title: Identification and characterization of Streptococcus pneumoniae Ffh, a homologue of SRP54 subunit of mammalian signal recognition particle.
abstract: Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.
authors.affiliation: Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zheng
authors.forename: Christopher
authors.initials: C
authors.lastname: Zook
authors.forename: Laura
authors.initials: L
authors.lastname: Campo
authors.forename: Martin
authors.initials: M
authors.lastname: Henault
authors.forename: Harold
authors.initials: H
authors.lastname: Watson
authors.forename: Q May
authors.initials: QM
authors.lastname: Wang
authors.forename: Sheng-Bin
authors.initials: SB
authors.lastname: Peng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial; D012335:RNA, Ribosomal / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D016415:Sequence Alignment; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: C409968:4.5S RNA; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D018160:Receptors, Cytoplasmic and Nuclear; D018271:Signal Recognition Particle; D020558:GTP Phosphohydrolases