Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP

YiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn2+ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn2+. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn2+ and validated its performance with known Zn2+-binding proteins. Using these tools, we find that, in the presence of Zn2+, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn2+ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that govern the transport cycle.

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Zinc Dependent Conformational Changes in the Cation Diffusion Facilitator YiiP from S. oneidensis

10.1101/2020.12.29.424758 ◽

2020 ◽

Author(s):

Maria Lopez-Redondo ◽

Shujie Fan ◽

Akiko Koide ◽

Shohei Koide ◽

Oliver Beckstein ◽

...

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Cytoplasmic Membrane ◽

Conformational Transition ◽

Conformational Dynamics ◽

Dynamics Simulation ◽

Membrane Interface ◽

Secondary Transporter ◽

Transport Cycle ◽

Alternating Access

AbstractYiiP is a secondary transporter that couples Zn2+ transport to the proton motive force. Structural studies of YiiP from prokaryotes as well as Znt8 from humans revealed three different Zn2+ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study effects of Zn2+ binding on the conformational transition, we have used a YiiP/Fab complex for single-particle cryo-EM together with Molecular Dynamics simulation to compare structures of YiiP from S. oneidensis in presence and absence of Zn2+. Without Zn2+, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be an intermediate state. The transition closes a hydrophobic gate and is controlled by the Zn2+ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn2+ binding sites and their role in the conformational dynamics that governs the transport cycle.

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Enhanced Sampling of Protein Conformational Transitions via Dynamically Optimized Collective Variables

10.1101/390278 ◽

2018 ◽

Author(s):

Z. Faidon Brotzakis ◽

Michele Parrinello

Keyword(s):

Degrees Of Freedom ◽

Conformational Transition ◽

Conformational Dynamics ◽

Computational Cost ◽

Md Simulations ◽

Conformational Transitions ◽

Enhanced Sampling ◽

Collective Variables ◽

Simulation Based ◽

Combined Data

AbstractProtein conformational transitions often involve many slow degrees of freedom. Their knowledge would give distinctive advantages since it provides chemical and mechanistic insight and accelerates the convergence of enhanced sampling techniques that rely on collective variables. In this study, we implemented a recently developed variational approach to conformational dynamics metadynamics to the conformational transition of the moderate size protein, L99A T4 Lysozyme. In order to find the slow modes of the system we combined data coming from NMR experiments as well as short MD simulations. A Metadynamics simulation based on these information reveals the presence of two intermediate states, at an affordable computational cost.

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Locking Two Rigid-body Bundles in an Outward-Facing Conformation: The Ion-coupling Mechanism in a LeuT-fold Transporter

Scientific Reports ◽

10.1038/s41598-019-55722-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jing Li ◽

Zhiyu Zhao ◽

Emad Tajkhorshid

Keyword(s):

State Transition ◽

Conformational Dynamics ◽

Md Simulations ◽

Substrate Affinity ◽

Coupling Mechanism ◽

Helical Bundles ◽

Secondary Transporter ◽

Substrate Binding Sites ◽

Kinetics Of ◽

Special Protocol

AbstractSecondary active transporters use electrochemical gradient of ions to fuel the “uphill” translocation of the substrate following the alternating-access model. The coupling of ions to conformational dynamics of the protein remains one of the least characterized aspects of the transporter function. We employ extended molecular dynamics (MD) simulations to examine the Na+-binding effects on the structure and dynamics of a LeuT-fold, Na+-coupled secondary transporter (Mhp1) in its major conformational states, i.e., the outward-facing (OF) and inward-facing (IF) states, as well as on the OF ↔ IF state transition. Microsecond-long, unbiased MD simulations illustrate that Na+ stabilizes an OF conformation favorable for substrate association, by binding to a highly conserved site at the interface between the two helical bundles and restraining their relative position and motion. Furthermore, a special-protocol biased simulation for state transition suggests that Na+ binding hinders the OF ↔ IF transition. These synergistic Na+-binding effects allosterically couple the ion and substrate binding sites and modify the kinetics of state transition, collectively increasing the lifetime of an OF conformation with high substrate affinity, thereby facilitating substrate recruitment from a low-concentration environment. Based on the similarity between our findings for Mhp1 and experimental reports on LeuT, we propose that this model may represent a general Na+-coupling mechanism among LeuT-fold transporters.

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Conformational Transition Pathways in Major Facilitator Superfamily Transporters

10.1101/708289 ◽

2019 ◽

Author(s):

Dylan Ogden ◽

Kalyan Immadisetty ◽

Mahmoud Moradi

Keyword(s):

Conformational Changes ◽

Glucose Transporter ◽

Conformational Transition ◽

Conformational Dynamics ◽

Salt Bridge ◽

Md Simulations ◽

Major Facilitator Superfamily ◽

Glucose Transporter 1 ◽

Major Facilitator ◽

Mfs Transporters

AbstractMajor facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

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CONFORMATIONAL DYNAMICS OF HIV-1 PROTEASE: A COMPARATIVE MOLECULAR DYNAMICS SIMULATION STUDY WITH MULTIPLE AMBER FORCE FIELDS

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720012500187 ◽

2012 ◽

Vol 10 (06) ◽

pp. 1250018 ◽

Cited By ~ 3

Author(s):

BISWA RANJAN MEHER ◽

MATTAPARTHI VENKATA SATISH KUMAR ◽

SMRITI SHARMA ◽

PRADIPTA BANDYOPADHYAY

Keyword(s):

Force Field ◽

Active Site ◽

Force Fields ◽

Conformational Dynamics ◽

Md Simulations ◽

Order Parameters ◽

Dynamics Simulation ◽

Active Site Cavity ◽

Amber Force Fields ◽

Hiv 1

Flap dynamics of HIV-1 protease (HIV-pr) controls the entry of inhibitors and substrates to the active site. Dynamical models from previous simulations are not all consistent with each other and not all are supported by the NMR results. In the present work, the effect of force field on the dynamics of HIV-pr is investigated by MD simulations using three AMBER force fields ff99, ff99SB, and ff03. The generalized order parameters for amide backbone are calculated from the three force fields and compared with the NMR S2 values. We found that the ff99SB and ff03 force field calculated order parameters agree reasonably well with the NMR S2 values, whereas ff99 calculated values deviate most from the NMR order parameters. Stereochemical geometry of protein models from each force field also agrees well with the remarks from NMR S2 values. However, between ff99SB and ff03, there are several differences, most notably in the loop regions. It is found that these loops are, in general, more flexible in the ff03 force field. This results in a larger active site cavity in the simulation with the ff03 force field. The effect of this difference in computer-aided drug design against flexible receptors is discussed.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Nature Communications ◽

10.1038/s41467-020-20032-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

10.1101/2020.04.03.023564 ◽

2020 ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

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Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

10.26434/chemrxiv.5496907 ◽

2017 ◽

Author(s):

Jana Shen ◽

Zhi Yue ◽

Helen Zgurskaya ◽

Wei Chen

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Transmembrane Domain ◽

Conformational Transition ◽

Conformational Dynamics ◽

Proton Release ◽

Intrinsic Resistance ◽

Constant Ph ◽

Wide Range ◽

Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood.

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Molecular Basis of Glucose Transport Mechanism in Plants

10.26434/chemrxiv.8049848.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Balaji Selvam ◽

Ya-Chi Yu ◽

Liqing Chen ◽

Diwakar Shukla

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Transport Mechanism ◽

Conformational Dynamics ◽

Site Directed Mutagenesis ◽

Free Energy Barrier ◽

Transport Cycle ◽

Dynamics Simulations

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

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Effect of Ionic Strength on the Aggregation Propensity of Aβ1-42 Peptide: An In-silico Study

Current Chemical Biology ◽

10.2174/2212796814999200818103157 ◽

2020 ◽

Vol 14 (3) ◽

pp. 216-226

Author(s):

Priyanka Borah ◽

Venkata S.K. Mattaparthi

Keyword(s):

Diffusion Coefficient ◽

Ionic Strength ◽

Marked Effect ◽

Computational Study ◽

Conformational Dynamics ◽

Rapid Change ◽

Md Simulations ◽

Radius Of Gyration ◽

Aggregation Propensity ◽

In Silico Study

Background: Aggregation of misfolded proteins under stress conditions in the cell might lead to several neurodegenerative disorders. Amyloid-beta (Aβ1-42) peptide, the causative agent of Alzheimer’s disease, has the propensity to fold into β-sheets under stress, forming aggregated amyloid plaques. This is influenced by factors such as pH, temperature, metal ions, mutation of residues, and ionic strength of the solution. There are several studies that have highlighted the importance of ionic strength in affecting the folding and aggregation propensity of Aβ1-42 peptide. Objective: To understand the effect of ionic strength of the solution on the aggregation propensity of Aβ1-42 peptide, using computational approaches. Materials and Methods: In this study, Molecular Dynamics (MD) simulations were performed on Aβ1-42 peptide monomer placed in (i) 0 M, (ii) 0.15 M, and (iii) 0.30 M concentration of NaCl solution. To prepare the input files for the MD simulations, we have used the Amberff99SB force field. The conformational dynamics of Aβ1-42 peptide monomer in different ionic strengths of the solutions were illustrated from the analysis of the corresponding MD trajectory using the CPPtraj tool. Results: From the MD trajectory analysis, we observe that with an increase in the ionic strength of the solution, Aβ1-42 peptide monomer shows a lesser tendency to undergo aggregation. From RMSD and SASA analysis, we noticed that Aβ1-42 peptide monomer undergoes a rapid change in conformation with an increase in the ionic strength of the solution. In addition, from the radius of gyration (Rg) analysis, we observed Aβ1-42 peptide monomer to be more compact at moderate ionic strength of the solution. Aβ1-42 peptide was also found to hold its helical secondary structure at moderate and higher ionic strengths of the solution. The diffusion coefficient of Aβ1-42 peptide monomer was also found to vary with the ionic strength of the solution. We observed a relatively higher diffusion coefficient value for Aβ1-42 peptide at moderate ionic strength of the solution. Conclusion: Our findings from this computational study highlight the marked effect of ionic strength of the solution on the conformational dynamics and aggregation propensity of Aβ1-42 peptide monomer.

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