Stochastic steps in secondary active sugar transport

Abstract

Secondary active transporters, such as those that adopt the leucinetransporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.T ransport of sugar molecules across membranes is virtually ubiquitous in biology, and it is of central importance in human health. The uptake of glucose is especially crucial due to its pivotal role in cellular metabolism and energy production. In mammals, glucose is absorbed in the small intestine and kidney via sodium-dependent glucose transporters (SGLTs), which localize to the apical membrane and concentrate glucose in the epithelia. SGLTs fall into the large leucine-transporter (LeuT) structural family of secondary active transporters that have evolved to concentrate a wide array of substrates across membranes using the energy stored in the Na + electrochemical potential gradient. For symporters in this family, transport occurs by an alternating access mechanism (1) in which the transporter first binds ligands in an outward-facing conformation, and then transitions to an inwardfacing conformation that releases the cargo to the cytoplasm. The order of ion and substrate binding and unbinding is likely tied to the function of the transporter, making it possible to convert the energy stored in the ion gradient into a substrate gradient, and vice versa when these proteins operate in reverse.Extensive biochemical uptake assays and electrophysiological studies of SGLTs have led to a view of Na + /glucose cotransport in which Na + binding precedes sugar binding on the external face of the transporter. Kinetic models adhering to this mechanism satisfactorily account fo...

Na + coordination at the Na2 site of the Na + /I − symporter

“…While this paper was being reviewed, Adelman et al (83) published a paper reporting the identification of residues in the Na + site of vSGLT corresponding to some of the residues discussed here.…”

Physiology of renal glucose handling via SGLT1, SGLT2 and GLUT2

“…Finally, the inner gate closes and the kinetic cycle continues to the initial starting position. We originally proposed an ordered internal dissociation of glucose and sodium, but recent experiments and molecular dynamic studies favour the simultaneous release of glucose and sodium [ 38 ]. One complete cycle takes about 20 ms.…”

“…Considerable progress has been made by using the structure of vSGLT to examine the molecular dynamics of SGLTs (interested readers are referred to the literature, e.g. [ 20 , 38 ]). These and other biophysical studies of human SGLT1 provide insights into the conformational changes associated with sodium binding and isomerisation of the transporter between the outward and inward facing structures (e.g.…”

Mechanism of Substrate Translocation in an Alternating Access Transporter

“…Molecular dynamics (MD) simulations reveal protein motions at an atomic level and thus appear particularly well suited to answer these questions, in conjunction with crystallography and other experimental techniques (Adelman et al, 2016; Dror et al, 2012; Faraldo-Gomez and Forrest, 2011; Fukuda et al, 2015; Lee et al, 2016; Li et al, 2015; Watanabe et al, 2010). Capturing critical events such as the translocation of a substrate through a transporter or the transition of a transporter between its outward-open and inward-open conformation, however, has proven challenging in simulation (Li et al, 2015; Shaikh et al, 2013).…”