mant-XDP binding signiﬁcantly, as seen in Figure 2B and C. This veriﬁes that the single mutants D134N and D337N allow for isolating nucleotide binding events at GD1 and GD2 and provide the ability to control the nucleotide states of both G-domains simultaneously, whereas wt-EngA preferentially binds mant-GDP (see Figure S1, Supplementary Material). In these, the domain carrying the mutation would bind xanthine nucleotides (when supplied) and allow guanine nucleotides to selectively bind the other. When D134N mutant was tested for 50S binding (Figure 2D, D4) in a co-fractionation experiment carried out at 50 mM NH4Cl and in presence of GMPPNP but not XMPPNP (i.e. EngA[Apo:GTP] state), it shows inter- actions with 50S, like the K28A mutant in Figure 2D (D2). As anticipated, upon increasing NH4Cl concentra- tion to 150 mM, the interaction (Figure 2D, D5) was com- pletely abolished like in Figure 2D (D3). Interestingly, the 50S interaction can be restored, as in the wt-protein, if XMPPNP is also provided to achieve EngA[GTP:GTP] state (Figure 2D, D6). This restoration was also veriﬁed using D337N mutant in lieu of D134N in a similar experi- ment. Here too, 50S binding is restored (Figure 2D, D7) as above. These experiments lend support to the observa- tions made from K28A, K228A mutants (Figure 2D, D1–D3), that GD2:GTP initiates EngA–50S interactions and GD1:GTP further stabilizes it. The role of GD1 in binding specific ribosomal subunits The above experiments show that both GD1:GTP and GD2:GTP are needed for eﬃcient 50S binding. Since the D134N and D337N mutants additionally facilitate varying nucleotides occupied by the two G-domains, we questioned if doing so would alter ribosome binding. The EngA[GDP:GTP] analogous state was achieved using D337N mutant, in co-fractionation experiments contain- ing GDP and XMPPNP. At 50 mM NH4Cl, this state, surprisingly, shows interactions not only with the 50S, but also with 30S and 70S subunits (Figure 3A, A1). To determine if the 30S and 70S interactions are speciﬁc to GD1:GDP, EngA[Apo:GTP] state was generated with the D337N mutant. Here, when GD1 is devoid of GDP, no interaction is observed with 30S and 70S (Figure 3A, A2), suggesting the importance of EngA[GDP:GTP] state for these interactions. These interactions could be restored upon supplying GDP at 150 mM NH4Cl too (Figure 3A, A3), indicating stable binding. So far it appears that EngA, by employing the GD1:GDP state (Figure 3A) generates additional interac- tions for binding 30S. To further understand the role of GD1 in regulating EngA–ribosome interactions, we generated a construct GD1–EngA, in which GD1 was truncated. When GD1–EngA was employed in co-fractionation experiments, the protein bound 30S, 50S and 70S, even at 150 mM NH4Cl (Figure 3B, B1), like EngA[GDP:GTP)]. Interestingly, another construct, GD1– GD2EngA, where both GD1 and GD2 were truncated (i.e. only the KH-domain was present), largely bound the 30S (Figure 3B, B2). Possibly, this experiment suggests that KH-domain possesses sites for 30S Nucleic Acids Research, 2009, Vol. 37, No. 7 2365 interaction, and taken together with Figure 3A (A–A3), it appears that the 30S site is accessible only in the GD1:GDP state. For ease of presentation, we deﬁne the EngA–ribosome interactions (discussed thus far) examined from the cells overexpressing EngA, as in vivo experiments. On the other hand, interactions described from now on, carried out using puriﬁed EngA and ribosomal subunits, would be referred as in vitro experiments. Unfastening inter-domain interactions facilitates EngA–ribosome interactions The in vitro EngA–ribosome interaction assays using pur- iﬁed EngA and ribosomal subunits, were carried out in presence of appropriate nucleotides. Surprisingly, the full length EngA constructs i.e. wt or the D to N mutants, failed to show any interaction in the EngA[GTP:GTP] or EngA[GDP:GTP] states. This led us to suspect the pres- ence of an unknown factor in the cell lysate enabling these interactions in the in-vivo studies. To examine this, the whole cell lysate devoid of ribosomes, was added to the reaction mixtures. However, this too did not result in any EngA–ribosome association (data not shown). Surprisingly, GD1– GD2EngA, where both GD1 and GD2 were truncated (i.e. only the KH-domain was present), or a construct GD1–EngA, where GD1 was truncated, when incubated with the puriﬁed ribosomal subunits, interacted with speciﬁc ribosomal subunits, as in the in vivo assays (Figure 3B). In these studies, GD1– GD2EngA associated with the 30S and GD1–EngA with 30S, 50S and 70S (Figure 3C, C1–C2). These experiments suggest that EngA needs to be devoid of GD1 to restore interactions with ribosomal subunits in the in vitro experiments. We reasoned that, in the full length protein, the inter-domain interactions pro- vided by GD1 may lock the enzyme in a conformational state inappropriate for binding the ribosome. Often, mild concentrations of urea, guanidium hydrochloride or SDS loosen inter-domain interactions, which may be facili- tated, in vivo, by a factor present in the cell lysate. To examine this, the full length D337N mutant was incubated on ice for 20 min with increasing concentrations of urea (from 500 mM to 2 M). From this, 100 pmol of protein was added to the crude ribosome in presence of appropriate nucleotides. The ﬁnal concentration of urea in reaction mixtures, following this dilution, was 15–45 mM. Surprisingly, incubating D337N mutant in 1.5–2 M of urea, restored interactions with 50S in the EngA[GTP:GTP] state (Figure 3C, C3) and with 30S, 50S and 70S in the EngA[GDP:GTP] state (Figure 3C, C4). However, it appears from Figure 3C that only a small fraction of the protein molecules associate with the ribosomal subunits, as most of the protein remains in the top fractions. Perhaps this could be associated to the mild urea concentrations ( 15–45 mM) employed here. To clarify if urea interferes with nucleotide binding, ﬂuores- cent nucleotide binding assays were conducted in the pres- ence and absence of 1.5 M urea. Emission spectra show that guanine and xanthine nucleotide binding to the