scholarly journals Reactivities of the Front Pocket N-Cap Cysteines in Human Kinases

2021 ◽  
Author(s):  
Ruibin Liu ◽  
Shaoqi Zhan ◽  
Ye Che ◽  
Jana Shen
Keyword(s):  
Electrostatic Interactions ◽  
Rational Design ◽  
Covalent Modification ◽  
Constant Ph ◽  
Covalent Inhibitors ◽  
Thiol Form ◽  
Reactive N ◽  
Systematic Understanding ◽  
Constant Ph Molecular Dynamics ◽  
Experimental Structure

Discovery of targeted covalent inhibitors directed at nucleophilic cysteines is attracting enormous interest. The front pocket (FP) N-cap cysteine has been the most popular site of covalent modification in kinases. Curiously, a long-standing hypothesis associates the N-cap position with cysteine hyper-reactivity; however, traditional computational methods suggest that the FP N-cap cysteines in all human kinases are predominantly unreactive at physiological pH. Here we applied a newly developed GPU-accelerated continuous constant pH molecular dynamics (CpHMD) tool to test the N-cap hypothesis and elucidate the cysteine reactivities. Simulations showed that the N-cap cysteines in BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 sample the reactive thiolate form to varying degrees at physiological pH; however, those in BLK and EGFR/ERBB2/ERBB4 which contain an Asp at the N-cap+3 position adopt the unreactive thiol form. The latter argues in favor of the base-assisted thiol-Michael addition mechanisms as suggested by the quantum mechanical calculations and experimental structure-function studies of EGFR inhibitors. Analysis revealed that the reactive N-cap cysteines are stabilized by hydrogen bond as well as electrostatic interactions, and in their absence a N-cap cysteine is unreactive due to desolvation. To test a corollary of the N-cap hypothesis, we also examined the reactivities of the FP N-cap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Additionally, our simulations predicted the reactive cysteine and lysine locations in all 15 kinases. Our findings offer a systematic understanding of cysteine reactivities in kinases and demonstrate the predictive power and physical insights CpHMD can provide to guide the rational design of targeted covalent inhibitors.

scholarly journals Charge Interactions in a Highly Charge-depleted Protein

2020 ◽  
Author(s):  
Stefan Hervø-Hansen ◽  
Casper Højgaard ◽  
Kristoffer Enøe Johansson ◽  
Yong Wang ◽  
Khadija Wahni ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Nmr Spectroscopy ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Protein Electrostatics ◽  
Surface Charges ◽  
Constant Ph ◽  
Charged Residues ◽  
Dynamics Simulations ◽  
Constant Ph Molecular Dynamics

ABSTRACTInteractions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.SignificanceElectrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.

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

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. <p><br></p>

scholarly journals Recent development and application of constant pH molecular dynamics

2014 ◽  
Vol 40 (10-11) ◽  
pp. 830-838 ◽  
Author(s):  
Wei Chen ◽  
Brian H. Morrow ◽  
Chuanyin Shi ◽  
Jana K. Shen
Keyword(s):  
Molecular Dynamics ◽  
Constant Ph ◽  
Constant Ph Molecular Dynamics

scholarly journals On the Use of the Discrete Constant pH Molecular Dynamics to Describe the Conformational Space of Peptides

Polymers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 99
Author(s):  
Cristian Privat ◽  
Sergio Madurga ◽  
Francesc Mas ◽  
Jaime Rubio-Martínez
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Md Simulations ◽  
Point Of View ◽  
Conformational Space ◽  
Conformational Sampling ◽  
Protonation State ◽  
Constant Ph ◽  
Biochemical Systems ◽  
Constant Ph Molecular Dynamics

Solvent pH is an important property that defines the protonation state of the amino acids and, therefore, modulates the interactions and the conformational space of the biochemical systems. Generally, this thermodynamic variable is poorly considered in Molecular Dynamics (MD) simulations. Fortunately, this lack has been overcome by means of the Constant pH Molecular Dynamics (CPHMD) methods in the recent decades. Several studies have reported promising results from these approaches that include pH in simulations but focus on the prediction of the effective pKa of the amino acids. In this work, we want to shed some light on the CPHMD method and its implementation in the AMBER suitcase from a conformational point of view. To achieve this goal, we performed CPHMD and conventional MD (CMD) simulations of six protonatable amino acids in a blocked tripeptide structure to compare the conformational sampling and energy distributions of both methods. The results reveal strengths and weaknesses of the CPHMD method in the implementation of AMBER18 version. The change of the protonation state according to the chemical environment is presumably an improvement in the accuracy of the simulations. However, the simulations of the deprotonated forms are not consistent, which is related to an inaccurate assignment of the partial charges of the backbone atoms in the CPHMD residues. Therefore, we recommend the CPHMD methods of AMBER program but pointing out the need to compare structural properties with experimental data to bring reliability to the conformational sampling of the simulations.

scholarly journals Anti-TNF Alpha Antibody Humira with pH-dependent Binding Characteristics: A constant-pH Molecular Dynamics, Gaussian Accelerated Molecular Dynamics, and In Vitro Study

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 334
Author(s):  
Shih-Ting Hong ◽  
Yu-Cheng Su ◽  
Yu-Jen Wang ◽  
Tian-Lu Cheng ◽  
Yeng-Tseng Wang
Keyword(s):  
Molecular Dynamics ◽  
Tnf Alpha ◽  
Complex Structures ◽  
Binding Characteristics ◽  
Accelerated Molecular Dynamics ◽  
Constant Ph ◽  
Ph Dependent ◽  
Constant Ph Molecular Dynamics ◽  
Antibody Drug

Humira is a monoclonal antibody that binds to TNF alpha, inactivates TNF alpha receptors, and inhibits inflammation. Neonatal Fc receptors can mediate the transcytosis of Humira–TNF alpha complex structures and process them toward degradation pathways, which reduces the therapeutic effect of Humira. Allowing the Humira–TNF alpha complex structures to dissociate to Humira and soluble TNF alpha in the early endosome to enable Humira recycling is crucial. We used the cytoplasmic pH (7.4), the early endosomal pH (6.0), and pKa of histidine side chains (6.0–6.4) to mutate the residues of complementarity-determining regions with histidine. Our engineered Humira (W1-Humira) can bind to TNF alpha in plasma at neutral pH and dissociate from the TNF alpha in the endosome at acidic pH. We used the constant-pH molecular dynamics, Gaussian accelerated molecular dynamics, two-dimensional potential mean force profiles, and in vitro methods to investigate the characteristics of W1-Humira. Our results revealed that the proposed Humira can bind TNF alpha with pH-dependent affinity in vitro. The W1-Humira was weaker than wild-type Humira at neutral pH in vitro, and our prediction results were close to the in vitro results. Furthermore, our approach displayed a high accuracy in antibody pH-dependent binding characteristics prediction, which may facilitate antibody drug design. Advancements in computational methods and computing power may further aid in addressing the challenges in antibody drug design.

scholarly journals Constant pH molecular dynamics of proteins in explicit solvent with proton tautomerism

2014 ◽  
Vol 82 (7) ◽  
pp. 1319-1331 ◽  
Author(s):  
Garrett B. Goh ◽  
Benjamin S. Hulbert ◽  
Huiqing Zhou ◽  
Charles L. Brooks
Keyword(s):  
Molecular Dynamics ◽  
Constant Ph ◽  
Explicit Solvent ◽  
Constant Ph Molecular Dynamics

Rational Design of Organic Electro-Optic Materials

2001 ◽  
Vol 708 ◽  
Author(s):  
Alex Jen ◽  
Robert Neilsen ◽  
Bruce Robinson ◽  
William H. Steier ◽  
Larry Dalton
Keyword(s):  
Wavelength Division Multiplexing ◽  
Material Properties ◽  
Electrostatic Interactions ◽  
Rational Design ◽  
Elevated Temperatures ◽  
Optical Loss ◽  
Quality Factors ◽  
Electro Optic ◽  
Lattice Hardening

ABSTRACTA number of material properties must be optimized before organic electro-optic materials can be used for practical device applications. These include electro-optic activity, optical transparency, and stability including both thermal and photochemical stability. Exploiting an improved understanding of the structure/function relationships, we have recently prepared materials exhibiting electro-optic coefficients of greater than 50 pm/V and optical loss values of less than 0.7 dB/cm at the telecommunication wavelengths of 1.3 and 1.55 microns. When oxygen is excluded to a reasonable extent, long-term photostability to optical power levels of 20 mW has been observed. Photostability is further improved by addition of scavengers and by lattice hardening. Long-term (greater than 1000 hours) thermal stability of poling-induced electro-optic activity is also observed at elevated temperatures (greater than 80°C) when appropriate lattice hardening is used. The successful improvement of organic electro-optic materials rests upon (1) attention to the design of chromophore structure including design to inhibit unwanted intermolecular electrostatic interactions and to improve chromophore instability and (2) attention to processing conditions including those involved in spin casting, electric field poling, and lattice hardening. A particularly attractive new direction has been the exploitation of dendrimer structures and particularly of multi-chromophore containing dendrimer structures. This approach has permitted the simultaneous improvement of all material properties. Development of new materials has facilitated the fabrication of a number of prototype devices and most recently has permitted investigation of the incorporation of electro-optic materials into photonic bandgap and microresonator structures. The latter are relevant to active wavelength division multiplexing (WDM). Significant quality factors (greater than 10,000) have been realized for such devices permitting wavelength discrimination at telecommunication wavelengths of 0.01 nm.

scholarly journals Mechanism of Covalent Binding of Ibrutinib to Bruton’s Tyrosine Kinase revealed by QM/MM Calculations

2020 ◽  
Author(s):  
Angus Voice ◽  
Gary Tresadern ◽  
Rebecca Twidale ◽  
Herman Van Vlijmen ◽  
Adrian Mulholland
Keyword(s):  
Tyrosine Kinase ◽  
Covalent Binding ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Mechanistic Study ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Covalent Inhibitors ◽  
Covalent Bond Formation

<p>Ibrutinib is the first covalent inhibitor of Bruton’s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition is crucial for the design of safer and more selective covalent inhibitors that target BTK. There are questions surrounding the precise mechanism of covalent bond formation in BTK as there is no appropriate active site residue that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. To address this, we have investigated several mechanistic pathways of covalent modification of C481 in BTK by ibrutinib using QM/MM reaction simulations. The lowest energy pathway we identified involves a direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (DG<sup>‡</sup>=10.5 kcal mol<sup>-1</sup>) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and related proteins. </p>

scholarly journals Adsorption of Lysozyme Into a Charged Confining Pore

2021 ◽  
Author(s):  
Sidney Carvalho ◽  
Ralf Metzler ◽  
Andrey Cherstvy ◽  
Daniel Caetano
Keyword(s):  
Electrostatic Interactions ◽  
Active Sites ◽  
Coarse Grained ◽  
Pore Surface ◽  
Solution Ph ◽  
Coarse Grained Model ◽  
Egg White Lysozyme ◽  
Constant Ph ◽  
Pka Shift ◽  
Effects Of Ph

Several applications arise from the confinement of proteins on surfaces since their stability and biological activity are enhanced. It is also known that the way a protein adsorbs on the surface is important for its biological function since its active sites should not be obstructed. In this study, the adsorption properties of hen egg-white Lysozyme, HEWL, into a negatively charged silica pore is examined employing a coarse-grained model and constant-pH Monte Carlo simulations. The role of electrostatic interactions is taken into account when including the Debye-Hueckel potentials into the Ca structure-based model. We evaluate the effects of pH, salt concentration, and pore radius on the protein preferential orientation and spatial distribution of its residues regarding the pore surface. By mapping the residues that stay closer to the pore surface, we find the increase of pH leads to orientational changes of the adsorbed protein when the solution pH gets closer to the HEWL isoelectric point. At these conditions, the pKa shift of these important residues caused by the adsorption into the charged confining surface results in a HEWL charge distribution that stabilizes the adsorption in the observed protein orientation. We compare our observations to the results of pKa shift for HEWL available in the literature and to some experimental data.

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