2368 Nucleic Acids Research, 2009, Vol. 37, No. 7 Figure 5. Two distinct ribosome-bound states of EngA. (A) The ﬁrst state that only binds the 50S, is generated by EngA[Apo:GTP] or EngA[GTP:GTP]. (B) The second state that binds 50S, 30S, and 70S is generated by EngA[GDP:GTP]. (C) A structural superposition of EngA homologues, Der (PDB:1MKY) and YphC (PDB:2HJG), showing a 60 A˚ movement of GD1 between the GTP- and GDP-bound forms. Here, GD2 is bound to GDP. The RNA binding site proposed by Muench et al. (17) is indicated by a circle. co-elution remains elusive. Driven by these facts, we rea- soned that EngA would bind both 50S and 30S ribosomal subunits if appropriate conditions were provided and that the two G-domains have a role in generating these. The importance of the G-domains is underscored by their contrasting GTP binding and hydrolyzing activities (16). Also, Brown and co-workers (13) suggest that both G-domains play critical roles for the cellular function of EngA. By using single mutants K28A or K228A that arrest GD1 or GD2, respectively, in a nucleotide-free state (Figure 2A), we deduce that GTP binding to GD2 is a primary requirement for any EngA–50S interaction (Figure 2D, D1–D2). The signiﬁcance of GD1 is revealed in experi- ments where it is held in a GTP-bound state. Using salt-sensitivity of an interaction as an estimated indicator of the binding strength (9,25), we infer that a weak inter- action with 50S in EngA[Apo:GTP] state, is further strengthened in EngA[GTP:GTP] state (Figure 2D). It appears that 50S interactions are initiated by the GD2:GTP conformation, but both GD1:GTP and GD2:GTP result in an eﬃcient and sustained EngA–50S interaction. The importance of nucleotide occupancies of the two G-domains is further revealed by mutants D134N and D337N, which allow speciﬁc binding to xanthine nucleo- tides (Figure 2B and C) (18). By segregating nucleotide binding events at the two G-domains using guanine and/ or xanthine nucleotides with these mutants, we could iden- tify conditions where EngA not only binds the 50S subunit but also the 30S (Figure 3A, A1–A3). The 30S interaction thus rationalizes 16S rRNA co-elution found in Figure 1A. In addition, it also accounts for the recent observation that EngA interacts with the structural pro- tein S7 of the 30S subunit (30). However, the most signif- icant ﬁnding based on these experiments is that EngA exists in two distinct ‘ribosome-bound states’ as depicted in Figure 5. The ﬁrst state (Figure 5A) provided by EngA[GTP:GTP)] or EngA[Apo:GTP] binds 50S, (Figure 2D) and the second (Figure 5B) provided by EngA[GDP:GTP] binds 30S, 50S and 70S (Figure 3A). These two ribosome-bound states of EngA are distin- guished by the GTP and GDP-bound forms of GD1 indicating an important regulatory role for it. A recently determined crystal structure of YphC reveals a large conformational change ( 60 A˚ ) of GD1 between its GTP and GDP-bound forms (Figure 5C) (17). Based on structural similarity to the C-terminal domain of S3 ribosomal protein, the authors deﬁne an ON state for EngA, where it binds rRNA via a region contributed by KH-domain and GD2 (indicated by a circle, Figure 5C). They speculate that EngA switches to an OFF state in GD1:GDP and results in a large conformational change (of GD1), which makes this region inaccessible to RNA. The proposed ON state is EngA[GTP:GDP] and the OFF state is EngA[GDP:GDP]. In contrast, our work shows that when GD2 is devoid of GTP, EngA does not bind any of the ribosomal subunits. Given this, the implications of the structural similarity and the proposed rRNA bind- ing site observed by Muench et al. (17) are intriguing. Interestingly, we ﬁnd that when GD1 is truncated, the construct GD1–EngA binds 30S, 50S and 70S subunits (Figure 3C, C1), essentially behaving like the EngA[GDP:GTP] state. Hence, this construct seems to