Simulated annealing reveals the kinetic activity of SGLT1, a member of the LeuT structural family

The Na+/glucose cotransporter (SGLT1) is the archetype of membrane proteins that use the electrochemical Na+ gradient to drive uphill transport of a substrate. The crystal structure recently obtained for vSGLT strongly suggests that SGLT1 adopts the inverted repeat fold of the LeuT structural family for which several crystal structures are now available. What is largely missing is an accurate view of the rates at which SGLT1 transits between its different conformational states. In the present study, we used simulated annealing to analyze a large set of steady-state and pre–steady-state currents measured for human SGLT1 at different membrane potentials, and in the presence of different Na+ and α-methyl-d-glucose (αMG) concentrations. The simplest kinetic model that could accurately reproduce the time course of the measured currents (down to the 2 ms time range) is a seven-state model (C1 to C7) where the binding of the two Na+ ions (C4→C5) is highly cooperative. In the forward direction (Na+/glucose influx), the model is characterized by two slow, electroneutral conformational changes (59 and 100 s−1) which represent reorientation of the free and of the fully loaded carrier between inside-facing and outside-facing conformations. From the inward-facing (C1) to the outward-facing Na-bound configuration (C5), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C5→C6) is electroneutral, the next step (C6→C7) carries 0.7 positive charges inside the cell. Alignment of the seven-state model with a generalized model suggested by the structural data of the LeuT fold family suggests that electrogenic steps are associated with the movement of the so-called thin gates on each side of the substrate binding site. To our knowledge, this is the first model that can quantitatively describe the behavior of SGLT1 down to the 2 ms time domain. The model is highly symmetrical and in good agreement with the structural information obtained from the LeuT structural family.

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The transport mechanism of the human sodium/myo-inositol transporter 2 (SMIT2/SGLT6), a member of the LeuT structural family

AJP Cell Physiology ◽

10.1152/ajpcell.00054.2014 ◽

2014 ◽

Vol 307 (5) ◽

pp. C431-C441 ◽

Cited By ~ 4

Author(s):

Louis J. Sasseville ◽

Jean-Philippe Longpré ◽

Bernadette Wallendorff ◽

Jean-Yves Lapointe

Keyword(s):

Steady State ◽

Conformational Changes ◽

Voltage Pulse ◽

Transport Mechanism ◽

Simulated Annealing Algorithm ◽

Transient Current ◽

Xenopus Laevis Oocytes ◽

Structural Family ◽

Novel Approach ◽

Steady State Current

The sodium/ myo-inositol transporter 2 (SMIT2) is a member of the SLC5A gene family, which is believed to share the five-transmembrane segment inverted repeat of the LeuT structural family. The two-electrode voltage-clamp (TEVC) technique was used to measure the steady-state and the pre-steady-state currents mediated by human SMIT2 after expression in Xenopus laevis oocytes. Phlorizin is first shown to be a poor inhibitor of pre-steady-state currents for depolarizing voltage pulse. From an up to threefold difference between the apparent ON and OFF transferred charges during a voltage pulse, we also show that a fraction of the transient current recorded for very negative potentials is not a true pre-steady-state current coming from the cotransporter conformational changes. We suggest that this transient current comes from a time-dependent leak current that can reach large amplitudes when external Na+ concentration is reduced. A kinetic model was generated through a simulated annealing algorithm. This algorithm was used to identify the optimal connectivity among 19 different kinetic models and obtain the numerical values of the associated parameters. The proposed 5-state model includes cooperative binding of Na+ ions, strong apparent asymmetry of the energy barriers, a rate-limiting step that is likely associated with the translocation of the empty transporter, and a turnover rate of 21 s−1. The proposed model is a proof of concept for a novel approach to kinetic modeling of electrogenic transporters and allows insight into the transport mechanism of members of the LeuT structural family at the millisecond timescale.

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Shaker potassium channel gating. III: Evaluation of kinetic models for activation.

The Journal of General Physiology ◽

10.1085/jgp.103.2.321 ◽

1994 ◽

Vol 103 (2) ◽

pp. 321-362 ◽

Cited By ~ 373

Author(s):

W N Zagotta ◽

T Hoshi ◽

R W Aldrich

Keyword(s):

Steady State ◽

Conformational Changes ◽

Time Course ◽

Single Channel ◽

Ionic Currents ◽

Single Step ◽

Gating Currents ◽

Open Probability ◽

Shaker Potassium Channel ◽

Single Channel Currents

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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An Accurate Accelerated Steady-State Model For Modular Multilevel Converters With Very High Voltage Levels

2020 IEEE Industry Applications Society Annual Meeting ◽

10.1109/ias44978.2020.9334761 ◽

2020 ◽

Author(s):

Ramadhani Kurniawan Subroto ◽

Yi Chun Chen ◽

Kuo Lung Lian ◽

Chia Chi Chu

Keyword(s):

Steady State ◽

High Voltage ◽

State Model ◽

Multilevel Converters ◽

Steady State Model ◽

Modular Multilevel Converters ◽

Very High

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Non-steady state model applicable to NMR studies for calculating flux rates in glycolysis, gluconeogenesis, and citric acid cycle

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)47155-4 ◽

1994 ◽

Vol 269 (42) ◽

pp. 26034-26039 ◽

Cited By ~ 1

Author(s):

G Martin ◽

M F Chauvin ◽

S Dugelay ◽

G Baverel

Keyword(s):

Steady State ◽

Citric Acid ◽

Citric Acid Cycle ◽

State Model ◽

Nmr Studies ◽

Acid Cycle ◽

Steady State Model

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A steady-state model for sulphur isotope fractionation in bacterial reduction processes

Geochimica et Cosmochimica Acta ◽

10.1016/0016-7037(73)90052-5 ◽

1973 ◽

Vol 37 (5) ◽

pp. 1141-1162 ◽

Cited By ~ 312

Author(s):

C.E. Rees

Keyword(s):

Steady State ◽

Isotope Fractionation ◽

State Model ◽

Bacterial Reduction ◽

Sulphur Isotope ◽

Reduction Processes ◽

Steady State Model

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A two state model study of photo‐detachment dynamics driven by an optimally designed polychromatic field: A simulated annealing based optimisation

International Journal of Quantum Chemistry ◽

10.1002/qua.26676 ◽

2021 ◽

Author(s):

Srijeeta Talukder ◽

Dipayan Seal ◽

Pulak Naskar ◽

Pinaki Chaudhury ◽

Subhasree Ghosh

Keyword(s):

Simulated Annealing ◽

State Model ◽

Model Study ◽

Two State Model

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Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Biochemical Society Transactions ◽

10.1042/bst20150138 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1023-1032 ◽

Cited By ~ 10

Author(s):

Thomas Stockner ◽

Anna Mullen ◽

Fraser MacMillan

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Abc Transporters ◽

Epr Spectroscopy ◽

Structural Information ◽

Dynamic Properties ◽

Molecular System ◽

Synergistic Effects ◽

Md Simulations ◽

Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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