Lactose-H +( −OH) transport system of Escherichia coli Multistate gated pore model based on half-sites stoichiometry for high-affinity substrate binding in a symmetrical dimer

Abstract

A model is proposed for the d-galactoside-H +( −OH) transporter of Escherichia coli that accounts for essentially all the experimental observations established for this system to date. In this model, the functional unit is postulated to be a dimer (consisting of two copies of lacY-specified polypeptide) which spans the membrane with a 2-fold symmetry axis in the membrane plane (Lancaster, J.R. (1978) J. Theor. Biol. 75, 35–50). The functional dimer is assumed to possess a single pore flanked by an inner gate (g i) and an outer gate (g o) and encompassing two oppositely oriented galactoside binding sites, designated m and μ. When g o is open and g i is closed under non-energized conditions, binding site m adopts a configuration defined as State A (i.e., m o A) exhibiting high affinity toward Class G a galactosides (thiodigalactoside, melibiose, α- p-nitrophenylgalactoside) but low affinity for Class G b galactosides (lactose, β- o-nitrophenylgalactoside, β-isopropylthiogalactoside), whereas binding site μ adopts State B (i.e., μ o B ) displaying relatively high affinity toward Class G b galactosides but comparatively low affinity for Class G a galactosides; further, each m o A : μ o B dimer contains one thiol group whose reaction with N-ethylmaleimide inactivates the transporter unless blocked by galactoside binding at site m o A, while the second homologous thiol of the dimer is unreactive toward thiol reagents. Translocation of the m o A : μ o B dimer involves closing of g o followed by opening of g i, and causes the two thiols (as well as sites m and μ) to interchange roles in a symmetrical fashion: m o A : μ o B ↔ m i B : μ i A. In the presence of a substantial (negative) transmembrane Δμ~ H + , the m : μ dimer is postulated to undergo an electrogenic protein conformational change to a second form, ∗(m : μ), in which both sites m and μ possess low affinity toward internal Class G b substrates; galactoside transport in both m : μ and ∗(m : μ) is assumed to be coupled to H +-symport ( −OH-antiport) with a stoichiometry of approximately 1 : 1. Finally, five characteristic predictions of the half-sites model are outlined for further tests of its validity.