scholarly journals Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex

2020 ◽  
Vol 6 (41) ◽  
pp. eabb9605 ◽  
Author(s):  
Soung-Hun Roh ◽  
Mrinal Shekhar ◽  
Grigore Pintilie ◽  
Christophe Chipot ◽  
Stephan Wilkens ◽  
...  
Keyword(s):  
Proton Transport ◽  
Specific Protein ◽  
Proton Pumping ◽  
Proton Channel ◽  
Ring Rotation ◽  
Adenosine Triphosphatases ◽  
Dynamics Simulations ◽  
Ordered Water ◽  
Membrane Scaffold

Rotary vacuolar adenosine triphosphatases (V-ATPases) drive transmembrane proton transport through a Vo proton channel subcomplex. Despite recent high-resolution structures of several rotary ATPases, the dynamic mechanism of proton pumping remains elusive. Here, we determined a 2.7-Å cryo–electron microscopy (cryo-EM) structure of yeast Vo proton channel in nanodisc that reveals the location of ordered water molecules along the proton path, details of specific protein-lipid interactions, and the architecture of the membrane scaffold protein. Moreover, we uncover a state of Vo that shows the c-ring rotated by ~14°. Molecular dynamics simulations demonstrate that the two rotary states are in thermal equilibrium and depict how the protonation state of essential glutamic acid residues couples water-mediated proton transfer with c-ring rotation. Our cryo-EM models and simulations also rationalize a mechanism for inhibition of passive proton transport as observed for free Vo that is generated as a result of V-ATPase regulation by reversible disassembly in vivo.

scholarly journals Stochastic rotational catalysis of proton pumping F-ATPase

2008 ◽  
Vol 363 (1500) ◽  
pp. 2135-2142 ◽  
Author(s):  
Mayumi Nakanishi-Matsui ◽  
Masamitsu Futai
Keyword(s):  
Proton Transport ◽  
Atp Synthesis ◽  
Energy Coupling ◽  
Proton Pumping ◽  
Proton Channel ◽  
Phosphate Binding ◽  
Stochastic Fluctuation ◽  
Rotational Catalysis ◽  
Α Helix

F-ATPases synthesize ATP from ADP and phosphate coupled with an electrochemical proton gradient in bacterial or mitochondrial membranes and can hydrolyse ATP to form the gradient. F-ATPases consist of a catalytic F 1 and proton channel F 0 formed from the α 3 β 3 γδϵ and ab 2 c 10 subunit complexes, respectively. The rotation of γϵ c 10 couples catalyses and proton transport. Consistent with the threefold symmetry of the α 3 β 3 catalytic hexamer, 120° stepped revolution has been observed, each step being divided into two substeps. The ATP-dependent revolution exhibited stochastic fluctuation and was driven by conformation transmission of the β subunit (phosphate-binding P-loop/α-helix B/loop/β-sheet4). Recent results regarding mechanically driven ATP synthesis finally proved the role of rotation in energy coupling.

scholarly journals Multiscale simulations reveal key features of the proton pumping mechanism in cytochrome c oxidase

2016 ◽  
Author(s):  
Ruibin Liang ◽  
Jessica M. J. Swanson ◽  
Yuxing Peng ◽  
Mårten Wikström ◽  
Gregory A. Voth
Keyword(s):  
Electron Transfer ◽  
Free Energy ◽  
Proton Transport ◽  
Atp Synthesis ◽  
Quantitative Agreement ◽  
Proton Pumping ◽  
Back Flow ◽  
Transport Dynamics ◽  
Hydrophobic Cavity ◽  
Dynamics Simulations

AbstractCytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane, contributing to the transmembrane proton electrochemical gradient that drives ATP synthesis. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free energy profiles and calculated rates) the internal proton transport events that enable pumping and chemistry during the A→PR→F transition in the aa3-type CcO. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored while the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates are in quantitative agreement with experimental measurements. The back flow of the pumped proton from the PLS to E286 and from E286 to the inner side of membrane are prevented by the fast reprotonation of E286 through the D-channel and large free energy barriers for the back flow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity (HC) and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways. This work presents a comprehensive description of the key steps in the proton pumping mechanism in CcO.SignificanceThe long studied proton pumping mechanism in cytochrome c oxidase (CcO) continues to be a source of debate. This work provides a comprehensive computational characterization of the internal proton transport dynamics, while explicitly including the role of Grotthuss proton shuttling, that lead to both pumping and catalysis. Focusing on the A to F transition, our results show that the transfer of both the pumped and chemical protons are thermodynamically driven by electron transfer, and explain how proton back leakage is avoided by kinetic gating. This work also explicitly characterizes the coupling of proton transport with hydration changes in the hydrophobic cavity and D-channel, thus advancing our understanding of proton transport in biomolecules in general.

scholarly journals Multiscale simulations reveal key features of the proton-pumping mechanism in cytochrome c oxidase

2016 ◽  
Vol 113 (27) ◽  
pp. 7420-7425 ◽  
Author(s):  
Ruibin Liang ◽  
Jessica M. J. Swanson ◽  
Yuxing Peng ◽  
Mårten Wikström ◽  
Gregory A. Voth
Keyword(s):  
Free Energy ◽  
Proton Transport ◽  
Proton Pumping ◽  
Strongly Coupled ◽  
Reactive Molecular Dynamics ◽  
Hydrophobic Cavity ◽  
Energy Profiles ◽  
Binuclear Center ◽  
Dynamics Simulations ◽  
Rate Limiting

Cytochrome c oxidase (CcO) reduces oxygen to water and uses the released free energy to pump protons across the membrane. We have used multiscale reactive molecular dynamics simulations to explicitly characterize (with free-energy profiles and calculated rates) the internal proton transport events that enable proton pumping during first steps of oxidation of the fully reduced enzyme. Our results show that proton transport from amino acid residue E286 to both the pump loading site (PLS) and to the binuclear center (BNC) are thermodynamically driven by electron transfer from heme a to the BNC, but that the former (i.e., pumping) is kinetically favored whereas the latter (i.e., transfer of the chemical proton) is rate-limiting. The calculated rates agree with experimental measurements. The backflow of the pumped proton from the PLS to E286 and from E286 to the inside of the membrane is prevented by large free-energy barriers for the backflow reactions. Proton transport from E286 to the PLS through the hydrophobic cavity and from D132 to E286 through the D-channel are found to be strongly coupled to dynamical hydration changes in the corresponding pathways and, importantly, vice versa.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

Quantitative Ranking of Ligand Binding Kinetics with a Multiscale Milestoning Simulation Approach

2018 ◽  
Author(s):  
Benjamin R. Jagger ◽  
Christoper T. Lee ◽  
Rommie Amaro
Keyword(s):  
Molecular Dynamics ◽  
Drug Discovery ◽  
Small Molecule ◽  
Brownian Dynamics ◽  
Model Simulation ◽  
Computational Cost ◽  
Lead Selection ◽  
Delta G ◽  
Dynamics Simulations

<p>The ranking of small molecule binders by their kinetic (kon and koff) and thermodynamic (delta G) properties can be a valuable metric for lead selection and optimization in a drug discovery campaign, as these quantities are often indicators of in vivo efficacy. Efficient and accurate predictions of these quantities can aid the in drug discovery effort, acting as a screening step. We have previously described a hybrid molecular dynamics, Brownian dynamics, and milestoning model, Simulation Enabled Estimation of Kinetic Rates (SEEKR), that can predict kon’s, koff’s, and G’s. Here we demonstrate the effectiveness of this approach for ranking a series of seven small molecule compounds for the model system, -cyclodextrin, based on predicted kon’s and koff’s. We compare our results using SEEKR to experimentally determined rates as well as rates calculated using long-timescale molecular dynamics simulations and show that SEEKR can effectively rank the compounds by koff and G with reduced computational cost. We also provide a discussion of convergence properties and sensitivities of calculations with SEEKR to establish “best practices” for its future use.</p>

Human Fibronectin Extra-Domain B (EDB)-Specific Aptide (APTEDB) Radiolabelling with Technetium-99m as a Potent Targeted Tumour-Imaging Agent

2018 ◽  
Vol 18 (2) ◽  
pp. 277-285 ◽  
Author(s):  
Mohsen Mohammadgholi ◽  
Nourollah Sadeghzadeh ◽  
Mostafa Erfani ◽  
Saeid Abediankenari ◽  
Seyed Mohammad Abedi ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Specific Binding ◽  
Specific Protein ◽  
Tumour Imaging ◽  
Specific Binding Ratio ◽  
Tumour Uptake ◽  
Technetium 99M ◽  
In Vivo Experiments ◽  
Human Fibronectin

Background: Human fibronectin extra-domain B (EDB) is particularly expressed during angiogenesis progression. It is, thus, a promising marker of tumour growth. Aptides are a novel class of peptides with high-affinity binding to specific protein targets. APTEDB is an antagonist-like ligand that especially interacts with human fibronectin EDB. Objective: This study was the first attempt in which the hydrazinonicotinamide (HYNIC)-conjugated APTEDB was labelled with technetium-99m (99mTc) as an appropriate radiotracer and tricine/EDDA exchange labeling. Methods: Radiochemical purity, normal saline, and serum stability were evaluated by HPLC and radio-isotope TLC scanner. Other examinations, such as protein-binding calculation, dissociation radioligand binding assay, and partition coefficient constant determination, were also carried out. The cellular-specific binding of 99mTc- HYNIC-conjugated APTEDB was assessed in two EDB-positive (U87MG) and EDB-negative (U373MG) cell lines. Bio-distribution was investigated in normal mice as well as in U87MG and U373MG tumour-bearing mice. Eventually, the radiolabelled APTEDB was used for tumour imaging using planar SPECT. Results: Radiolabelling was achieved with high purity (up to 97%) and accompanied by high solution (over 90% after overnight) and serum (80% after 2 hours) stability. The obtained cellular-specific binding ratio was greater than nine-fold. In-vivo experiments showed rapid blood clearance with mainly renal excretion and tumour uptake specificity (0.48±0.03% ID/g after 1h). The results of the imaging also confirmed considerable tumour uptake for EDB-positive cell line compared with the EDB-negative one. Conclusion: Aptides are considered to be a potent candidate for biopharmaceutical applications. They can be modified with imaging or therapeutic agents. This report shows the capability of 99mTc-HYNIC-APTEDB for human EDB-expressing tumours detection.

scholarly journals Proton Transport Mechanism and Pathways in the Superprotonic Phase of M3H(AO4)2 Solid Acids from Ab Initio Molecular Dynamics Simulations

2021 ◽  
Author(s):  
Boris Merinov ◽  
Sergey Morozov
Keyword(s):  
Molecular Dynamics ◽  
Ab Initio ◽  
Proton Transport ◽  
Transport Mechanism ◽  
Solid Acids ◽  
Ab Initio Molecular Dynamics ◽  
Theoretical Studies ◽  
Dynamics Simulations ◽  
Experimental And Theoretical Studies ◽  
Superprotonic Phase

The proton transport mechanism in superprotonic phases of solid acids is a subject of experimental and theoretical studies for a number of years. Despite this, details of the mechanism still...

scholarly journals The transmembrane domain and the proton channel in proton-pumping transhydrogenases

2000 ◽  
Vol 1459 (2-3) ◽  
pp. 284-290 ◽  
Author(s):  
Tania Bizouarn ◽  
Johan Meuller ◽  
Magnus Axelsson ◽  
Jan Rydström
Keyword(s):  
Transmembrane Domain ◽  
Proton Pumping ◽  
Proton Channel

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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