Nucleotide binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations.
SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.
Schmidt
M
M
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Ding
H
H
Ramamurthy
V
V
Mukerji
I
I
Oliver
D
D
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007700:Kinetics; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013816:Thermodynamics
D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins