B and Supplementary Fig. 2b). Electron density was clearly interpretable for
B and Supplementary Fig. 2b). Electron density was clearly interpretable for the SSM and `RBD’5 but not for amino acids 39702 that constitute the linker (39306) involving SSM and `RBD’5 (Fig. 1a,b and Supplementary Fig. 1a). Two conformations have been observed at the Cterminal or `RBD’5 side of the linker, every hinged at L405 in order that the position of P404 wasNat Struct Mol Biol. Author manuscript; accessible in PMC 2014 July 14.Gleghorn et al.Pagevariable (Supplementary Fig. 2c). The observed variability raises the possibility that SSM might interact with `RBD’5 as a monomer (cis), dimer (trans), or both in the crystal structure (Fig. 1b), but we can’t correlate either linker conformation using a monomeric or dimeric state. Every 649 interface is created when the `V’-shape formed by SSM 1 and 2 straddles `RBD’5 1, when the `V’-shape designed by `RBD’5 1 and 2 straddles SSM 1 (Fig. 1b ). The intramolecular interactions of an SSM and an `RBD’5 form a core composed of residues with hydrophobic side chains (Fig. 1c). The external solvent boundary of this core is defined by Thr371 of your longer from the two SSM -helices, 1; Ser384 of SSM 2; Gln411, Tyr414, and Gln419 of `RBD’5 1; and Lys470 of `RBD’5 two (Fig. 1c). Every of those residues amphipathically contributes hydrophobic portions of their side chains to the core, with their polar BMP-2, Human/Mouse/Rat (His) component pointed outward. Val370, Ile374, Ala375, Leu378 and Leu379 of SSM 1 also contribute to the hydrophobic core as do Ala387, Ile390 and Leu391 of SSM 2; `RBD’5 1 constituents Pro408 (which begins 1), Leu412, Leu415 and Val418; and Phe421 of L1 (Fig. 1c). Also, `RBD’5 two contributes Leu466, Leu469, Leu472 and Leu475 (Fig. 1c). In the two polar interactions in the SSM RBD’5 interface, a single a basic charge is contributed by SSM Arg376: its two -amine groups hydrogen-bond with two carboxyl groups of the citrate anion present in the crystal structure, though its – and -amines interact using the main-chain oxygens of, respectively, Glu474 and Ser473 which might be positioned close to the C-terminus of `RBD’5 2 (Fig. 1d). SSM Arg376 is conserved in these vertebrates analyzed except for D. rerio, where the residue is Asn, and Glu474 and Ser473 are invariant in vertebrates that contain the `RBD’5 two C-terminus (Supplementary Fig. 1a). In the other polar interaction, the side-chain hydroxyl group of SSM Thr371 and also the main-chain oxygen of Lys367 hydrogen-bond with the amine group of `RBD’5 Gln419, though the -amine of Lys367 hydrogen-bonds with the hydroxyl group of Gln419 (Fig. 1c). SSM residues lacking strict conservation, i.e., Met373, Tyr380, Gly381, Thr383 and Pro385, are positioned around the solvent-exposed side, opposite towards the interface that interacts with `RBD’5 (Supplementary Fig. 2d). Comparison of `RBD’5 to an RBD that binds dsRNA We were surprised that the 3 RBD structures identified by the Dali server28 to be structurally most equivalent to `RBD’5 do bind dsRNA (Supplementary Table 1). Of the 3, Aquifex aeolicus RNase III RBD29 delivers essentially the most comprehensive comparison. A structurebased sequence alignment of this RBD with hSTAU1 `RBD’5 revealed that while the two structures are nearly identical, hSTAU1 `RBD’5 features a slightly shorter loop (L)1, an FSH Protein Purity & Documentation altered L2, and a longer L3 (Fig. 2a,b). Moreover, hSTAU1 `RBD’5 lacks key residues that typify the three RNA-binding regions (Regions 1, 2 and 3) of canonical RBDs23 and which might be present inside the A. aeolicus RNase III RBD (Fig. 2b). Probably the most apparent differences reside in Area 2.
