The Structure of the EF‐Tu · GDP · Me²⁺ Complex

Abstract

The structure of the MgGDP complex at the active site of elongation factor (EF-Tu) has been investigated by using phosphorothioate analogs of G D P in the absence and presence of various metal ions, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) measurements. The high stereoselectivity of EF-Tu for the diastereomers of guanosine 5'-0-( 1-thiodiphosphate) (GDP[xS]) is independent of the nature of the metal ion and is caused by the interaction of the protein with the a-phosphate of GDP. By using G D P analogs where the oxygens at either thea-phosphate or thep-phosphate have been selectively labelled with 170 and measuring their effect on the EPR spectrum of EF-Tu-bound manganese we are able to show that only the /?-phosphate of G D P is coordinated to the metal ion in the EF-Tu . Me2+ . GDPcomplex. 31P-NMR studies on G D P and guanosine 5'-0-(2-thiodiphosphate) (GDP[/?S]) bound to EF-Tu indicate that in the EF-Tu Me2+ G D P complex Mgz+ interacts more strongly with the /?-phosphate than with the a-phosphate. Together with binding studies using GDP[/?S] our N M R results also indicate that the protein is complexed to the b-phosphorous of G D P via two oxygens.Pure diastereomeric thiophosphate analogs of ADP and ATP have been prepared [I] and it has been found that enzymes which interact with these analogs usually have a pronounced specificity for one diastereomer [2,3]. Recently Goody and Leberman [4] have prepared GDP [BS] and the diastereoisomers of GDP [aS] and studied their interaction with polypeptide elongation factor (EF-Tu) from Eschevichia coli. From their results they concluded that a a,p-bidentate MgGDP complex is the substrate recognized by EF-Tu and that the interaction of G D P with the enzyme at the /?-phosphate might involve two non-bridging oxygens. Here we describe a study of the affinity of these thiophosphate analogs to EF-Tu from Bacillus stearothermophilus which, because of its inherent stability in the nucleotide and metalfree form ), is better suited than EF-Tu from E. cofi for these measurements. The results obtained in the absence and presence of various metal ions render the first conclusion of Goody and Leberman unlikely, but support the second one. We conclude from these studies that Mg . G D P is bound as its P-monodentate complex.By making use of the spectrum of manganese ion bound to EF-Tu in the presence of 170-enriched analogs of GDP, we confirm that our model is correct and that in the active site of EF-Tu there is an interaction of the G D P with Mn2+ only via the /?-phosphate group. Further confirmation of our model is provided by 3'P-NMR studies on complexes between EF-Tu, nucleotides, and magnesium ions. MATERIALS AND METHODSUnless otherwise stated, all materials were commercially available. The G D P thiophosphate analogs GDP[xS] isomers A and B and GDP[jS] were prepared as described [4].

Stereochemistry of the elongation factor Tu . GTP complex

“…In the EFTu . GDP[aS] complex, the S, isomer, whose configuration is identical with that of GTP[aS] S,, is preferred by a factor of 25-30 over the R, isomer; for the EF-Tu.GDP complex it has been confirmed by EPR measurements that the a-phosphate is not coordinated to the metal ion [9,28]. Thus we propose that the active-site geometry around the a-phosphorous is very similar in the EF-Tu .…”

“…Replacing Mg(I1) by Mn(I1) does not produce any change in affinities for the a-phosphate isomers, but abolishes the specifity for the /l diastereomers. Cd(II), coordinating preferentially to sulfur, decreases the S,/R, ratio of the GTP[aS] nucleotides, just as it decreased the S,/R, ratio of the GDP[crS] isomers [9]. The effect of Cd(I1) on the GTP[PS] isomers is pronounced, causing a reversal of specifity.…”

“…The S,/R, ratio, i.e. the reciprocal of the ratio of concentrations of the respective isomers which cause 50 inhibition [I], is 19.2, which is very close to the S,/R, ratio for the binding of GDP [crS] isomers to EF-Tu [9]. For the GTP[PS] isomers ( Fig.…”

“…We have proposed earlier [9] that in EF-Tu . G D P all oxygen atoms of the [Gphosphate are involved in binding to either the protein or the metal ion.…”

“…Using thiophosphatc analogs of GTP, we have studied the active-site structure of EF-Tu. GTP and compared this with rcsults obtained carlier using the corresponding analogs of G D P [9] to obtain insight into the stereochemical course of the GTPase reaction and to -~/,hi.c,f.rtrtrorz,s. EF-Tu, if not indicated otherwise, is the protein synthcsis elongation factor Tu from Brrci1lu.s sterurother/nophilus; EF-T is the complex between EF-Tu and protein synthesis elongation factor Ts (EF-Ts); EF-Tu, is the metal-free and nucletide-free EF-Tu.…”