Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase.
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.
Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology Hellas and Department of Biology, University of Crete, PO Box 1527, GR711 10 Iraklio, Crete, Greece.
Efrosyni
E
Papanikou
Spyridoula
S
Karamanou
Catherine
C
Baud
Giorgos
G
Sianidis
Miriam
M
Frank
Anastassios
A
Economou
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D016415:Sequence Alignment
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins