NMR studies of the GTP/GDP binding domain of translation initiation factor IF2

Abstract

Translation Initiation Factor 2 (IF2) plays an important role in the initiation stage of bacterial protein biosynthesis. This protein binds both fMet-tRNA and 30S ribosomal subunit in the presence of GTP, and it stimulates the formation of the 70S initiation complex. The NMR samples of the 15N-, 15N,13C- and 2H,15N,13C-labelled GTP/GDP-binding domain of IF2 (IF2G2) from Bacillus stearothermophilus were prepared. The structures of IF2G2 have been determined using NMR spectroscopy for the free and GDP-bound states. The IF2G2 structure reveals a typical GTPase fold, strikingly similar to that of the small GTPase Ras and of the G-domain of EF-G. Both IF2G2 and IF2G2-GDP structures contain a well defined core (backbone RMSD to the average structure 0.5 A and 0.4 A for free and GDP-bound forms, respectively) and unstructured switch 1 and 2 regions, which likely undergo conformational exchange and possible become more structured in the intact IF2. The following nucleotide-binding elements: P-loop, G4- and G5-boxes are situated in the well-ordered regions of the free IF2G2 and are in a conformation close to that of the GDP-bound form of IF2G2 implying that nucleotide ring- and diphosphate-binding parts of the IF2 are in a proper conformation for GDP binding. Classic GTPase-nucleotide interactions between the GDP and IF2G2 are detected in the IF2G2-GDP complex: (i) a protein P-loop which interacts with the phosphate group of GDP and (ii) the IF2G2 G4- and G5-boxes with the guanosine base.

Compared to the solution structure of the free IF2G2, the structure of the IF2G2-GDP shows a significant difference (backbone RMSD between the core regions ~A). This difference can also be observed by a dramatic change in 15N-HSQC spectra of the IF2G2 upon GDP binding. This large conformational change, much larger than noted previously for other GTPase domains, is likely to be transduced to the rest of IF2 and therefore might be important in IF2 function during the 70S complex formation.

NMR titration experiments with slowly hydrolysable GTP analog GDPNP showed that upon binding the GTP mimic there is increased mobility in IF2G2 caused by the interaction. This different IF2G2 response on various nucleotides is particularity interesting since it was proposed recently that the GTP/GDP binding site of IF2 is a sensor for another nucleotide, the starvation alarmone ppGpp (Guanosine 3',5'-(bis) diphosphate or guanosine tetraphosphate) rather than a genuine GTPase center. In this light it is interesting that the 15N-HSQC spectra of the IF2G2-GDP and IF2G2-ppGpp complexes revealed minor differences which might be indicative of possible structural changes that might be of functional importance and could be involved in regulation of biosynthesis according to different cellular metabolic states.