2364 Nucleic Acids Research, 2009, Vol. 37, No. 7 Figure 2. The importance of GD1 and GD2 in binding 50S. Nucleotide binding was assayed by recording emission spectra ( ex 355 nm) (A–C) upon incubating 8 mM of protein with 0.4 mM ﬂuorescent mant-nucleotides (mant-GDP and/or mant-XDP). Spectra corresponding to various nucleotide- bound forms of the protein are indicated in diﬀerent colors. The protein–nucleotide combinations used are indicated in the insets in corresponding colors and numbers. (A) GDP binding to K28A and K228A mutants is reduced, when compared with the binding to wt-EngA, due to impaired nucleotide binding in the mutated G-domains. (B and C) Proteins carrying D134N and D337N mutations, preferentially bind mant-XDP even in presence of a 100-fold excess of GDP (over mant-XDP). The mutant proteins also bind mant-GDP since the mutation to preferentially bind xanthine nucleotides is created only in one of the G-domains, allowing the other to bind mant-GDP. (D) These mutants were subjected to ribosome co-fractionation experiments as in Figure 1D, with varying salt concentrations and nucleotides, as indicated. (D1) K228A mutant in presence of GMPPNP, which results in EngA[GTP:Apo] state, shows no interaction with ribosomal subunits at 50 mM NH4Cl. Whereas EngA[Apo:GTP] state, generated using K28A, shows an association with 50S (D2) at 50 mM NH4Cl which is abolished (D3) at 150 mM NH4Cl. EngA[Apo:GTP] state was also generated using the mutant D134N. An association with 50S seen (D4) at 50 mM NH4Cl, is abolished (D5) at 150 mM NH4Cl. EngA– 50S interaction can be restored (D6) if XMPPNP, too, is provided to the D134N mutant to achieve EngA [GTP:GTP] state. (D7) This eﬀect is veriﬁed using D337N mutant, which under similar conditions binds the 50S. EngA[GTP:GTP] state, i.e. when GD1 also binds GTP. To substantiate this, we designed the following mutants that allow simultaneous variation of the nucleotide-bound states of GD1 and GD2, as the lysine mutants K28A or K228A only lead to a nucleotide-free (Apo) state. Five conserved signature motifs (G1–G5) in the G-domain participate in stabilizing the bound nucleotide and its hydrolysis. Of these, a conserved Asp in G4 motifs (N/TKxD) renders speciﬁcity to bind guanine nucleotides by making bifurcated hydrogen bonds to the guanine base of GTP/GDP. In the structure of YphC too, the corre- sponding residues D134, D337 in G4 motifs make similar interactions with the guanine ring (17). Hence, D134N and D337N mutants of GD1 and GD2, respectively, were generated, as they would change speciﬁcity from guanine to xanthine nucleotides [(18) and references therein]. Like with the lysine mutants, D134N and D337N mutants were employed in ﬂuorescent nucleotide binding experiments using mant-XDP and mant-GDP to verify nucleotide binding and change in speciﬁcity towards xanthine nucleo- tides. Figure 2B indicates D134N to bind mant-XDP at GD1, where the mutation was created and mant-GDP at GD2, which is not mutated. Similarly, Figure 2C indicates binding of mant-GDP at GD1 and mant-XDP at GD2, in D337N mutant. The selective binding to xanthine nucleotides by these mutants was further conﬁrmed by measuring mant-XDP binding in presence of excess GDP (GDP:mant-XDP = 100:1). This does not aﬀect