Stochastic steps in secondary active sugar transport.
Secondary active transporters, such as those that adopt the leucine-transporter 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.
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260;
Joshua L
JL
Adelman
Department of Physiology, Geffen School of Medicine, University of California, Los Angeles, CA 90095;
Chiara
C
Ghezzi
Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158;
Paola
P
Bisignano
Department of Physiology, Geffen School of Medicine, University of California, Los Angeles, CA 90095;
Donald D F
DD
Loo
School of Basic Science, College of Convergence, Daegu Gyeongbuk Institute of Science and Technology, Daegu 42988, Korea;
Seungho
S
Choe
Department of Physiology, Geffen School of Medicine, University of California, Los Angeles, CA 90095; The Institute for Stem Cell Biology and Regenerative Medicine (instem), National Centre for Biological Sciences - Tata Institute of Fundamental Research, Bangalore 560065, Karnataka, India.
Jeff
J
Abramson
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260;
John M
JM
Rosenberg
Department of Physiology, Geffen School of Medicine, University of California, Los Angeles, CA 90095; michael.grabe@ucsf.edu ewright@mednet.ucla.edu.
Ernest M
EM
Wright
Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; michael.grabe@ucsf.edu ewright@mednet.ucla.edu.
Michael
M
Grabe
D005947:Glucose / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008390:Markov Chains; D056004:Molecular Dynamics Simulation; D009010:Monte Carlo Method; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000378:metabolism*
C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D005947:Glucose
SGLT; kinetics; simulation; symporter; transporter