Long-Range Electrostatic Interactions Significantly Modulate the Affinity of Dynein for Microtubules

The dynein family of microtubule minus-end directed motor proteins drives diverse functions in eukaryotic cells, including cell division, intracellular transport, and flagellar beating. Motor protein processivity, which characterizes how far a motor walks before detaching from its filament, depends on the interaction between its microtubule-binding domain (MTBD) and the microtubule. Dynein's MTBD switches between high- and low-binding affinity states as it steps. Significant structural and functional data show that specific salt bridges within the MTBD and between the MTBD and the microtubule govern these affinity state shifts. However, recent computational work suggests that non-specific, long-range electrostatic interactions between the MTBD and the microtubule may also play a significant role in the processivity of dynein. To investigate this hypothesis, we mutated negatively charged amino acids remote from the dynein MTBD-microtubule-binding interface to neutral residues and measured the binding affinity using microscale thermophoresis and optical tweezers. We found a significant increase in the binding affinity of the mutated MTBDs for microtubules. Furthermore, we found that charge screening by free ions in solution differentially affected the binding and unbinding rates of MTBDs to microtubules. Together, these results demonstrate a significant role for long-range electrostatic interactions in regulating dynein-microtubule affinity. Moreover, these results provide insight into the principles that potentially underlie the biophysical differences between molecular motors with various processivities and protein-protein interactions more generally.

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Mechanistic basis of propofol-induced disruption of kinesin processivity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023659118 ◽

2021 ◽

Vol 118 (5) ◽

pp. e2023659118

Author(s):

Mandira Dutta ◽

Susan P. Gilbert ◽

José N. Onuchic ◽

Biman Jana

Keyword(s):

Binding Affinity ◽

Molecular Motors ◽

Free Energy Calculations ◽

General Anesthetic ◽

Forward Movement ◽

Microtubule Binding ◽

Linker Region ◽

Binding Interface ◽

Binding Surface ◽

Mechanistic Basis

Propofol is a widely used general anesthetic to induce and maintain anesthesia, and its effects are thought to occur through impact on the ligand-gated channels including the GABAA receptor. Propofol also interacts with a large number of proteins including molecular motors and inhibits kinesin processivity, resulting in significant decrease in the run length for conventional kinesin-1 and kinesin-2. However, the molecular mechanism by which propofol achieves this outcome is not known. The structural transition in the kinesin neck-linker region is crucial for its processivity. In this study, we analyzed the effect of propofol and its fluorine derivative (fropofol) on the transition in the neck-linker region of kinesin. Propofol binds at two crucial surfaces in the leading head: one at the microtubule-binding interface and the other in the neck-linker region. We observed in both the cases the order–disorder transition of the neck-linker was disrupted and kinesin lost its signal for forward movement. In contrast, there was not an effect on the neck-linker transition with propofol binding at the trailing head. Free-energy calculations show that propofol at the microtubule-binding surface significantly reduces the microtubule-binding affinity of the kinesin head. While propofol makes pi–pi stacking and H-bond interactions with the propofol binding cavity, fropofol is unable to make a suitable interaction at this binding surface. Therefore, the binding affinity of fropofol is much lower compared to propofol. Hence, this study provides a mechanism by which propofol disrupts kinesin processivity and identifies transitions in the ATPase stepping cycle likely affected.

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E484K mutation in SARS-CoV-2 RBD enhances binding affinity with hACE2 but reduces interactions with neutralizing antibodies and nanobodies: Binding free energy calculation studies

10.1101/2021.02.17.431566 ◽

2021 ◽

Author(s):

Wei Bu Wang ◽

Yu Liang ◽

Yu Qin Jin ◽

Jing Zhang ◽

Ji Guo Su ◽

...

Keyword(s):

Hydrogen Bonds ◽

Binding Affinity ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Electrostatic Interactions ◽

Tight Binding ◽

Energy Calculation ◽

Binding Affinities ◽

Loop Region ◽

Binding Interface

AbstractThe pandemic of the COVID-19 disease caused by SARS-CoV-2 has led to more than 100 million infections and over 2 million deaths worldwide. The progress in the developments of effective vaccines and neutralizing antibody therapeutics brings hopes to eliminate the threat of COVID-19. However, SARS-CoV-2 continues to mutate, and several new variants have been emerged. Among the various naturally-occurring mutations, the E484K mutation shared by both the 501Y.V2 and 501Y.V3 variants attracted serious concerns, which may potentially enhance the receptor binding affinity and reduce the immune response. In the present study, the molecular mechanism behind the impacts of E484K mutation on the binding affinity of the receptor-binding domain (RBD) with the receptor human angiotensin-converting enzyme 2 (hACE2) was investigated by using the molecular dynamics (MD) simulations combined with the molecular mechanics-generalized Born surface area (MMGBSA) method. Our results indicate that the E484K mutation results in more favorable electrostatic interactions compensating the burial of the charged and polar groups upon the binding of RBD with hACE2, which significantly improves the RBD-hACE2 binding affinity. Besides that, the E484K mutation also causes the conformational rearrangements of the loop region containing the mutant residue, which leads to more tight binding interface of RBD with hACE2 and formation of some new hydrogen bonds. The more tight binding interface and the new hydrogen bonds formation also contribute to the improved binding affinity of RBD to the receptor hACE2. In addition, six neutralizing antibodies and nanobodies complexed with RBD were selected to explore the effects of E484K mutation on the recognition of these antibodies to RBD. The simulation results show that the E484K mutation significantly reduces the binding affinities to RBD for most of the studied neutralizing antibodies, and the decrease in the binding affinities is mainly owing to the unfavorable electrostatic interactions caused by the mutation. Our studies revealed that the E484K mutation may improve the binding affinity between RBD and the receptor hACE2, implying more transmissibility of the E484K-containing variants, and weaken the binding affinities between RBD and the studied neutralizing antibodies, indicating reduced effectiveness of these antibodies. Our results provide valuable information for the effective vaccine development and antibody drugs design.

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Single Molecule Study of Long-Range Electrostatic Binding Affinity of Cytoplasmic Dynein's Microtubule Binding Domain

Biophysical Journal ◽

10.1016/j.bpj.2017.11.2800 ◽

2018 ◽

Vol 114 (3) ◽

pp. 512a

Author(s):

Subash C. Godar ◽

Hailey Lovelace ◽

Jared Eller ◽

Mattheu Spencer ◽

Lin Li ◽

...

Keyword(s):

Binding Affinity ◽

Long Range ◽

Single Molecule ◽

Binding Domain ◽

Microtubule Binding ◽

Microtubule Binding Domain ◽

Electrostatic Binding

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The molecular basis for SARS-CoV-2 binding to dog ACE2

Nature Communications ◽

10.1038/s41467-021-24326-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zengyuan Zhang ◽

Yanfang Zhang ◽

Kefang Liu ◽

Yan Li ◽

Qiong Lu ◽

...

Keyword(s):

Binding Affinity ◽

Molecular Basis ◽

Domestic Animals ◽

Receptor Binding Domain ◽

Salt Bridges ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Binding Interface ◽

Protein Receptor ◽

Contact Residues

AbstractSARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.

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The molecular basis for SARS-CoV-2 recognized by dog ACE2

10.21203/rs.3.rs-263301/v1 ◽

2021 ◽

Author(s):

Zengyuan Zhang ◽

Yanfang Zhang ◽

Kefang Liu ◽

Yan Li ◽

Qiong Lu ◽

...

Keyword(s):

Binding Affinity ◽

Molecular Basis ◽

Domestic Animals ◽

Salt Bridges ◽

Binding Region ◽

Angiotensin Converting Enzyme 2 ◽

Binding Interface ◽

Protein Receptor ◽

Contact Residues ◽

Receptor Binding Region

Abstract SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin converting enzyme 2 (dACE2) can bind to SARS-CoV-2 spike (S) protein receptor binding region (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. we solved the crystal structure of RBD in complex with dACE2 and found that the total numbers of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that to hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2, and need intense monitoring and controlling.

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Probing Anticoagulant Fondaparinux for Interference with Prothrombotic B2GPI/Anti-B2GPI Antibody Complexes

Blood ◽

10.1182/blood.v118.21.1209.1209 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1209-1209

Author(s):

Alexey Kolyada ◽

Alfredo De Biasio ◽

Natalia Beglova

Keyword(s):

Binding Site ◽

Binding Affinity ◽

Electrostatic Interactions ◽

Conflicts Of Interest ◽

Annexin V ◽

Site Directed Mutagenesis ◽

Lipoprotein Receptors ◽

Protective Function ◽

Anionic Phospholipids ◽

Binding Interface

Abstract Abstract 1209 Background: The presence of autoimmune antibodies directed to beta2-glycoprotein-I (B2GPI) often leads to thrombosis in antiphospholipid syndrome (APS). Heparin, low molecular weight heparin (LMWH) and fondaparinux are commonly used for prophylaxis and treatment of thromboses in APS. These drugs bind and activate antithrombin III to inactivate blood clotting proteases. Fondaparinux is a synthetic pentasaccharide matching a specific sequence within heparin interacting with antithrombin. Aim: We investigated if fondaparinux can bind B2GPI and ameliorate prothrombotic properties of B2GPI/anti-B2GPI antibody complexes. Results: We found that fondaparinux interacts with B2GPI and that the binding is dominated by electrostatic interactions. We measured the binding affinity by monitoring changes in the intrinsic fluorescence of domain V of B2GPI (B2GPI-DV) upon titration with fondaparinux. In the presence of 100 mM NaCl, the binding affinity was about 1.5 uM and stoichiometry of the binding is 1:1. Using solution NMR spectroscopy, we determined that the binding interface of the complex is centered on Lys251 of B2GPI-DV. This observation was confirmed by site-directed mutagenesis. The Lys251/Asp mutant fails to bind B2GPI-DV. Interestingly, the binding site for fondaparinux on B2GPI does not overlap with the major binding site for heparin. Cellular activation by the binding of B2GPI/anti-B2GPI antibody complexes with cell-surface receptors (among them ApoER2, a lipoprotein receptor from the LDLR family) and interference with the protective function of annexin V on anionic phospholipids expressed on the surfaces of activated cells are two potential prothrombotic mechanisms of B2GPI/antibody complexes. We found that fondaparinux does not prevent the association of the ligand-binding modules from ApoER2 with B2GPI-DV. Therefore, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes with lipoprotein receptors. Neither fondaparinux, nor heparin and LMWH were effective in inhibiting the binding of B2GPI/anti-B2GPI antibody complexes to cardiolipin-coated plates suggesting that these drugs do not prevent the destructive effect of B2GPI/antibody complexes on antithrombotic function of annexin V. Conclusions: At therapeutic concentrations, fondaparinux forms only small number of complexes with B2GPI, given that the binding affinity of the complex is in a micromolar range. When bound to B2GPI, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes to lipoprotein receptors and anionic phospholipids. Disclosures: No relevant conflicts of interest to declare.

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Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes

Molecular Biology of the Cell ◽

10.1091/mbc.e17-05-0324 ◽

2017 ◽

Vol 28 (23) ◽

pp. 3315-3322 ◽

Cited By ~ 5

Author(s):

Serapion Pyrpassopoulos ◽

Henry Shuman ◽

E. Michael Ostap

Keyword(s):

Optical Tweezers ◽

Lipid Membranes ◽

Electrostatic Interactions ◽

Intracellular Transport ◽

Adhesion Force ◽

Pleckstrin Homology Domain ◽

Adhesion Forces ◽

Px Domain ◽

C Terminus ◽

Early Endosomes

KIF16B is a highly processive kinesin-3 family member that participates in the trafficking and tubulation of early endosomes along microtubules. KIF16B attaches to lipid cargoes via a PX motif at its C-terminus, which has nanomolar affinity for bilayers containing phosphatidylinositol-3-phosphate (PI[3]P). As the PX domain has been proposed to be a primary mechanical anchor for the KIF16B-cargo attachment, we measured the adhesion forces and detachment kinetics of the PX domain as it interacts with membranes containing 2% PI(3)P and 98% phosphatidylcholine. Using optical tweezers, we found that the adhesion strength of a single PX domain ranged between 19 and 54 pN at loading rates between 80 and 1500 pN/s. These forces are substantially larger than the interaction of the adhesion of a pleckstrin homology domain with phosphatidylinositol 4,5-bisphosphate. This increased adhesion is the result of the membrane insertion of hydrophobic residues adjacent to the PI(3)P binding site, in addition to electrostatic interactions with PI(3)P. Attachment lifetimes under load decrease monotonically with force, indicating slip-bond behavior. However, the lifetime of membrane attachment under load appears to be well matched to the duration of processive motility of the KIF16B motor, indicating the PX domain is a suitable mechanical anchor for intracellular transport.

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Improved binding affinity of the Omicron's spike protein with hACE2 receptor is the key factor behind its increased virulence

10.1101/2021.12.28.474338 ◽

2021 ◽

Author(s):

Rajender Kumar ◽

Murugan Natarajan Arul ◽

Vaibhav Srivastava

Keyword(s):

Free Energy ◽

Hydrogen Bonding ◽

Binding Affinity ◽

Electrostatic Interactions ◽

Binding Free Energy ◽

Free Energy Calculations ◽

Spike Protein ◽

Strong Interactions ◽

Salt Bridges ◽

New Variant

The new variant of SARS-CoV-2, Omicron, has been quickly spreading in many countries worldwide. Compared to the original virus, Omicron is characterized by several mutations in its genomic region, including spike protein's receptor-binding domain (RBD). We have computationally investigated the interaction between RBD of both wild-type and omicron variants with hACE2 receptor using molecular dynamics and MM-GBSA based binding free energy calculations. The mode of the interaction between Omicron's RBD to the human ACE2 (hACE2) receptor is similar to the original SARS-CoV-2 RBD except for a few key differences. The bind-ing free energy difference shows that the spike protein of Omicron has increased binding affinity for the hACE-2 receptor. The mutated residues in the RBD showed strong interactions with a few amino acid residues of the hACE2. More specifically, strong electrostatic interactions (salt bridges) and hydrogen bonding were observed between R493 and R498 residues of the Omicron RBD with D30/E35 and D38 residues of the hACE2, respectively. Other mutated amino acids in the Omicron RBD, e.g. S496 and H505, also exhibited hydrogen bonding with the hACE2 receptor. The pi-stacking interaction was also observed between tyrosine residues (RBD-Tyr501: hACE2-Tyr41) in the complex, which contributes majorly to binding free energies suggesting this as one of the key interactions stabilizing the complex formation. The structural insights of RBD:hACE2 complex, their binding mode information and residue wise contributions to binding free energy provide insight on the increased transmissibility of Omicron and pave the way to design and optimize novel antiviral agents.

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Polarizable embedding for simulating redox potentials of biomolecules

Physical Chemistry Chemical Physics ◽

10.1039/c9cp01533g ◽

2019 ◽

Vol 21 (22) ◽

pp. 11642-11650 ◽

Cited By ~ 4

Author(s):

Ruslan N. Tazhigulov ◽

Pradeep Kumar Gurunathan ◽

Yongbin Kim ◽

Lyudmila V. Slipchenko ◽

Ksenia B. Bravaya

Keyword(s):

Long Range ◽

Electrostatic Interactions ◽

Theoretical Description ◽

Biological Macromolecules ◽

Classical Approach ◽

Redox Potentials ◽

Computational Protocol ◽

Polarizable Embedding

We present a computational protocol exploiting polarizable embedding hybrid quantum-classical approach and resulting in accurate estimates of redox potentials of biological macromolecules. A special attention is paid to fundamental aspects of the theoretical description such as the effects of environment polarization and of the long-range electrostatic interactions on the computed energetic parameters.

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Molecular Motors: Force and Movement Generated by Single Myosin II Molecules

Physiology ◽

10.1152/nips.01389.2002 ◽

2002 ◽

Vol 17 (5) ◽

pp. 213-218 ◽

Cited By ~ 3

Author(s):

Caspar Rüegg ◽

Claudia Veigel ◽

Justin E. Molloy ◽

Stephan Schmitz ◽

John C. Sparrow ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Molecular Motors ◽

Molecular Motor ◽

Myosin Ii ◽

Force Production ◽

Myosin Isoforms ◽

Single Molecule Level ◽

Recent Developments ◽

Muscle Myosin

Muscle myosin II is an ATP-driven, actin-based molecular motor. Recent developments in optical tweezers technology have made it possible to study movement and force production on the single-molecule level and to find out how different myosin isoforms may have adapted to their specific physiological roles.

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