2022
DOI: 10.1101/2022.05.01.490196
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Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

Abstract: VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5- carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predicti… Show more

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Cited by 8 publications
(6 citation statements)
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“…While the structures of the unliganded protein and the protein bound to Neu5Ac are in the inward-open conformation, the density of the binding site of Neu5Ac is unambiguous ( Figure 5A ). The architecture of the Neu5Ac binding site is similar to that of citrate/malate/fumarate in the di/tricarboxylate transporter of V. cholerae ( Vc INDY) (Kinz-Thompson et al, 2022). Neu5Ac binds to the transport domain and there are no direct interactions with the residues in the scaffold domain.…”
Section: Resultsmentioning
confidence: 99%
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“…While the structures of the unliganded protein and the protein bound to Neu5Ac are in the inward-open conformation, the density of the binding site of Neu5Ac is unambiguous ( Figure 5A ). The architecture of the Neu5Ac binding site is similar to that of citrate/malate/fumarate in the di/tricarboxylate transporter of V. cholerae ( Vc INDY) (Kinz-Thompson et al, 2022). Neu5Ac binds to the transport domain and there are no direct interactions with the residues in the scaffold domain.…”
Section: Resultsmentioning
confidence: 99%
“…The polar groups bind to both the C1-caboxylate side of the molecule and the C8-C9 carbonyls, suggesting that SSS transporters have probably evolved to transport nine-carbon sugars such as Neu5Ac (Wahlgren et al, 2018). Interestingly, even the dicarboxylate transporter from V. cholerae ( Vc INDY) binds to its ligand via electrostatic interactions with both carboxylate groups ( Figure 6C ) (Kinz-Thompson et al, 2022). The high affinity of the substrate-binding component ( Fn SiaP) to Neu5Ac is physiologically relevant because it sequesters Neu5Ac in a volume where the concentration of Neu5Ac is very low.…”
Section: Resultsmentioning
confidence: 99%
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“…To demonstrate the use of KDA we are fo- cusing on the biophysics of secondary active transporters, [27][28][29] an area covered originally in Hill's work 11,12 with many recent applications of kinetic models. [1][2][3][4][5][6][30][31][32][33] These membrane proteins use the free energy stored in a transmembrane ionic gradient to drive energetically uphill transport of a substrate (another ion or small molecule) by alternating between multiple protein conformations in a manner that is coupled to ion and substrate binding. Secondary active transporters are prime examples of free energy transduction in biology 12 and serve as systems to study non-equilibrium behavior at the molecular scale.…”
Section: Introductionmentioning
confidence: 99%
“…The level of defocusing mentioned in (iii) for each trajectory was quantified by the level of maximum intensity. Trajectories with maximum normalized intensities that were greater than 1.1 and lasted for more than one continuous frame were not included.The normalized, AF546 fluorescence intensity vs. time trajectories from each movie were analyzed together using a variational Bayesian Hidden Markov Model (HMM) to yield a global model of the single-molecule observations and dynamics present in each replicate movie of each experiment58,59 . Two-state HMMs were used to analyze the B12 binding data in Fig.S5aand the resulting transition matrices were used estimate the values of kbinding and krelease .…”
mentioning
confidence: 99%