If some of the experimentally determined
If some of the experimentally determined tRNAGlu identity elements are unique to E. coli, then there also must be some unique phylum-specific residues in the corresponding GluRS (E. coli in particular, and proteobacteria in general) as well. In this paper we focus on identifying such residues in E. coli GluRS. Our strategy is to utilize whole EG00229 mg bacterial sequences from a large number of bacteria and compare the GluRS sequence vis-à-vis tRNAGlu sequences. Following this strategy, Arg266 in E. coli GluRS was identified to be unique in proteobacterial GluRS, changing mostly to Leu in non-proteobacteria. Arg266 is conserved in bacteria that possess both the augmented D-helix and the acceptor arm identity elements (Fig. 1a). An Arg266Leu mutant of E. coli GluRS was constructed to explore structural and functional role of Arg266. Replacement of Arg266 by Leu drastically altered the catalytic efficiency of GluRS in addition to slightly altering the secondary structure and the stability of the protein. The implications of our results are discussed with a focus on phylum-specific tRNAGlu–GluRS interaction in bacteria.
Materials and methods
Results and discussion
Conclusion We have identified a conserved catalytic domain residue in proteobacterial GluRS, present at the tRNAGlu–GluRS interface. The conserved residue (Arg266 in E. coli GluRS) is a proteobacterial signature and is correlated with the presence of a select set of E. coli tRNAGlu identity elements (augmented D-helix and U2·A71 in the acceptor stem). In non-proteobacteria the residue position is mostly occupied by Leu (but never by Arg). An R266L mutant of E. coli GluRS exhibited impaired glutamylation efficiency, slightly reduced helical secondary structure and enhanced stability, establishing that unlike non-proteobacterial GluRS, Leu is structurally and functionally (possibly due to local structural changes) incompatible with E. coli GluRS. Sequence analysis of a large database of bacterial GluRS sequences showed that Arg266 is always accompanied by a unique loop sequence that follows it, when compared to the loop that follows Leu266. Since D-helix containing tRNAGlu is not only present in bacteria that possess GluRS with Arg266, but also in bacteria that possess GluRS with Leu266, we propose that the true role of Arg266 is not to optimize interactions of GluRS with an augmented D-helix of tRNAGlu. Some other unique proteobacteria-specific evolutionary pressure led to the appearance of Arg266 in proteobacterial GluRS.
Introduction Glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type mediate fast neurotransmission in the central nervous system and are assembled from four subunits: GluR-A-D (GluR1-4, see Seeburg et al., 2001, for review). In contrast to the GluR-A, -C, and -D subunits, GluR-B contains an arginine at a critical position in the pore-forming M2 segment, which is not genetically encoded. Instead this arginine codon is introduced by RNA editing in the GluR-B transcript. Incorporation of GluR-B into heteromeric AMPA receptor strongly reduces the permeability of the receptor channel to Ca2+ ions and modifies current rectification and macroscopic channel conductance (Burnashev et al., 1992). Since GluR-B is widely expressed in the central nervous system (CNS; Monyer et al. 1991, Petralia et al. 1997), an overwhelming majority of AMPA receptors in the CNS has low permeability for Ca2+. Only a subset of neurons, predominantly GABAergic interneurons, and glia cells express AMPA receptors with high Ca2+ permeability Monyer et al. 1991, Geiger et al. 1995. In recent years, AMPA receptors have emerged as key elements of long-lasting changes in the efficacy of central synapses, which are believed to constitute cellular correlates of complex behaviors such as learning and memory Malinow and Malenka 2002, Reisel et al. 2002, Zamanillo et al. 1999, Lee et al. 2003, addiction, and reward Thomas et al. 2001, Sutton et al. 2003. In particular, synaptic activity regulates AMPA receptor function via modulation of ion channel properties of the receptor and its targeting to the postsynaptic membrane. GluR-A and GluR-B serve as key substrates for activity-induced regulation of synaptic transmission via rapid and selective modifications in their phosphorylation status and binding to scaffolding proteins involved in membrane trafficking, which, in turn, determine their synaptic availability and function Shi et al. 2001, Liu and Cull-Candy 2000, Malinow and Malenka 2002, Lee et al. 2003. Thus, the Ca2+ permeability and ion conductance properties of synaptic AMPA receptors are not static features of a particular neuron but are modified dynamically by synaptic activity via rapid alterations in GluR-A and GluR-B subunits.