Dataset Information

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

ABSTRACT: The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new compounds to identify key structural features that specify recognition by AtSUC2. Our analysis of the structure-activity relationship revealed that the presence of a free hydroxyl group on the coumarin moiety is essential for binding by AtSUC2 and subsequent phloem mobility. Structural modeling of the AtSUC2 substrate-binding pocket explains some important structural requirements for the interaction of coumarin glucosides with the AtSUC2 transporter.

SUBMITTER: De Moliner F

PROVIDER: S-EPMC5920547 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

De Moliner Fabio F Knox Kirsten K Reinders Anke A Ward John M JM McLaughlin Paul J PJ Oparka Karl K Vendrell Marc M

Journal of experimental botany 20180401 10

The phloem sucrose transporter, AtSUC2, is promiscuous with respect to substrate recognition, transporting a range of glucosides in addition to sucrose, including naturally occurring coumarin glucosides. We used the inherent fluorescence of coumarin glucosides to probe the specificity of AtSUC2 for its substrates, and determined the structure-activity relationships that confer phloem transport in vivo using Arabidopsis seedlings. In addition to natural coumarin glucosides, we synthesized new com ...[more]

PMID: 29506213

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Project description:Binding specificity is an important consideration in drug design. An effective drug molecule often must bind with high specificity to its intended target in the body; lower specificity implies the possibility of significant binding to unintended partners, which could instigate deleterious side effects. However, if the target is a rapidly mutating agent, a drug that is too specific will quickly lose its efficacy by not binding well to functional mutants. Therefore, in molecular design, it is crucial to tailor the binding specificity of a drug to the problem at hand. In practice, specificity is often studied on a case-by-case basis, and it is difficult to create general understanding of the determinants of specificity from the union of such available cases. In this work, we undertook a comprehensive, general study of molecular binding with emphasis on understanding the determinants of specificity from a physical standpoint. By extending a theoretical framework grounded in continuum electrostatics and creating an abstracted lattice model that captures key physical aspects of binding interactions, we systematically explored the relationship between a molecule's physical characteristics and its binding specificity toward potential partners. The theory and simulated binding interactions suggested that charged molecules are more specific binders than their hydrophobic counterparts for several reasons. First, the biological spectrum of possible binding characteristics includes more partners that bind equally well to hydrophobic ligands than to charged ligands. Also, charged ligands, whose electrostatic potentials have strong orientational dependence, are more sensitive to shape complementarity than their hydrophobic counterparts. Ligand conformational and orientational flexibility can further influence a charged molecule's ability to bind specifically. Interestingly, we found that conformational flexibility can increase the specificity of polar and charged ligands, by allowing them to greatly lower the binding free energy to a select few partners relative to others. Additionally, factors such as a molecule's size and the ionic strength of the solution were found to predictably affect binding specificity. Taken together, these results, all of which stem from a unified theoretical framework, provide valuable physical insight into the general determinants of binding specificity and promiscuity in a biological environment. The general principles discussed here could prove useful in the design of molecules with tailored specificities, leading to more effective therapeutics.

Project description:BackgroundIn contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.Methodology/principal findingsTo unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H(+) co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H(+) transport was associated with a decrease in membrane capacitance (C(m)). In addition to sucrose C(m) was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these C(m) changes, presteady-state currents (I(pre)) of ZmSUT1 transport were analyzed. Decay of I(pre) could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to I(pre), was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed C(m) changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.ConclusionsTaken together our results indicate that in the absence of sucrose, 'trapped' protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to C(m) changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.

Dataset Information

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

Publications

Probing binding specificity of the sucrose transporter AtSUC2 with fluorescent coumarin glucosides.

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