Mechanistic study of classical translocation-dead SpoIIIE36 reveals the functional importance of the hinge within the SpoIIIE motor.
SpoIIIE/FtsK ATPases are central players in bacterial chromosome segregation. It remains unclear how these DNA translocases harness chemical energy (ATP turnover) to perform mechanical work (DNA movement). Bacillus subtilis sporulation provides a dramatic example of intercompartmental DNA transport, in which SpoIIIE moves 70% of the chromosome across the division plane. To understand the mechanistic requirements for DNA translocation, we investigated the DNA translocation defect of a classical nontranslocating allele, spoIIIE36. We found that the translocation phenotype is caused by a single substitution, a change of valine to methionine at position 429 (V429M), within the motor of SpoIIIE. This substitution is located at the base of a hinge between the RecA-like β domain and the α domain, which is a domain unique to the SpoIIIE/FtsK family and currently has no known function. V429M interferes with both protein-DNA interactions and oligomer assembly. These mechanistic defects disrupt coordination between ATP turnover and DNA interaction, effectively uncoupling ATP hydrolysis from DNA movement. Our data provide the first functional evidence for the importance of the hinge in DNA translocation.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Marina
M
Besprozvannaya
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Valerie L
VL
Pivorunas
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA bburton@mcb.harvard.edu.
Briana M
BM
Burton
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D014644:Genetic Variation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013171:Spores, Bacterial / Q000502:physiology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C030069:spore-specific proteins, Bacillus