The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

Abstract

Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophilus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP.

Fig. 1.

Expression and purification of SiaPQM. (A) Coomassie-stained SDS PAGE gel of ligand-free, N-terminally decahistidine tagged SiaP purified by using Ni-affinity chromatography, followed by anion-exchange chromatography. Lane 1, molecular mass ladder with sizes of relevant proteins indicated in kDa; lane 2, purified SiaP. (B) Western blot using anti-His antibodies of membranes expressing SiaQM with an C- or N-terminal decahistidine tag from L. lactis (Lanes 1 and 2, respectively) or E. coli (Lanes 3 and 4, respectively). (C) Coomassie-stained SDS PAGE of fractions from the Ni-affinity chromatographic purification of SiaQM from E. coli membrane vesicles. Lane 1, molecular mass ladder; lane 2, membrane vesicles; lane 3, column flowthrough; lane 4, 40 mM imidazole wash; lanes 5 and 6, 500 mM imidazole elution fractions. (D) Coomassie-stained SDS PAGE gel of SiaQM containing proteoliposomes. Lane 1, molecular mass ladder; lane 2, SiaQM-containing proteoliposomes.

Fig. 2.

Analysis of the energetic requirements for Neu5Ac uptake. (A) Uptake of 5 μM [¹⁴C]-Neu5Ac into SiaQM-containing proteoliposomes in the presence of a sodium gradient (Δμ_Na) and either 5 μM SiaP (closed triangles), 5 μM VC1779 (closed circles), no SBP (open circles), or in the presence of 5 μM SiaP but no Δμ_Na (open triangles); (B) Uptake of 5 μM [¹⁴C]-Neu5Ac into SiaQM-containing proteoliposomes in the presence of 5 μM SiaP and the following different energetic conditions: Δμ_Na + ΔΨ (closed squares), Δμ_Na alone (closed triangles), Δμ_Na + ΔpH (open circles), ΔΨ (open triangles), and ΔpH (closed circles). (C) Comparison of the [¹⁴C]-Neu5Ac uptake properties of SiaQM-containing proteoliposomes in the presence of a ΔΨ plus either equimolar Na⁺ across the membrane (closed circles) or an inwardly directed sodium gradient (open circles).

Fig. 3.

Chase with unlabeled Neu5Ac and Neu5Ac counterflow. (A) Uptake of 5 μM [¹⁴C]-Neu5Ac into SiaQM-containing proteoliposomes in the presence of 5 μM SiaP, Δμ_Na, and ΔΨ. At t = 100 s (indicated with an arrow) 1 mM unlabeled Neu5Ac (open circles) or the same volume of dH₂O (closed circles) was added. (B) Uptake of 1 μM [¹⁴C]-Neu5Ac into E. coli BW25113ΔnanAT expressing NanT (open symbols) or SiaPQM (filled symbols). At t = 100 s (indicated with an arrow), either 1 mM unlabeled Neu5Ac (triangular symbols) or an equal volume of buffer (circular symbols) was added to the reactions. (C) Substrate counterflow activity of SiaQM-containing proteoliposomes (closed circles). Control curve of [¹⁴C]-Neu5Ac uptake into SiaQM-containing proteoliposomes in the presence of 5 μM SiaP and Δμ_Na + ΔΨ (open circles).

Fig. 4.

Dependence of Neu5Ac transport on the concentration of SiaP. (A) Uptake of 5 μM [¹⁴C]-Neu5Ac into SiaQM-containing proteoliposomes in the presence of Δμ_Na + ΔΨ and 2.5 μM (open circles), 5 μM (open triangles), 10 μM (closed squares), or 20 μM (closed diamonds) SiaP. (B) Effect on uptake of [¹⁴C]-Neu5Ac into SiaQM-containing proteoliposomes upon the addition of 17.5 μM unliganded SiaP (open circles), 17.5 μM unliganded VC1779 (filled triangles), or buffer (50 mM KPi, pH7, filled circles) at 100 s. The SiaQM-containing proteoliposomes were used in the presence of 2.5 μM SiaP and Δμ_Na. The point of addition is indicated by an arrow.

Fig. 5.

Model of Na⁺-dependent transport of sialic acid by SiaPQM. The Upper image (Import Cycle) shows the uptake of sialic acid (denoted as asterisk), driven by a (electro)chemical Na⁺ gradient (Δμ_Na + FΔΨ; F, Faraday constant). After binding of sialic acid (asterisk) to SiaP, the liganded complex docks onto SiaQM. A minimum of two sodium ions (black dots) bind to the complex and drive the translocation of sialic acid across the membrane; the sodium ions are cotransported with sialic acid, after which the system relaxes back to the initial conformation. The Lower image (Export Cycle) shows the efflux of sialic acid under conditions that an excess of unliganded SiaP is available. The critical point is that efflux of sialic acid only occurs when unliganded SiaP docks onto SiaQM with bound substrate. Assuming tight coupling in the transport reaction, two or more Na⁺ ions will be exported together with sialic acid.