompassing gND7-506 stem/loops I and II (gND7- 506(I-II)), corresponding to nucleotides 1�44 in Figure 3A (Experimental Procedures). The structure was solved by Molecular Replacement and the ASU contains one MRP1, one MRP2, and one gND7-506(I�II). The heterotetramer, identical to that in the apo structures, was generated by crystallographic symmetry. 5 p90629-mrp2-3 1 3 unspecified role unspecified role neutral component neutral component region stem/loop II (oligo i) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain fluorescein 5'-fluorescein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei physical association physical association The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. We found that the MRP1/ MRP2 complex binds the stem/loop II (oligo I) and stem/loop I + stem/loop II (oligo II) of gND7-506 (Figure 3B) with apparent binding affinities (Kds) of 7.0 � 0.5 nM and 5.5 � 0.9 nM, respectively, at salt concentrations of 50mM NaCl. Only weak binding was observed to the oligo(U)-tail (Kd approx. 150 nM) and essentially no binding to stem/loop I alone, except when these molecules were heated to unfold them. 3 B p90629-mrp2-4 1 3 unspecified role unspecified role neutral component neutral component region stem/loopI-stem/loopII (oligo II) sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain fluorescein 5'-fluorescein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei physical association physical association The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. We found that the MRP1/ MRP2 complex binds the stem/loop II (oligo I) and stem/loop I + stem/loop II (oligo II) of gND7-506 (Figure 3B) with apparent binding affinities (Kds) of 7.0 � 0.5 nM and 5.5 � 0.9 nM, respectively, at salt concentrations of 50mM NaCl. Only weak binding was observed to the oligo(U)-tail (Kd approx. 150 nM) and essentially no binding to stem/loop I alone, except when these molecules were heated to unfold them. The oligonucleotide was 5'-fluorescinated. Increasing the salt concentration decreased the binding and at concentrations of NaCl above 0.2 M binding is quickly abrogated. (Kds - 5.1 E-9 at 0.05 M NaCl, 8.5 E-9 M at 0.1 M NaCl, 2.7 E-8 M in 0.15 M NaCl, 2.06 E-8 in 0.2 M NaCl and no saturable binding at 0.3 and 0.4 M NaCl.) 3 B, C, S1 Magnesium is not required for high affinity binding (Kd 6.9 E-9 in the presence of 5.0 E-3 M magnesium ions versus 5.1 E-3 M in its absence). p90629-mrp2-5 1 3 unspecified role unspecified role neutral component neutral component region stem/loop I sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain fluorescein 5'-fluorescein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei 4 unspecified role unspecified role neutral component neutral component 9tryp Trypanosoma brucei physical association physical association The sequence of the protein MRP1 used here has a Glu instead of a Leu at position 43, unlike the Uniprot sequence (P90629). The MRP1 sequence used had the N-terminal mito-targeting sequence and a C-terminal region found to cause insolubility removed. The MRP2 sequence used had the N-terminal mito-targeting sequence removed. The oligonucleotide was 5'-fluorescinated. S3 B The Kds after unfolding for the oligo(U) tail and stem/loop I were 26.6 � 5.0 nM and 51.1 � 4.1 nM, respectively. The RNA molecule was heated to 100 degrees C to unfold it.