TY - JOUR
T1 - Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB
AU - Larrea, Delfina
AU - de Paz, Héctor D.
AU - Matilla, Inmaculada
AU - Guzmán-Herrador, Dolores L.
AU - Lasso, Gorka
AU - de la Cruz, Fernando
AU - Cabezón, Elena
AU - Llosa, Matxalen
N1 - Publisher Copyright:
© 2017, Springer-Verlag GmbH Germany.
PY - 2017/10/1
Y1 - 2017/10/1
N2 - Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening–closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.
AB - Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening–closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.
KW - Bacterial conjugation
KW - DNA transport
KW - Molecular motors
KW - Type IV secretion systems
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U2 - 10.1007/s00438-017-1331-3
DO - 10.1007/s00438-017-1331-3
M3 - Article
C2 - 28597316
AN - SCOPUS:85020419029
SN - 1617-4615
VL - 292
SP - 1037
EP - 1049
JO - Molecular Genetics and Genomics
JF - Molecular Genetics and Genomics
IS - 5
ER -