Membrane transport is generally thought to occur via an alternating access mechanism in which the transporter adopts at least two states, accessible from two different sides of the membrane to exchange substrates from the extracellular environment and the cytoplasm or from the cytoplasm and the intracellular matrix of the organelles (only in eukaryotes). In recent years, a number of high resolution structures have supported this general framework for a wide class of transport molecules, although additional states along the transport pathway are emerging as critically important. Given that substrate binding is often weak in order to enhance overall transport rates, there exists the distinct possibility that transporters may transport the incorrect substrate. This is certainly the case for many pharmaceutical compounds that are absorbed in the gut or cross the blood brain barrier through endogenous transporters. Docking studies on the bacterial sugar transporter vSGLT reveal that many highly toxic compounds are compatible with binding to the orthosteric site, further motivating the selective pressure for additional modes of selectivity. Motivated by recent work in which we observed failed substrate delivery in a molecular dynamics simulation where the energized ion still goes down its concentration gradient, we hypothesize that some transporters evolved to harness this 'slip' mechanism to increase substrate selectivity and reduce the uptake of toxic molecules. Here, we test this idea by constructing and exploring a kinetic transport model that includes a slip pathway. While slip reduces the overall productive flux, when coupled with a second toxic molecule that is more prone to slippage, the overall substrate selectivity dramatically increases, suppressing the accumulation of the incorrect compound. We show that the mathematical framework for increased substrate selectivity in our model is analogous to the classic proofreading mechanism originally proposed for tRNA synthase; however, because the transport cycle is reversible we identified conditions in which the selectivity is essentially infinite and incorrect substrates are exported from the cell in a 'detoxification' mode. The cellular consequences of proofreading and membrane slippage are discussed as well as the impact on future drug development.

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest.

MOTIVATION: A large fraction of the entries contained in the Protein Data Bank describe proteins in complex with low molecular weight molecules such as physiological compounds or synthetic drugs. In many cases, the same molecule is found in distinct protein-ligand complexes. There is an increasing interest in Medicinal Chemistry in comparing protein binding sites to get insight on interactions that modulate the binding specificity, as this structural information can be correlated with other experimental data of biochemical or physiological nature and may help in rational drug design. RESULTS: The web service protein-ligand interaction presented here provides a tool to analyse and compare the binding pockets of homologous proteins in complex with a selected ligand. The information is deduced from protein-ligand complexes present in the Protein Data Bank and stored in the underlying database. AVAILABILITY: Freely accessible at http://bioinformatics.istge.it/pli/.