TitleStructural and dynamic properties of translocase motor SecA
NameKeramisanou, Dimitra (author), Charalambos, Kalodimos (chair), Frank, Jordan (internal member), Phillip, Huskey (internal member), John, Hunf (outside member), Rutgers University, Graduate School - Newark,
Cellular control mechanisms
DescriptionSecA is a large, 204 kDa, homodimeric, helicase-like protein that is a key component of the bacteria protein secretion machinery. SecA, being a motor protein, couples the translocation of polypeptide segments across or into biological membranes with the expenditure of metabolic energy extracted from ATP hydrolysis. SecA adopts a compact conformation in the cytoplasm but switches to a relaxed one when is engaged to translocation at the membrane. Specific interaction of SecA with SecYEG induces large conformational changes to both partners that result in the stimulation of SecA’s ATPase activity and trigger the opening of the channel.
We use a combination of NMR spectroscopy, Isothermal Titration Calorimetry (ITC) and biochemical techniques to characterize E. coli SecA along the protein secretion pathway. Recent advances in isotope labeling and NMR methodology (methyl-TROSY) enabled the NMR study of SecA.
We found that the nucleotide binding cleft of SecA exists in a metastable state that undergoes a disorder-order transition upon nucleotide binding. Our data show that SecA uses a novel mechanism wherein conserved regions lining the cleft undergo cycles of disorder-order transitions while switching among functional catalytic states. The structural relation of SecA to helicases suggests that these proteins may utilize similar mechanisms to convert the ATP binding/hydrolysis energy to mechanical work.
Our data reveal that C domain undergoes cycles of detachment and rebinding to the motor that are linked with the ATPase activation of SecA. When the contacts with the C domain are loosened, SecA becomes activated and this process is probably facilitated by the membrane. Moreover, allosteric communication between the preprotein binding and the motor domain of SecA is regulated by nucleotide and signal peptide binding. Finally, we found that the extreme C terminus of SecY (C6 loop) contacts both the motor and the C domain of SecA and we identified important residues from the loop C6 that mediate this interaction.
In summary, our studies revealed the intimate relation between flexibility and catalytic efficiency in SecA as well the allosteric communication among the domains.
NoteIncludes bibliographical references (p. 131-144)
Noteby Dimitra Keramisanou
CollectionGraduate School - Newark Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.