Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K-D value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K-D = 160(+/-40) muM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme. (C) 2004 Elsevier Ltd. All rights reserved.