scholarly journals Proton movement and coupling in the POT family of peptide transporters

2017 ◽  
Vol 114 (50) ◽  
pp. 13182-13187 ◽  
Author(s):  
Joanne L. Parker ◽  
Chenghan Li ◽  
Allete Brinth ◽  
Zhi Wang ◽  
Lutz Vogeley ◽  
...  
Keyword(s):  
Transport Systems ◽  
Coupled Transport ◽  
Defense Compounds ◽  
Proton Binding ◽  
Peptide Transporters ◽  
The Family ◽  
Peptide Binding Site ◽  
Extracellular Side ◽  
Proton Movement ◽  
Dynamics Simulations

POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.

scholarly journals Proton Coupling and the Multiscale Kinetic Mechanism of a Peptide Transporter

2021 ◽  
Author(s):  
Chenghan Li ◽  
Zhi Yue ◽  
Simon Newstead ◽  
Gregory A. Voth
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Kinetic Mechanism ◽  
Peptide Transport ◽  
Peptide Transporter ◽  
Coupled Transport ◽  
Proton Binding ◽  
Drug Molecules ◽  
Proton Coupling ◽  
Proton Movement

ABSTRACTThe proton electrochemical gradient drives substrate transport across the cell membrane via a diverse set of secondary active transporters. Proton coupled peptide transporters (POTs) are important for peptide transport in prokaryotes and eukaryotic cells, where they mediate the uptake of di- and tri-peptides in addition to drug and pro-drug molecules. Previously, we captured a POT transporter from Staphylococcus hominis, PepTSh, in a cytoplasm-facing, inward open state (Minhas et al., 2018). Biochemical experiments have further revealed several critical residues for proton coupled transport; however, the precise role played by these residues in coupling proton binding to conformational changes as well as the timescales for proton transfers have remained obscure. Here, we employed multiscale modeling, including classical molecular dynamics, reactive molecular dynamics, and enhanced free energy sampling to characterize proton coupling within this transporter. We show directly that proton binding to a glutamate on TM7 opens the extracellular gate. The inward proton flow is found to induce movement of the peptide towards the cytosol by varying the protonation state of a second conserved glutamate on TM10. We also show that proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible, revealing a mechanism for proton movement inside the transporter.

scholarly journals Alternative proton-binding site and long-distance coupling inEscherichia colisodium–proton antiporter NhaA

2020 ◽  
Vol 117 (41) ◽  
pp. 25517-25522 ◽  
Author(s):  
Jack A. Henderson ◽  
Yandong Huang ◽  
Oliver Beckstein ◽  
Jana Shen
Keyword(s):  
Binding Site ◽  
Transport Mechanism ◽  
Coupled Transport ◽  
Long Distance ◽  
Electrogenic Transport ◽  
Proton Binding ◽  
Binding Residue ◽  
Constant Ph ◽  
Dynamics Simulations

Escherichia coliNhaA is a prototypical sodium–proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.

scholarly journals BK channel inhibition by strong extracellular acidification

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yu Zhou ◽  
Xiao-Ming Xia ◽  
Christopher J Lingle
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Extracellular Acidification ◽  
Channel Inhibition ◽  
Wide Range ◽  
The Family ◽  
Voltage Dependent ◽  
Intracellular Organelles ◽  
Extracellular Side ◽  
Key Residues

Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.

scholarly journals Gas Sensing by Bacterial H-NOX Proteins: An MD Study

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2882 ◽  
Author(s):  
Ahmed M. Rozza ◽  
Dóra K. Menyhárd ◽  
Julianna Oláh
Keyword(s):  
Gas Sensing ◽  
Hydrophobic Interactions ◽  
Transport Systems ◽  
Alternative Mode ◽  
Channel System ◽  
Gas Molecule ◽  
O2 Diffusion ◽  
Dynamics Simulations ◽  
And Storage ◽  
Kinetics Of

Gas sensing is crucial for both prokaryotes and eukaryotes and is primarily performed by heme-based sensors, including H-NOX domains. These systems may provide a new, alternative mode for transporting gaseous molecules in higher organisms, but for the development of such systems, a detailed understanding of the ligand-binding properties is required. Here, we focused on ligand migration within the protein matrix: we performed molecular dynamics simulations on three bacterial (Ka, Ns and Cs) H-NOX proteins and studied the kinetics of CO, NO and O2 diffusion. We compared the response of the protein structure to the presence of ligands, diffusion rate constants, tunnel systems and storage pockets. We found that the rate constant for diffusion decreases in the O2 > NO > CO order in all proteins, and in the Ns > Ks > Cs order if single-gas is considered. Competition between gases seems to seriously influence the residential time of ligands spent in the distal pocket. The channel system is profoundly determined by the overall fold, but the sidechain pattern has a significant role in blocking certain channels by hydrophobic interactions between bulky groups, cation–π interactions or hydrogen bonding triads. The majority of storage pockets are determined by local sidechain composition, although certain functional cavities, such as the distal and proximal pockets are found in all systems. A major guideline for the design of gas transport systems is the need to chemically bind the gas molecule to the protein, possibly joining several proteins with several heme groups together.

scholarly journals Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters

2011 ◽  
Vol 137 (2) ◽  
pp. 239-251 ◽  
Author(s):  
Ehud Ohana ◽  
Nikolay Shcheynikov ◽  
Dongki Yang ◽  
Insuk So ◽  
Shmuel Muallem
Keyword(s):  
Spatial Orientation ◽  
In Silico ◽  
Unique Feature ◽  
Structural Motif ◽  
Coupled Transport ◽  
Sequence Identity ◽  
Anion Transporters ◽  
The Family ◽  
In Silico Modeling ◽  
Minimal Sequence

Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu−; Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu− in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu− charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl−/1HCO3− to 1Cl−/1HCO3−, while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.

scholarly journals Molecular Dynamics Simulations of resistin and RELMβ proteins: Insight into structural dynamics

2021 ◽  
Author(s):  
L. América Chi Uluac ◽  
M. Cristina Vargas González
Keyword(s):  
Molecular Dynamics ◽  
High Temperature ◽  
Molecular Dynamics Simulations ◽  
Structural Dynamics ◽  
Protein Structures ◽  
Globular Domain ◽  
The Family ◽  
Network Of Hydrogen Bonds ◽  
Dynamics Simulations ◽  
Metabolic Dysfunctions

Diabetes mellitus and high levels of resistin are risk factors for COVID-19, suggest- ing a shared mechanism for their contribution to the increased severity of COVID-19. Resistin belongs to the family of resistin-like molecules (RELMs) whose implications for inflammatory and metabolic dysfunctions warrant its study in order to shed light on the etiology of these concerning pathologies. In this work, our objective is to char- acterize the structural dynamics of the reported crystallized resistin-like molecules. We performed molecular dynamics simulations of all-atom solvated protein at physiological and high temperatures for the three mouse structures reported so far. We found that in all the structures studied, there is a loss of helicity as a first step of protein denat- uration. There is a high stability of the globular β-sheet domain in resistin protein structures that is not conserved for RELMβ. At high temperature, we found a partial interconversion of α-helices into β-sheets in all proteins, indicating that this propensity is not only found during aggregation but also heating. We had been able to identify a largely persistent hydrogen-bond network shared by all the proteins in the interchain globular domain at room temperature. This network of hydrogen bonds is conserved considerably at high temperature in resistin structures, but not in RELMβ. These findings may guide future studies to increase our understanding of the different and shared mechanisms of action of RELMs.

scholarly journals Mechanism of activation at the selectivity filter of the KcsA K+ channel

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Florian T Heer ◽  
David J Posson ◽  
Wojciech Wojtas-Niziurski ◽  
Crina M Nimigean ◽  
Simon Bernèche
Keyword(s):  
Conformational Changes ◽  
Selectivity Filter ◽  
K Channel ◽  
Ion Permeation ◽  
K Channels ◽  
Narrow Region ◽  
Gating Mechanism ◽  
Structural Fluctuations ◽  
Extracellular Side ◽  
Dynamics Simulations

Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.

Approach to the patient with salt-wasting tubulopathies

2018 ◽  
Author(s):  
Detlef Bockenhauer ◽  
Robert Kleta
Keyword(s):  
Sodium Concentration ◽  
Systemic Blood Pressure ◽  
Transport Systems ◽  
Sodium Reabsorption ◽  
Coupled Transport ◽  
Salt Diet ◽  
Salt Wasting ◽  
Low Salt ◽  
Sodium Coupled Transport ◽  
Parallel Action

Sodium is the main ion of the extracellular compartments, and it is through control of sodium reabsorption that the kidneys maintain volume homoeostasis and systemic blood pressure. The amount of sodium that is first filtered by the glomerulus and then reabsorbed in the tubule is quite staggering: assuming a glomerular filtration rate of 100 mL/min and a serum sodium concentration of 140 mmol/L, an average-sized person filters about 20,000 mmol of sodium per day, equivalent to the amount in 1.2 kg of cooking salt. In the steady state, the amount of sodium excreted is equal to the amount ingested. An average Western diet contains about 8–10 g of salt per day; a low-salt diet may be around 2 g per day. Under physiological conditions, the tubules reabsorb about 99% of filtered sodium. This enormous task is accomplished by a combination of distinct and sequentially oriented sodium or sodium-coupled transport systems along the nephron and the concerted and parallel action of some of these systems within the kidney. These are described, along with the consequences of disorders of the processes. A diagnostic approach to salt-losing states such as Fanconi, Bartter Gitelman and other syndromes, and hypoaldosteronism, is described.

scholarly journals Differential Regulation and Substrate Preferences in Two Peptide Transporters of Saccharomyces cerevisiae

2007 ◽  
Vol 6 (10) ◽  
pp. 1805-1813 ◽  
Author(s):  
Houjian Cai ◽  
Melinda Hauser ◽  
Fred Naider ◽  
Jeffrey M. Becker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transport Systems ◽  
Transport Pathways ◽  
Substrate Preferences ◽  
Peptide Transporters ◽  
Yeast Strains ◽  
The Common ◽  
Regulatory Controls ◽  
Dipeptide Transport ◽  
Common Laboratory

ABSTRACT Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [14C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [14C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.

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