Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins

Abstract

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na -coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581 T , and compare it with the substrate-bound structure previously reported. Conformational freeenergy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.binding thermodynamics | periplasmic binding protein | secondary transporter | ABC transporter | replica-exchange metadynamics

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

“…3). These results are in agreement with MD simulations, were no stable intermediate states were predicted for the P domain of the ectoine TRAP transporter TeaABC (12). The slightly different open conformations that were present in our crystal structure (Fig.…”

“…The slightly different open conformations that were present in our crystal structure (Fig. S6) fit to the relatively broad energetic minimum for the open structure that was observed in those calculations (12). Considering the current hypothesis for the transport cycle of TRAP transporters (4), large concentrations of closed ligand-free P domains would trigger unproductive closing and opening of the transporter.…”

“…S6 shows that chain A fits almost perfectly to the previously published wild-type structure, while chain B is in a slightly more closed conformation. Such slightly closed states of unliganded P domains were predicted by MD simulations (12). While both spin labels are clearly visible in the electron density, the 54R1 side chain is better defined and therefore apparently less mobile (Fig.…”

“…1). In the substrate-bound state, the two ab-domains close around the substrate reminiscent of a Venus flytrap and the reverse motion is thought to occur when the substrate is channeled into the transporter (4), likely by an allosteric mechanism through conformational changes in the membrane domains (12). The overwhelming majority of P domains have a conserved arginine in the substrate binding cleft (position 147 in HiSiaP).…”

Two-state dynamics of the SH3–SH2 tandem of Abl kinase and the allosteric role of the N-cap

“…All bias-exchange metadynamics calculations were carried out with GROMACS/PLUMED (45,46) and the CHARMM22/CMAP protein force field, using analogous simulation systems and conditions as above, except for the introduction of a virtual hydrogen scheme, which enable us to use a time step of 4 fs. In bias-exchange metadynamics, a series of concurrent simulations or replicas are carried out in which the protein is adiabatically driven from one conformer to another, through historydependent biasing forces that oppose sampling of conformations previously explored (47,48). At a given frequency, attempts are made to exchange the biases accumulated in each replica, using a Metropolis Monte Carlo acceptance criterion, as in other Hamiltonian exchange schemes (49).…”