Published online 26 February 2009 Nucleic Acids Research, 2009, Vol. 37, No. 7 2359–2370 doi:10.1093/nar/gkp107 Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding Sushil Kumar Tomar, Neha Dhimole, Moon Chatterjee and Balaji Prakash* Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India Received January 10, 2009; Revised and Accepted February 9, 2009 ABSTRACT EngA, a unique GTPase containing a KH-domain preceded by two consecutive G-domains, displays distinct nucleotide binding and hydrolysis activities. So far, Escherichia coli EngA is reported to bind the 50S ribosomal subunit in the guanosine- 5’-trihosphate (GTP) bound state. Here, for the first time, using mutations that allow isolating the activ- ities of the two G-domains, GD1 and GD2, we show that apart from 50S, EngA also binds the 30S and 70S subunits. We identify that the key requirement for any EngA–ribosome association is GTP binding to GD2. In this state, EngA displays a weak 50S association, which is further stabilized when GD1 too binds GTP. Exchanging bound GTP with guanosine-5’-diphosphate (GDP), at GD1, results in interactions with 50S, 30S and 70S. Therefore, it appears that GD1 employs GTP hydrolysis as a means to regulate the differential specificity of EngA to either 50S alone or to 50S, 30S and 70S subunits. Furthermore, using constructs lacking either GD1 or both GD1 and GD2, we infer that GD1, when bound to GTP and GDP, adopts distinct conformations to mask or unmask the 30S binding site on EngA. Our results suggest a model where distinct nucleotide-bound states of the two G-domains regulate formation of specific EngA– ribosome complexes. INTRODUCTION Guanosine-50-trihosphate (GTP) binding proteins or GTPases make use of conformational changes associated with GTP binding and hydrolysis and thereby switch between three distinct nucleotide-bound states, i.e. an ‘empty’ nucleotide-free state, a GTP-bound ‘ON’ state and a guanosine-50-diphosphate (GDP)-bound ‘OFF’ state. In doing so, several well characterized GTPases act as molecular switches and impart a tight control over important biological processes ranging from signal transduction, translation, intracellular transport and so on (1–3). On the other hand, little is known about the function of 11 universally conserved bacterial GTPases (4). Although initially thought to be present in prokar- yotes, some of these are well conserved in eukaryotes too. Several recent reports suggest that these GTPases bind ribosomal subunits and possibly play key roles in their biogenesis or assembly (4–14). Of these, Escherichia coli Obg is implicated in the process of ribosome assembly where it interacts with both the 30S and 50S subunits (7). HﬂX was recently shown to bind the 50S (8). Of the cir- cularly permuted GTPases, YjeQ and YloQ bind the 30S ribosomal subunit (9,10), RbgA or YlqF participates in the late step of 50S subunit assembly in Bacillus subtilis (11) and YqeH binds 30S in the GTP-bound form (B. Anand et al. unpublished results) to participate in its assembly (12). Era is known to bind the 30S ribosomal subunit in a nucleotide-free state (6). EngA has been shown to bind the 50S subunit (13–15) and it has been reported that YphC (EngA homologue in B. subtilis) and YsxC function together in 50S assembly (14). Of the aforementioned proteins, EngA is unique as it is the only GTPase known to possess two contiguous G-domains, GD1 and GD2. A KH-domain that follows the G-domains is usually known to be involved in RNA binding or participate in protein–protein interactions (16,17). Based on the crystal structure of Der, an EngA homologue in Thermotoga maritima, it was postulated that the KH-domain participates in protein–protein inter- actions (16). A recently determined structure of YphC from B. subtilis, suggests its role in binding the ribosomal RNA (17). As several prokaryotic GTPases bind riboso- mal subunits, we began our investigations to ﬁnd out if EngA too would bind the ribosome. Reports by Hwang and Inouye (15), Bharat et al. (13) and Schaefer et al. (14) concur with our observation that EngA binds the 50S ribosomal subunit speciﬁcally when bound to a non- hydrolysable GTP analogue, guanosine-5-[b,g-imido]tri- phosphate (GMPPNP). Bharat et al. (13) further suggest that both G-domains are important for ribosome binding. The crystal structure of YphC too, suggests the involve- ment of GD1 in ribosome binding, wherein a large con- formational change ( 60 A˚ ) is observed in GD1 between *To whom correspondence should be addressed. Tel: +91 512 259 4013; Fax: +91 512 259 4010; Email: bprakash@iitk.ac.in 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.