Molecular Dynamics Simulations And Hydrogen Bond Network Modeling Of Sugar Transporters

GLUT1 facilitates the down-gradient translocation of D-glucose across cell membrane in mammals.XylE,an Escherichia coli homolog of GLUT1,utilizes proton gradient as an energy source to drive uphill D-xylose transport.GLUT1 and XylE are members of sugar porter family and share about 40% sequence similarity.However,the former works as a uniporter for facilitated diffusion while the later catalyzes active transport.Previous studies of XylE and GLUT1 suggest that the variation between an acidic residue(Asp27in XylE)and a neutral one(Asn29 in GLUT1)is a key element for their mechanistic divergence.In this work,we combined computational and biochemical approaches to investigate the mechanism of proton coupling by XylE and the functional divergence between GLUT1 and XylE.Using molecular dynamics simulations,we evaluated the free energy profiles of the transition between inward-and outward-facing conformations.We constructed three systems without substrates: GLUT1,XylE_H(Asp27 protonated),and XylE_noH(Asp27deprotonated),to study the mechanism of proton coupling and functional distinction.Our results revealed the correlation between the protonation state and conformational preference in XylE,which is supported by the crystal structures.In particular,GLUT1 possesses a rather flat free energy landscape ensuring rapid turnover,while protonated XylE has a high energy transition state at occluded conformation which could presumably prevent proton leak without sugar binding.In addition,our simulations suggested a thermodynamic difference between XylE and GLUT1 that cannot be explained by the single residue variation at the protonation site.To understand the molecular basis,we applied Bayesian network models to analyze the alteration in the architecture of the hydrogen bond networks during conformational transition.The models and subsequent experimental validation suggest that multiple residue substitutions are required to produce the thermodynamic and functional distinction between XylE and GLUT1.These computational and biochemical characterizations provide unprecedented insight into the mechanistic difference between proton symporters and uniporters.