High concentrations of GTP induce conformational changes in the essential bacterial GTPase EngA and enhance its binding to the ribosome

FEBS J. 2018 Jan;285(1):160-177. doi: 10.1111/febs.14333. Epub 2017 Dec 1.

Abstract

EngA is a conserved bacterial GTPase involved in ribosome biogenesis. While essential in bacteria, EngA does not have any human orthologue and can thus be an interesting target for new antibacterial compounds. EngA is the only known GTPase bearing two G domains, making unique its catalytic cycle and the induced modulation of its conformation and interaction with the ribosome. We have investigated nucleotide-induced conformational changes in EngA in order to unveil their role in ribosome binding. SAXS and limited proteolysis were used to probe EngA conformational changes, and revealed a change in protein structure and a distinct rate of proteolysis induced by GTP. Structure analysis showed that the conformation adopted in solution in the presence of GTP does not match any known EngA structure, while the SAXS data measured in the presence of GDP are in perfect agreement with two crystal structures (i.e. 2HGJ and 4DCU). Combination of mass spectrometry and N-terminal sequencing for the analysis of the fragmentation pattern upon proteolytic cleavage gave insights into which regions become more or less accessible in the different nucleotide-bound states. Interactions studies confirmed a stronger binding of EngA to the bacterial ribosome in the presence of GTP and suggest that the induced change in conformation of EngA plays a key role for ribosome binding.

Keywords: EngA; GTPase; conformational change; limited proteolysis; mass spectrometry; ribosome; small-angle X-ray scattering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Crystallography, X-Ray
  • GTP Phosphohydrolases / chemistry*
  • GTP Phosphohydrolases / metabolism
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • Guanosine Triphosphate / pharmacology*
  • Models, Molecular
  • Protein Binding / drug effects
  • Protein Conformation / drug effects*
  • Proteolysis / drug effects
  • Ribosomes / chemistry*
  • Ribosomes / metabolism
  • Scattering, Small Angle
  • X-Ray Diffraction

Substances

  • Bacterial Proteins
  • Guanosine Triphosphate
  • GTP Phosphohydrolases

Associated data

  • PDB/2HGJ
  • PDB/4DCU
  • PDB/1MKY
  • PDB/2HJG
  • PDB/4DCV
  • PDB/3J8G
  • PDB/4KYU