Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding.
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 activities 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.
Tomar
Sushil Kumar
SK
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India.
Dhimole
Neha
N
Chatterjee
Moon
M
Prakash
Balaji
B
D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D054681:Ribosome Subunits, Large, Bacterial / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C541027:EngA protein, E coli; D019204:GTP-Binding Proteins