Abstract
The pilA gene of Neisseria gonorrhoeae encodes the response regulator of a two-component regulatory system that controls pilin gene expression. Examination of the primary sequence of PilA indicates that the protein contains at least two functional domains. The N-terminal region has a proposed helix-turn-helix motif thought to be involved in DNA binding. This region also contains the residues that are presumed to form the acidic pocket involved in phosphorylation by PilB, the sensor kinase of the system. The C terminus of the protein has extensive homology to the G (GTP-binding) domains of the eukaryotic signal recognition particle (SRP) 54-kDa protein and the alpha subunit of the SRP receptor, or docking protein. This homology also extends to similar regions of the bacterial SRP homologs Ffh and FtsY. Here, we demonstrate that purified PilA has significant GTPase activity, and that this activity has an absolute requirement for MgCl2 and is sensitive to KCl and low pH. We also show that PilA has a strict specificity for GTP, and that GTP hydrolysis follows first order kinetics, with a maximum velocity (Vmax) of 1900 pmol of Pi produced per min per mg of protein and a Km for GTP of 9.6 microM at 37 degrees C.
Highlights
Neisseria gonorrhoeae (GC) pili undergo phase variation at a fairly high frequency in vitro (10Ϫ4 to 10Ϫ3 per cell per generation; Ref. 1)
The SRP54 protein is associated with the 7 S RNA in the complex and binds to the signal sequence of the nascent protein as it emerges from the ribosome [18]
The SRP54 proteins have two domains: an “M” domain, which interacts with the signal peptide of nascent proteins as well as to the 7 S (4.5 S in E. coli) RNA, and a “G” domain, which has the GTPase activity required for the interaction of the complex with the docking protein [19]
Summary
(Received for publication, August 8, 1995, and in revised form, August 28, 1995). From the Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201. The C terminus of the protein has extensive homology to the G (GTP-binding) domains of the eukaryotic signal recognition particle (SRP) 54-kDa protein and the ␣ subunit of the SRP receptor, or docking protein. The carboxyl-terminal part of PilA has significant homology to the N-terminal G (GTP-binding) domains of the 54-kDa subunit of the eukaryotic signal recognition particle (SRP)1 [11]. Homologs of this protein (SRP54) have been identified in bacteria and are designated Ffh In this report we present evidence that purified preparations of PilA have significant GTPase activity and characterize this activity biochemically
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