Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Aug;5(8):807-11.
doi: 10.1038/sj.embor.7400206. Epub 2004 Jul 23.

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

Efrosyni Papanikou  1 , Spyridoula Karamanou, Catherine Baud, Giorgos Sianidis, Miriam Frank, Anastassios Economou
Affiliations

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

Efrosyni Papanikou et al. EMBO Rep. 2004 Aug.

Abstract

The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Helicase Motif III is essential for protein translocation. (A) Model of a SecA protomer. CTD, C-terminal domain; IRA, intramolecular regulator of ATPase; NBD, nucleotide-binding domain; SD, scaffold domain; SSD, substrate specificity domain; WD, wing domain. The grey square represents Motif III. (B) SF2 motifs (Roman numerals; Sianidis et al, 2001; Caruthers & McKay, 2002) are indicated on a SecA linear map. (C) Comparison of SF2 Motif III sequences from RNA helicases eIF4A (yeast) and NS3 (hepatitis C virus), DNA repair protein UvrB (Bacillus caldotenax) and SecA (Escherichia coli, Mycobacterium tuberculosis and Bacillus subtilis), below a secondary structure schematic (cylinder, α-helix; arrow, βstrand). Conserved residues are in bold. Generated substitutions are shown below. (D) Complementation of the secAts strain BL21.19 by pET5 or pET5 carrying cloned secAs. Indicated culture dilutions (grown at 30°C) were spotted on LB/ampicillin plates and incubated (42°C). (E) proOmpA (pOA) translocation in SecYEG proteoliposomes catalysed by WT SecA or SecA Motif III mutants (40 μg/ml) as described (Karamanou et al, 1999). Lane 1, 30% of undigested input [35S]proOmpA; lanes 2–7, treated with proteinase K. Translocated proOmpA became protease-accessible when Triton X-100 (1% v/v) was added before proteolysis (lane 4).
Figure 2
Figure 2
Preprotein binding to SecAT393N. (A,B) SecA and SecAT393N binding to a signal peptide (3K7L (A); Baud et al, 2002) or a mature domain (CH5EE (B); Papanikou et al, in preparation) optical biosensor. MBP, maltose-binding protein, used as a control. (C,D) N68 or N68T393N (buffer B, 1 mM ATP) was supplemented with increasing amounts of 3K7L (C) or CH5EE (D). Basal ATPase was determined (30 min; 37°C; Lill et al, 1990). ATPase activities are expressed as a percentage of the activity of N68 in the absence of peptides.
Figure 3
Figure 3
Poor stimulation of SecAT393N ATPase by preprotein. (A) Basal (b), membrane (m) and translocation (t) ATPase activities of SecA or N68 derivatives (Lill et al, 1990). (B) Genetic complementation of the secAts strain BL21.19 (as in Fig 1D). (C) In vitro proOmpA (pOA) translocation in SecYEG proteoliposomes catalysed by SecA or derivatives (as in Fig 1E).
Figure 4
Figure 4
Ligand-induced conformation of SecA and SecAT393N (20 μg) were trypsinized (16 μg/ml; buffer B; 2.5 min; 4°C) in the absence of ligands or in the presence of CH5EE (19.5 μM) or ADP (10 μM), or both. Trypsin was inactivated by Pefabloc (9 mM). Polypeptides were separated by SDS–polyacrylamide gel electrophoresis and immunostained by anti-IRA2 (p16) or antisSD (p12) domain-specific antibodies (Baud et al, 2002).
Figure 5
Figure 5
Interactions between Motif III and other SF2 motifs in SecA (Hunt et al, 2002; Sharma et al, 2003). See text for details.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baud C, Karamanou S, Sianidis G, Vrontou E, Politou AS, Economou A (2002) Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain. J Biol Chem 277: 13724–13731 - PubMed
    1. Caruthers JM, McKay DB (2002) Helicase structure and mechanism. Curr Opin Struct Biol 12: 123–133 - PubMed
    1. Cho HS, Ha NC, Kang LW, Chung KM, Back SH, Jang SK (1998) Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA. J Biol Chem 273: 15045–15052 - PubMed
    1. Hartl FU, Lecker S, Schiebel E, Hendrick JP, Wickner W (1990) The binding of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli membrane. Cell 63: 269–279 - PubMed
    1. Hunt JF, Weinkauf S, Henry L, Fak JJ, McNicholas P, Oliver DB, Deisenhofer J (2002) Nucleotide control of interdomain interactions in the conformational reaction cycle of SecA. Science 297: 2018–2026 - PubMed

Publication types

MeSH terms

LinkOut - more resources