Binding of GTP and GDP induces a significant conformational change in the GTPase domain of Ffh, a bacterial homologue of the SRP 54 kDa subunit.
The bacterial Ffh protein is homologous to the SRP54 subunit of the signal recognition particle. Ffh plays a key role in the targeting of proteins to the membrane and it is composed of a N-terminal domain (N), a middle GTPase (G) domain and a C-terminal M domain which has binding sites for SRP RNA and signal peptide. The GTP binding and hydrolysis of Ffh is critical to its function. We have used protease digestion to probe the conformation of the Mycoplasma mycoides Ffh N+G domain. In the absence of nucleotide the protein was comparatively sensitive to protease cleavage and we identified sites particularly prone to cleavage in a region near the C-terminus of the GTPase domain. However, in the presence of GTPgammaS or GDP this region is stabilized and the protein adopts a more ordered structure. The pattern of cleavage with GTPgammaS was indistinguishable from that when GDP was bound, indicating that the conformation of the nucleotide-free form is distinct from that when either GTPgammaS or GDP is bound to the protein. The possible functional role of this significant conformational change is discussed.
Farmery
M
M
Department of Medical Biochemistry, Göteborg University Medicinaregatan 9A, S-413 90 Göteborg, Sweden.
Macao
B
B
Larsson
T
T
Samuelsson
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D009176:Mycoplasma mycoides / Q000201:enzymology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D011994:Recombinant Proteins; D018271:Signal Recognition Particle; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014357:Trypsin; D020558:GTP Phosphohydrolases