provide analogous interactions and accomplish a state similar to the ‘second ribosome-bound state’ of EngA. However, GD1– GD2EngA, where both GD1 and GD2 are truncated, binds the 30S, implying that KH-domain alone is suﬃcient to provide the 30S bind- ing interactions (Figure 3C, C2). As GD1–EngA and EngA[GDP:GTP] bind 30S, while EngA[GTP:GTP] does not, it is possible that GD1, upon GTP binding undergoes a conformational change (see arrows in Figure 5A) and masks the 30S site on the KH-domain. In addition to this, in vitro experiments, carried out using E. coli EngA and B. subtilis YphC, indicate a need for the disruption of GD1–KH interface to promote ribo- some binding, together with GTP hydrolysis at GD1. The wt-proteins, GST–EngA and His-YphC, fail to interact with ribosomal subunits, unless supplied with a mild con- centration of urea (Figure 3C) or till the inter-domain interactions are compromised by mutating Tyr134 at the GD1–KH interface (Figure 4). Perhaps, this indicates the need for an additional factor in the cellular environment to facilitate the disruption of GD–KH interface and appropriately position GD1 to promote ribosome binding. Together with the inferences drawn based on this study, and the available structural data (16,17), the rRNA bind- ing proposed by Muench et al. may be further clariﬁed as follows. Assuming there would be no large conforma- tional change between GD2:GTP and GD2:GDP, the site proposed by Muench et al. is likely responsible for 50S binding, as it is proposed to be at the interface of GD2 and KH (17). Furthermore, GD1 makes use of GTP binding to mask the 30S binding site provided by the KH-domain and also stabilizes 50S interaction. The GTP hydrolysis at GD1 would then result in unmask- ing the 30S binding site (see arrows in Figure 5B). On the other hand, Muench et al. do not ﬁnd any other exposed and conserved region to bind rRNA (17) that can account for a 30S site, necessitating further crystal structure analysis. Our work, for the ﬁrst time, presents evidence that EngA binds both 50S and 30S, and most importantly, depicts two distinct conformational states of EngA with varied speciﬁcity for ribosomal subunits. Taken together with the large conformational change in GD1 (17) and the role of EngA in ribosome biogenesis (14), it is tempting to speculate that EngA, having completed the 50S assembly in the EngA[GTP:GTP] state, promotes 50S–30S interac- tion in the EngA[GDP:GTP] state to assemble the com- plete 70S particles. Evidently, rigorous experimentation will be needed to examine the biological signiﬁcance of GD1 in enabling EngA to switch between the two ‘ribosome- bound states’. Determining the crystal structures of EngA in the various nucleotide states of its G-domains could relate the large conformational changes to the function of the protein, i.e. ribosome biogenesis and/or assembly. Our attempts so far have led to poorly diﬀracting crystals and eﬀorts are underway to improve their quality to elucidate the structural mechanisms prevalent in EngA. Nucleic Acids Research, 2009, Vol. 37, No. 7 2369 SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Prof S. Sarkar and Mr M. Saxena for sharing facilities to carry out Fluoroscence nucleotide binding studies. We thank Neeraj Kumar, Deepak Joshi and Navdeep Yadav for excellent technical and secretarial assistance. We also thank S. K. Verma, S. Mathew, A. Baskaran, D. Dey, Drs A. Bandopadhaya, S. Ramaswamy and V. K. Nandicoori for constructive criticism and suggestions. S.K.T. acknowledges Ministry of Human Resource Development (MHRD) India and N.D. acknowledges Council of Scientiﬁc and Industrial Research (CSIR) for ﬁnancial assistance. FUNDING Wellcome Trust, UK (in the form of International Senior Research Fellowship to B.P., grant number 73616); Department of Biotechnology, Government of India; CARE funds, IIT Kanpur. Funding for open access charge: Wellcome Trust,UK. Conﬂict of interest statement. None declared REFERENCES 1. Bourne,H.R., Sanders,D.A. and McCormick,F. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature, 348, 125–132. 2. Sprang,S.R. (1997) G protein mechanisms: insights from structural analysis. Annu. Rev. Biochem., 66, 639–678. 3. Mohr,D., Wintermeyer,W. and Rodnina,M.V. (2002) GTPase activation of elongation factors Tu and G on the ribosome. Biochemistry, 41, 12520–12528. 4. Caldon,C.E. and March,P.E. (2003) Function of the universally conserved bacterial GTPases. Curr. Opin. 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