PMC:4504005 / 7358-10286
Annnotations
TEST0
{"project":"TEST0","denotations":[{"id":"25982113-151-159-8319010","span":{"begin":206,"end":210},"obj":"[\"24115156\", \"24115156\", \"24115156\"]"},{"id":"25982113-234-242-8319011","span":{"begin":347,"end":351},"obj":"[\"21625560\", \"21625560\", \"21625560\"]"},{"id":"25982113-226-234-8319012","span":{"begin":365,"end":369},"obj":"[\"20966082\", \"20966082\", \"20966082\"]"},{"id":"25982113-234-242-8319013","span":{"begin":387,"end":391},"obj":"[\"18801728\", \"18801728\", \"18801728\"]"},{"id":"25982113-226-234-8319014","span":{"begin":393,"end":397},"obj":"[\"19674967\", \"19674967\", \"19674967\"]"},{"id":"25982113-232-240-8319015","span":{"begin":399,"end":403},"obj":"[\"20172855\", \"20172855\", \"20172855\"]"},{"id":"25982113-234-242-8319016","span":{"begin":421,"end":425},"obj":"[\"18375760\", \"18375760\", \"18375760\"]"},{"id":"25982113-233-241-8319017","span":{"begin":1457,"end":1461},"obj":"[\"22102369\", \"22102369\", \"22102369\"]"},{"id":"25982113-221-229-8319018","span":{"begin":2252,"end":2256},"obj":"[\"9438028\", \"9438028\", \"9438028\"]"}],"text":"Engineering CLR:RAMP ECD Complexes for Crystallization\nWe previously reported a tethered fusion protein approach to engineer the CLR:RAMP1 and CLR:RAMP2 ECD complexes for crystallization (Moad and Pioszak, 2013), inspired by previous successes using maltose binding protein (MBP) as a “crystallization module” for class B GPCR ECDs (Kumar et al., 2011; Pal et al., 2010; Pioszak et al., 2008, 2009, 2010; Pioszak and Xu, 2008). MBP-RAMP1 or MBP-RAMP2 ECD-CLR ECD fusion proteins in which the two ECDs were covalently tethered with a flexible (Gly-Ser)5 linker were designed to ensure complex stability and enforce 1:1 CLR:RAMP stoichiometry. The tethered RAMP1-CLR ECD fusion was a monomer, whereas the tethered RAMP2-CLR ECD fusion purified as a dimer, but the physiological relevance of oligomerization is unknown. Both proteins selectively bound their respective peptides but failed to yield crystals in the presence of peptides.\nWe reasoned that tether flexibility and oligomerization of the AM1 receptor ECD complex hindered the crystallization efforts. We produced new constructs with a (Gly-Ser-Ala)3 tether designed to decrease flexibility and we identified a single amino acid substitution in the RAMP2 ECD, L106R, which prevented dimerization of the tethered RAMP2-CLR fusion protein (Figure S1A) by disrupting a putative oligomerization interface identified by examining crystal packing in the ligand-free CLR:RAMP2 ECD structure (Kusano et al., 2012). The monomeric RAMP2 L106R-tethered construct retained selectivity for AM over CGRP and bound AM(22-52)NH2 essentially identical to the wild-type tethered fusion in an AlphaScreen competition binding assay (IC50 ∼5–15 μM) (Figures S1B, S1C, and S1H). In a cell-based cAMP signaling assay the full-length AM1 receptor with RAMP2 [L106R] exhibited wild-type response to AM (Figure S1D; Table S4). High-quality crystals of MBP-RAMP2 ECD [L106R]-(GSA)3-CLR ECD grown in the presence of AM(25-52)NH2 were readily obtained (Figure S1E). Crystals of MBP-RAMP1 ECD-(GSA)3-CLR ECD grown in the presence of CGRP(20-37)NH2 diffracted poorly (data not shown); fortunately, high-quality crystals were obtained in the presence of a high-affinity CGRP analog CGRP(27-37)NH2 [D31, P34, F35] (Rist et al., 1998) (Figure S1F). In the competition assay, the CGRP receptor crystallization construct was selective for CGRP over AM and bound the CGRP analog with higher affinity (IC50 ∼0.46 μM) than CGRP(8-37)NH2 (IC50 ∼2 μM) (Figure S1G). The CGRP analog also bound the AM1 receptor crystallization construct with higher affinity than wild-type CGRP but was still lower affinity than AM (Figure S1H). The crystallized proteins thus exhibited peptide selectivity consistent with the intact receptors. The peptides in both crystal forms are antagonist fragments that lack the N-terminal 7TM domain-activating region (Figure S1I). The CGRP analog will hereafter be referred to as CGRPmut."}
2_test
{"project":"2_test","denotations":[{"id":"25982113-24115156-118216112","span":{"begin":206,"end":210},"obj":"24115156"},{"id":"25982113-24115156-118216112","span":{"begin":206,"end":210},"obj":"24115156"},{"id":"25982113-21625560-118216113","span":{"begin":347,"end":351},"obj":"21625560"},{"id":"25982113-21625560-118216113","span":{"begin":347,"end":351},"obj":"21625560"},{"id":"25982113-20966082-118216114","span":{"begin":365,"end":369},"obj":"20966082"},{"id":"25982113-20966082-118216114","span":{"begin":365,"end":369},"obj":"20966082"},{"id":"25982113-18801728-118216115","span":{"begin":387,"end":391},"obj":"18801728"},{"id":"25982113-18801728-118216115","span":{"begin":387,"end":391},"obj":"18801728"},{"id":"25982113-19674967-118216116","span":{"begin":393,"end":397},"obj":"19674967"},{"id":"25982113-19674967-118216116","span":{"begin":393,"end":397},"obj":"19674967"},{"id":"25982113-20172855-118216117","span":{"begin":399,"end":403},"obj":"20172855"},{"id":"25982113-20172855-118216117","span":{"begin":399,"end":403},"obj":"20172855"},{"id":"25982113-18375760-118216118","span":{"begin":421,"end":425},"obj":"18375760"},{"id":"25982113-18375760-118216118","span":{"begin":421,"end":425},"obj":"18375760"},{"id":"25982113-22102369-118216119","span":{"begin":1457,"end":1461},"obj":"22102369"},{"id":"25982113-22102369-118216119","span":{"begin":1457,"end":1461},"obj":"22102369"},{"id":"25982113-9438028-118216120","span":{"begin":2252,"end":2256},"obj":"9438028"},{"id":"25982113-9438028-118216120","span":{"begin":2252,"end":2256},"obj":"9438028"},{"id":"25982113-24115156-71076112","span":{"begin":206,"end":210},"obj":"24115156"},{"id":"25982113-24115156-71076112","span":{"begin":206,"end":210},"obj":"24115156"},{"id":"25982113-21625560-71076113","span":{"begin":421,"end":425},"obj":"21625560"},{"id":"25982113-21625560-71076113","span":{"begin":421,"end":425},"obj":"21625560"},{"id":"25982113-20966082-71076113","span":{"begin":421,"end":425},"obj":"20966082"},{"id":"25982113-20966082-71076113","span":{"begin":421,"end":425},"obj":"20966082"},{"id":"25982113-18801728-71076113","span":{"begin":421,"end":425},"obj":"18801728"},{"id":"25982113-18801728-71076113","span":{"begin":421,"end":425},"obj":"18801728"},{"id":"25982113-19674967-71076113","span":{"begin":421,"end":425},"obj":"19674967"},{"id":"25982113-19674967-71076113","span":{"begin":421,"end":425},"obj":"19674967"},{"id":"25982113-20172855-71076113","span":{"begin":421,"end":425},"obj":"20172855"},{"id":"25982113-20172855-71076113","span":{"begin":421,"end":425},"obj":"20172855"},{"id":"25982113-18375760-71076113","span":{"begin":421,"end":425},"obj":"18375760"},{"id":"25982113-18375760-71076113","span":{"begin":421,"end":425},"obj":"18375760"},{"id":"25982113-22102369-71076114","span":{"begin":1457,"end":1461},"obj":"22102369"},{"id":"25982113-22102369-71076114","span":{"begin":1457,"end":1461},"obj":"22102369"},{"id":"25982113-9438028-71076115","span":{"begin":2252,"end":2256},"obj":"9438028"},{"id":"25982113-9438028-71076115","span":{"begin":2252,"end":2256},"obj":"9438028"}],"text":"Engineering CLR:RAMP ECD Complexes for Crystallization\nWe previously reported a tethered fusion protein approach to engineer the CLR:RAMP1 and CLR:RAMP2 ECD complexes for crystallization (Moad and Pioszak, 2013), inspired by previous successes using maltose binding protein (MBP) as a “crystallization module” for class B GPCR ECDs (Kumar et al., 2011; Pal et al., 2010; Pioszak et al., 2008, 2009, 2010; Pioszak and Xu, 2008). MBP-RAMP1 or MBP-RAMP2 ECD-CLR ECD fusion proteins in which the two ECDs were covalently tethered with a flexible (Gly-Ser)5 linker were designed to ensure complex stability and enforce 1:1 CLR:RAMP stoichiometry. The tethered RAMP1-CLR ECD fusion was a monomer, whereas the tethered RAMP2-CLR ECD fusion purified as a dimer, but the physiological relevance of oligomerization is unknown. Both proteins selectively bound their respective peptides but failed to yield crystals in the presence of peptides.\nWe reasoned that tether flexibility and oligomerization of the AM1 receptor ECD complex hindered the crystallization efforts. We produced new constructs with a (Gly-Ser-Ala)3 tether designed to decrease flexibility and we identified a single amino acid substitution in the RAMP2 ECD, L106R, which prevented dimerization of the tethered RAMP2-CLR fusion protein (Figure S1A) by disrupting a putative oligomerization interface identified by examining crystal packing in the ligand-free CLR:RAMP2 ECD structure (Kusano et al., 2012). The monomeric RAMP2 L106R-tethered construct retained selectivity for AM over CGRP and bound AM(22-52)NH2 essentially identical to the wild-type tethered fusion in an AlphaScreen competition binding assay (IC50 ∼5–15 μM) (Figures S1B, S1C, and S1H). In a cell-based cAMP signaling assay the full-length AM1 receptor with RAMP2 [L106R] exhibited wild-type response to AM (Figure S1D; Table S4). High-quality crystals of MBP-RAMP2 ECD [L106R]-(GSA)3-CLR ECD grown in the presence of AM(25-52)NH2 were readily obtained (Figure S1E). Crystals of MBP-RAMP1 ECD-(GSA)3-CLR ECD grown in the presence of CGRP(20-37)NH2 diffracted poorly (data not shown); fortunately, high-quality crystals were obtained in the presence of a high-affinity CGRP analog CGRP(27-37)NH2 [D31, P34, F35] (Rist et al., 1998) (Figure S1F). In the competition assay, the CGRP receptor crystallization construct was selective for CGRP over AM and bound the CGRP analog with higher affinity (IC50 ∼0.46 μM) than CGRP(8-37)NH2 (IC50 ∼2 μM) (Figure S1G). The CGRP analog also bound the AM1 receptor crystallization construct with higher affinity than wild-type CGRP but was still lower affinity than AM (Figure S1H). The crystallized proteins thus exhibited peptide selectivity consistent with the intact receptors. The peptides in both crystal forms are antagonist fragments that lack the N-terminal 7TM domain-activating region (Figure S1I). The CGRP analog will hereafter be referred to as CGRPmut."}