carried out with cells overexpressing the GST vector alone (as a negative control) and the cells overexpressing GST– EngA. The cells were lysed in the presence of appropriate nucleotides, whenever indicated. The immuno-blots in Figure 1D, using an anti-GST antibody, identify the pro- tein present in the various fractions obtained following a sucrose density gradient centrifugation (see Materials and methods section). The negative control, i.e. cells expres- sing GST alone, shows no interaction with the ribosomal subunits (Figure 1D, D1) and thus negates the possibility of the GST-tag generating interactions with the ribosome. In addition, the pGEX vector employed here was modiﬁed to have a TEV protease cleavage site following the GST and thrombin cleavage site, resulting in a large linker region ( 15 residues) between GST and the protein. This allows suﬃcient domain movements between the two. Therefore, GST–EngA was employed for all further experiments. EngA, from here on, implies GST–EngA. The experiment with cells overexpressing EngA reveals that it co-fractionates with the 50S subunit only in pres- ence of non-hydrolysable GTP analogue, GMPPNP (Figure 1D, D4), but not GDP (Figure 1D, D3) or in absence of nucleotides (Figure 1D, D2). In concurrence with existing reports (13,15) EngA seems to be a GTP- dependent 50S binding protein. As the strength of riboso- mal interactions can be assessed by increasing the amount of salt used in the co-fractionation experiments (9,25), we employed the same to assess EngA–50S interaction in presence of GMPPNP. Figure 1D (D5 and D6) show that the interactions are stable even at 150 and 350 mM NH4Cl. While 50S binding accounts for 23S rRNA co-elution (Figure 1A), 16S rRNA co-elution remains unexplained. Hence, further experiments were designed to explore conditions that promote EngA–30S interactions. GTP binding to GD2 is a primary requirement for ribosome binding The two G-domains of EngA have contrasting nucleotide binding aﬃnities and GTP hydrolysis rates. Robinson et al. (16) suggest that GD2 binds nucleotides with higher aﬃnity and has slow GTP hydrolysis rates in com- parison to GD1, which has lower nucleotide binding aﬃn- ity and faster GTP hydrolysis rates. Hence, it could be that the two G-domains of EngA render distinct aﬀects towards ribosome binding. To investigate the same, ribo- some binding by EngA was tested while varying the nucleotide occupied states of the two G-domains. From hereon, for enhanced clarity, the GTP or GDP-bound states of GD1 and GD2 are denoted as GD1:GTP, GD1:GDP and GD2:GTP, GD2:GDP, respectively. Similarly, the nucleotide state of EngA, which is a conse- quence of the nucleotide-bound states of its two G-domains, is denoted as follows: for example, EngA [Apo:GTP] indicates that GD1 is in a nucleotide-free state and GD2 is in a GTP-bound state, while EngA[GDP:GTP] implies GD1 is in a GDP-bound state and GD2 is in a GTP-bound state, and so on. In order to assess the signiﬁcance of the two G-domains towards 50S binding, we generated single mutants K28A Nucleic Acids Research, 2009, Vol. 37, No. 7 2363 and K228A. These residues are present in the P-loops of GD1 and GD2, respectively. Typically, the lysine residues in the P-loops (GxxxxGKS/T) of GTPases coordinate the phosphates of GTP and GDP and their mutation is known to disrupt nucleotide binding (26,27). In the crystal structure of YphC (EngA homologue in B. subtilis) too, these lysines show similar interactions with the nucleotide (17). Hence, GD1 or GD2 can be arrested in a nucleotide- free state, by single mutants K28A or K228A, respectively, while simultaneously controlling the nucleotide-bound state of the other G-domain (without a mutation) by supplying appropriate nucleotides during cell lysis. However, to conﬁrm the nucleotide-free states of GD1 and GD2 in K28A and K228A mutants, respectively, ﬂu- orescence nucleotide binding experiments were carried out using mant-nucleotides. The mant-nucleotides are sensi- tive ﬂuorescent probes which show an increase in ﬂuores- cence when present in a hydrophobic environment (28) i.e. when the mant-nucleotide binds to the protein in our experiments. Figure 2A shows a decrease in mant-GDP binding by mutants K28A and K228A in comparison to wt-EngA that has two intact G-domains to bind the nucleotide. Interestingly, mutant K28A, that disrupts nucleotide binding at GD1, shows a higher binding in comparison with K228A, in which binding is disrupted at GD2. This is in agreement with the reported higher nucleotide binding aﬃnity of GD2 (16). These experi- ments conﬁrm the nucleotide-free states generated by K28A and K228A. Co-fractionation experiments were carried out with cells overexpressing mutants K28A or K228A, and lysed in presence of GMPPNP to ensure either the EngA[Apo:GTP] or EngA[GTP:Apo] state. Compared with the ﬂuorescent nucleotide binding experiments where the protein is in excess (protein:nucleotide ratio 20:1), in all the co-fractionation experiments, the nucleo- tides ( 1 mM) were provided in excess (over the protein mM) to ensure that a majority of protein molecules attain the desired nucleotide-bound states. Figure 2D (D1), corresponding to K228A mutant, representing EngA[GTP:Apo] state, in presence of 50 mM NH4Cl, shows no interactions with the ribosome. This suggests an importance of GD2:GTP in ribosome binding. In con- trast, the K28A mutant (Figure 2D, D2) representing the converse state, i.e. EngA[Apo:GTP], shows an interaction with 50S at 50 mM NH4Cl. It is surprising that while the wt-protein retains 50S interaction even at 350 mM salt (Figure 1D, D6), K28A mutant representing EngA [Apo:GTP] state, shows no interaction at 150 mM NH4Cl (Figure 2D, D3), suggesting a weak interaction. Hence, these experiments suggest that it is GTP binding to GD2, but not to GD1, which is a primary requirement to observe any 50S interaction. GTP binding to GD1 stabilizes 50S binding The above data suggests that EngA–50S interaction requires a GD2:GTP state. However, the strong interac- tion seen for the wt-protein (Figure 1D, D5 and D6) sug- gests that the weak interactions found in EngA[Apo:GTP] state (Figure 2D, D2) may be further stabilized in the