2366 Nucleic Acids Research, 2009, Vol. 37, No. 7 Figure 3. GD1 plays a role in EngA binding the 30S subunit. (A) The D337N mutant and constructs GD1–EngA, GD1– GD2EngA were used in ribosome co-fractionation experiments as in Figure 1D. An EngA[GDP:GTP] state was achieved using D337N mutant, with GDP and XMPPNP. It associates with 50S, 30S and 70S subunits (A1) at 50 mM NH4Cl. In this mutant, when GD1 was not supplied with GDP, resulting in EngA[Apo:GTP] state, it showed no interaction (A2) with 30S. Stable interactions with 30S can be restored (A3) even at 150 mM NH4Cl, if GDP is supplied to GD2, i.e. EngA[GDP:GTP] state. (B) GD1–EngA interacts with 30S, 50S and 70S in presence of GMPPNP at 150 mM NH4Cl (B1), while GD1– GD2EngA binds only the 30S (B2). (C) In the in vitro ribosome interaction assays, proteins were incubated with crude ribosome (ﬁve A254) and appropriate nucleotides as described in Materials and methods section. The reaction mixtures were resolved on a manually prepared 17 ml step gradient of sucrose and 30 fractions were collected from top to bottom while monitoring A254. RNA was isolated from each of the fractions and analysed by formaldehyde–agarose gel for the presence of 16S and 23S rRNA. Based on this, top fraction devoid of ribosomes, peak fractions corresponding to 30S, 50S and 70S subunits were analysed on a SDS–PAGE. Immuno-blots, using anti-GST antibodies, for the ‘top’, 30S, 50S and 70S fractions are shown. The GD1– GD2EngA associates with the 30S (C1) and GD1–EngA with 30S, 50S and 70S (C2). Urea treated D337N mutant incubated with GMPPNP, XMPPNP, restored 50S interactions (C3) and also the interactions with 30S, 50S and 70S when incubated with GDP and XMPPNP (C4). Fluorescent nucleotide binding assays were conducted (as in Figure 2) with the D337N mutant in presence of various nucleotides and urea (1.5 M), as indicated. Emission spectra show that urea does not alter nucleotide binding to the protein. protein (D337N mutant) remains unaltered even at 1.5 M urea (Figure 3C). Role of GD1–KH interactions in ribosome association In vitro experiments with urea (Figure 3C) made us exam- ine interactions at the domain interfaces of EngA, which may be disrupted by urea to achieve an appropriate con- formational state of EngA that binds the ribosome. In the crystal structure of YphC (EngA homologue in B. subtilis), GD1, bound to GDP, provides hydrophobic interactions for the GD1–KH interface (Figure 4A). GTP binding to GD1 disrupts these interactions as evident from the large conformational change ( 60 A˚ ) in the position of GD1 (Figure 4A). This hydrophobic nature of GD1–KH interface is conserved in several homologs of EngA. We, therefore, reasoned that in the aforementioned in vitro experiments (Figure 3C), urea may disrupt these interac- tions at GD1–KH interface and allow ribosome binding. To examine this, Tyr134, a key residue contributed by GD1 to this interface (Figure 4A), was mutated (see Figure S2, Supplementary Material). A His-YphC double mutant (Y134A, D297N) and the wt-His-YphC were employed in the in vitro interactions. In YphC, D297 is identical to D337 of E. coli EngA and hence its mutation to N would alter the speciﬁcity of GD2 from guanine to xanthine. When wt-HisYphC was incubated with puriﬁed ribosomal subunits, it did not display any interactions in EngA[GTP:GTP] state as noted for GST–EngA in the in vitro experiments. As anticipated, the YphC double