The TpsB translocator HMW1B of haemophilus influenzae forms a large conductance channel.
The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane beta-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.
Duret
Guillaume
G
Department of Biology & Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Szymanski
Michal
M
Choi
Kyoung-Jae
KJ
Yeo
Hye-Jeong
HJ
Delcour
Anne H
AH
D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D006193:Haemophilus influenzae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology
D018829:Adhesins, Bacterial; C117431:HMW1 protein, bacteria; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins