Structure-function relationships in Escherichia coli translational elongation factor G: modification of lysine residues by the site-specific reagent pyridoxal phosphate

Abstract

Translational elongation factor G (EF-G) of Escherichia coli was modified with the selective, site-specific lysine reagent pyridoxal phosphate (PLP). The reaction results in the modification of a maximum of 12 lysine residues, one of which is essential for guanosine 5'-triphosphate (GTP) binding and whose modification is inhibited by the presence of GTP. Formation of a reversible adduct between 2,3-butanedione and an essential arginine similarly located in the GTP binding site [Rohrbach, M.S., & Bodley, J. W. (1977) Biochemistry 16, 1360-1363] also protects EF-G from PLP inactivation, suggesting that these two residues are spatially close to each other in the native factor. The essential lysine residue was found in the trypsin-resistant fragment T4 (Mr 41 000). In addition to the lysine essential for GTP binding, at least one further lysine was found to be important for EF-G function, since GTP-protected, PLP-modified EF-G molecules fully competent in binding to 50S ribosomal subunits showed decreased activity in 50S- and 70S-dependent GTP hydrolysis. It is likely that a PLP-modified lysine impairs the interaction of the factor with 30S ribosomal subunits and/or a conformational change of the factor required for the hydrolysis of GTP.

Crystal structure of a mutant elongation factor G trapped with a GTP analogue

“…In contrast, in all structures of wild-type EF-G and eEF2 the interaction between the P-loop lysine and the threonine or serine in switch II is conserved. According to chemical modification studies [36] one lysine is involved in nucleotide binding in EF-G. GTP affinity is abolished when this lysine is modified by pyridoxal phosphate, but this effect can be eliminated by protecting the lysine through pre-binding of GTP. The K25A mutation in T. thermophilus EF-G decreases the GTPase activity (ATG et al, in preparation) and a similar mutation in EF-Tu (K24E) abolishes the GTP affinity [37].…”

Structural Insights into Fusidic Acid Resistance and Sensitivity in EF-G

“…It is known that at least one lysine residue is required for GTP binding in EF-G from E. coli. 37 Of the 39 lysine residues present in EF-G from T. thermophilus, Lys25 is almost completely conserved in all known GTPases, ATPases and even in some kinases. 38,39 It is quite excluded from the solvent in the wild-type EF-G structure and is in the vicinity of the nucleotide-binding site.…”

Protein Thioacylation. 1. Reagents Design and Synthesis

“…Improved procedures allow us to process at room temperature without opening disulfide cross-links. , Carbonyl reagents may be acetone, cyclohexanone or benzaldehyde. , Methanal (formaline) react twice with each lysine residue . Other reagents involved in alkylation reactions are prop-2-enal 18 and α-lactose (α-disaccharide) 19 as well as monosaccharides 20 and 5-pyridoxal phosphate. −

2 Protein reductive alkylation.

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“…17 Other reagents involved in alkylation reactions are prop-2-enal 18 and R-lactose (R-disaccharide) 19 as well as monosaccharides 20 and 5-pyridoxal phosphate. [21][22][23][24] Reversible alkylation is observed with an R-hydroxyaldehyde or ketone. 25 As previously indicated TNBS is used to phenylate amines in lysine residues, [26][27][28][29] thus grafting a large hydrophobic moiety (Figure 3).…”