Publications by authors named "Wonpil Im"

192 Publications

Dynamic Interactions of Fully Glycosylated SARS-CoV-2 Spike Protein with Various Antibodies.

bioRxiv 2021 May 11. Epub 2021 May 11.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.
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http://dx.doi.org/10.1101/2021.05.10.443519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132224PMC
May 2021

Additive CHARMM36 Force Field for Nonstandard Amino Acids.

J Chem Theory Comput 2021 Jun 19;17(6):3554-3570. Epub 2021 May 19.

Laboratoire d'Optique et Biosciences (CNRS UMR7645, INSERM U1182), Ecole Polytechnique, Institut Polytechnique de Paris, F-91128 Palaiseau, France.

Nonstandard amino acids are both abundant in nature, where they play a key role in various cellular processes, and can be synthesized in laboratories, for example, for the manufacture of a range of pharmaceutical agents. In this work, we have extended the additive all-atom CHARMM36 and CHARMM General force field (CGenFF) to a large set of 333 nonstandard amino acids. These include both amino acids with nonstandard side chains, such as post-translationally modified and artificial amino acids, as well as amino acids with modified backbone groups, such as chromophores composed of several amino acids. Model compounds representative of the nonstandard amino acids were parametrized for protonation states that are likely at the physiological pH of 7 and, for some more common residues, in both d- and l-stereoisomers. Considering all protonation, tautomeric, and stereoisomeric forms, a total of 406 nonstandard amino acids were parametrized. Emphasis was placed on the quality of both intra- and intermolecular parameters. Partial charges were derived using quantum mechanical (QM) data on model compound dipole moments, electrostatic potentials, and interactions with water. Optimization of all intramolecular parameters, including torsion angle parameters, was performed against information from QM adiabatic potential energy surface (PES) scans. Special emphasis was put on the quality of terms corresponding to PES around rotatable dihedral angles. Validation of the force field was based on molecular dynamics simulations of 20 protein complexes containing different nonstandard amino acids. Overall, the presented parameters will allow for computational studies of a wide range of proteins containing nonstandard amino acids, including natural and artificial residues.
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http://dx.doi.org/10.1021/acs.jctc.1c00254DOI Listing
June 2021

Conformationally flexible core-bearing detergents with a hydrophobic or hydrophilic pendant: Effect of pendant polarity on detergent conformation and membrane protein stability.

Acta Biomater 2021 Apr 29. Epub 2021 Apr 29.

Department of Bionano Engineering, Center for Bionano Intelligence Education and Research, Hanyang University, Ansan 155-88, South Korea. Electronic address:

Membrane protein structures provide atomic level insight into essential biochemical processes and facilitate protein structure-based drug design. However, the inherent instability of these bio-macromolecules outside lipid bilayers hampers their structural and functional study. Detergent micelles can be used to solubilize and stabilize these membrane-inserted proteins in aqueous solution, thereby enabling their downstream characterizations. Membrane proteins encapsulated in detergent micelles tend to denature and aggregate over time, highlighting the need for development of new amphiphiles effective for protein solubility and stability. In this work, we present newly-designed maltoside detergents containing a pendant chain attached to a glycerol-decorated tris(hydroxymethyl)methane (THM) core, designated GTMs. One set of the GTMs has a hydrophobic pendant (ethyl chain; E-GTMs), and the other set has a hydrophilic pendant (methoxyethoxylmethyl chain; M-GTMs) placed in the hydrophobic-hydrophilic interfaces. The two sets of GTMs displayed profoundly different behaviors in terms of detergent self-assembly and protein stabilization efficacy. These behaviors mainly arise from the polarity difference between two pendants (ethyl and methoxyethoxylmethyl chains) that results in a large variation in detergent conformation between these sets of GTMs in aqueous media. The resulting high hydrophobic density in the detergent micelle interior is likely responsible for enhanced efficacy of the M-GTMs for protein stabilization compared to the E-GTMs and a gold standard detergent DDM. A representative GTM, M-GTM-O12, was more effective for protein stability than some recently developed detergents including LMNG. This is the first case study investigating the effect of pendant polarity on detergent geometry correlated with detergent efficacy for protein stabilization. STATEMENT OF SIGNIFICANCE: This study introduces new amphiphiles for use as biochemical tools in membrane protein studies. We identified a few hydrophilic pendant-bearing amphiphiles such as M-GTM-O11 and M-GTM-O12 that show remarkable efficacy for membrane protein solubilization and stabilization compared to a gold standard DDM, the hydrophobic counterparts (E-GTMs) and a significantly optimized detergent LMNG. In addition, detergent results obtained in the current study reveals the effect of detergent pendant polarity on protein solubility and stability. Thus, the current study represents both significant chemical and conceptual advance. The detergent tools and design principle introduced here advance protein science and facilitate structure-based drug design and development.
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http://dx.doi.org/10.1016/j.actbio.2021.04.043DOI Listing
April 2021

Location and Conformational Ensemble of Menaquinone and Menaquinol, and Protein-Lipid Modulations in Archaeal Membranes.

J Phys Chem B 2021 05 29;125(18):4714-4725. Epub 2021 Apr 29.

Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Halobacteria, a type of archaea in high salt environments, have phytanyl ether phospholipid membranes containing up to 50% menaquinone. It is not understood why a high concentration of menaquinone is required and how it influences membrane properties. In this study, menaquinone-8 headgroup and torsion parameters of isoprenoid tail are optimized in the CHARMM36 force field. Molecular dynamics simulations of archaeal bilayers containing 0 to 50% menaquinone characterize the distribution of menaquinone-8 and menaquinol-8, as well as their effects on mechanical properties and permeability. Menaquinone-8 segregates to the membrane midplane above concentrations of 10%, favoring an extended conformation in a fluid state. Menaquinone-8 increases the bilayer thickness but does not significantly alter the area compressibility modulus and lipid chain ordering. Counterintuitively, menaquinone-8 increases water permeability because it lowers the free energy barrier in the midplane. The thickness increase due to menaquinone-8 may help halobacteria ameliorate hyper-osmotic pressure by increasing the membrane bending constant. Simulations of the archaeal membranes with archaerhodopsin-3 show that the local membrane surface adjusts to accommodate the thick membranes. Overall, this study delineates the biophysical landscape of 50% menaquinone in the archaeal bilayer, demonstrates the mixing of menaquinone and menaquinol, and provides atomistic details about menaquinone configurations.
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http://dx.doi.org/10.1021/acs.jpcb.1c01930DOI Listing
May 2021

Site-Specific Lipidation Enhances IFITM3 Membrane Interactions and Antiviral Activity.

ACS Chem Biol 2021 05 22;16(5):844-856. Epub 2021 Apr 22.

Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States.

Interferon-induced transmembrane proteins (IFITMs) are -palmitoylated proteins in vertebrates that restrict a diverse range of viruses. -palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how -palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific -palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific -palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.
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http://dx.doi.org/10.1021/acschembio.1c00013DOI Listing
May 2021

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

Structure 2021 Apr 13. Epub 2021 Apr 13.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.
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http://dx.doi.org/10.1016/j.str.2021.03.018DOI Listing
April 2021

Structural Insight into Phospholipid Transport by the MlaFEBD Complex from P. aeruginosa.

J Mol Biol 2021 Jun 11;433(13):166986. Epub 2021 May 11.

National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing100101, China. Electronic address:

The outer membrane (OM) of Gram-negative bacteria, which consists of lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet, plays a key role in antibiotic resistance and pathogen virulence. The maintenance of lipid asymmetry (Mla) pathway is known to be involved in PL transport and contributes to the lipid homeostasis of the OM, yet the underlying molecular mechanism and the directionality of PL transport in this pathway remain elusive. Here, we reported the cryo-EM structures of the ATP-binding cassette (ABC) transporter MlaFEBD from P. areuginosa, the core complex in the Mla pathway, in nucleotide-free (apo)-, ADP (ATP + vanadate)- and ATP (AMPPNP)-bound states as well as the structures of MlaFEB from E. coli in apo- and AMPPNP-bound states at a resolution range of 3.4-3.9 Å. The structures show that the MlaFEBD complex contains a total of twelve protein molecules with a stoichiometry of MlaFEBD, and binds a plethora of PLs at different locations. In contrast to canonical ABC transporters, nucleotide binding fails to trigger significant conformational changes of both MlaFEBD and MlaFEB in the nucleotide-binding and transmembrane domains of the ABC transporter, correlated with their low ATPase activities exhibited in both detergent micelles and lipid nanodiscs. Intriguingly, PLs or detergents appeared to relocate to the membrane-proximal end from the distal end of the hydrophobic tunnel formed by the MlaD hexamer in MlaFEBD upon addition of ATP, indicating that retrograde PL transport might occur in the tunnel in an ATP-dependent manner. Site-specific photocrosslinking experiment confirms that the substrate-binding pocket in the dimeric MlaE and the MlaD hexamer are able to bind PLs in vitro, in line with the notion that MlaFEBD complex functions as a PL transporter.
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http://dx.doi.org/10.1016/j.jmb.2021.166986DOI Listing
June 2021

CHARMM-GUI Polymer Builder for Modeling and Simulation of Synthetic Polymers.

J Chem Theory Comput 2021 Apr 2;17(4):2431-2443. Epub 2021 Apr 2.

Departments of Biological Sciences, Chemistry, Bioengineering, and Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Molecular modeling and simulations are invaluable tools for polymer science and engineering, which predict physicochemical properties of polymers and provide molecular-level insight into the underlying mechanisms. However, building realistic polymer systems is challenging and requires considerable experience because of great variations in structures as well as length and time scales. This work describes in CHARMM-GUI (http://www.charmm-gui.org/input/polymer), a web-based infrastructure that provides a generalized and automated process to build a relaxed polymer system. not only provides versatile modeling methods to build complex polymer structures, but also generates realistic polymer melt and solution systems through the built-in coarse-grained model and all-atom replacement. The coarse-grained model parametrization is generalized and extensively validated with various experimental data and all-atom simulations. In addition, the capability of for generating relaxed polymer systems is demonstrated by density calculations of 34 homopolymer melt systems, characteristic ratio calculations of 170 homopolymer melt systems, a morphology diagram of poly(styrene--methyl methacrylate) block copolymers, and self-assembly behavior of amphiphilic poly(ethylene oxide--ethylethane) block copolymers in water. We hope that is useful to carry out innovative and novel polymer modeling and simulation research to acquire insight into structures, dynamics, and underlying mechanisms of complex polymer-containing systems.
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http://dx.doi.org/10.1021/acs.jctc.1c00169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078172PMC
April 2021

Structure, Dynamics, Receptor Binding, and Antibody Binding of the Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein in a Viral Membrane.

J Chem Theory Comput 2021 Apr 10;17(4):2479-2487. Epub 2021 Mar 10.

Departments of Biological Sciences, Chemistry, Bioengineering, and Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2) and is considered the major target for drug and vaccine development. We previously built fully glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of the receptor-binding domain (RBD) and different templates for the stalk region. In this work, multiple μs-long all-atom molecular dynamics simulations were performed to provide deeper insights into the structure and dynamics of S protein and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of the RBD and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of commonly used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibodies. Overall, our observations offer structural and dynamic insights into the SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.
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http://dx.doi.org/10.1021/acs.jctc.0c01144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047829PMC
April 2021

Biomechanical characterization of SARS-CoV-2 spike RBD and human ACE2 protein-protein interaction.

Biophys J 2021 03 17;120(6):1011-1019. Epub 2021 Feb 17.

Department of Bioengineering; Department of Mechanical Engineering and Mechanics. Electronic address:

The current COVID-19 pandemic has led to a devastating impact across the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus causing COVID-19) is known to use the receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has a higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approaches to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between SARS-CoV-2 RBD and ACE2 range from 70 to 105 pN and are 30-40% higher than those of SARS-CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that SARS-CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the SARS-CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After removing N-linked glycans on ACE2, its mechanical binding strength with SARS-CoV-2 RBD decreases to a similar level of the SARS-CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1 and could help develop new strategies to block SARS-CoV-2 entry.
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http://dx.doi.org/10.1016/j.bpj.2021.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886630PMC
March 2021

Influences of Lipid A Types on LPS Bilayer Properties.

J Phys Chem B 2021 03 18;125(8):2105-2112. Epub 2021 Feb 18.

Departments of Biological Sciences, Chemistry, Bioengineering, and Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Lipopolysaccharides (LPS) present in the outer leaflet of Gram-negative bacterial outer membranes protect the bacteria from external threats and influence antibiotic permeability as well as immune system recognition. The structure of lipid A, the anchor of an LPS molecule to the outer membrane, can make direct influences on membrane properties. Particularly, in a Gram-negative bacterium responsible for cholera, a severe diarrheal disease, modifications of lipid A structures grant antibiotic resistance and are a primary factor that led to the current cholera pandemic. However, the difference in structural properties incurred by such modifications has not been fully explored. In this work, five symmetric bilayer systems comprised of distinct lipid A structures of LPS with O1 O-antigen were modeled and simulated to explore influences of different lipid A types on membrane properties. All-atom molecular dynamics simulations reveal that membrane properties such as hydrophobic thickness, acyl chain order parameter, and area per lipid are largely impacted by lipid A modifications due to differences in composition and acyl chain distortions. The modified lipid A is also less negatively charged, which possibly reveals a resistance mechanism to cationic antimicrobial peptide evasion. These findings present a possible explanation for 's immune system evasion properties and establish the differences between the lipid A types, which should be of use for any future study of the Gram-negative bacteria.
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http://dx.doi.org/10.1021/acs.jpcb.0c09144DOI Listing
March 2021

CHARMM-GUI Supports Hydrogen Mass Repartitioning and Different Protonation States of Phosphates in Lipopolysaccharides.

J Chem Inf Model 2021 Feb 14;61(2):831-839. Epub 2021 Jan 14.

Department of Biological Sciences, Department of Chemistry, Department of Bioengineering, and Department of Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Hydrogen mass repartitioning (HMR) that permits time steps of all-atom molecular dynamics simulation up to 4 fs by increasing the mass of hydrogen atoms has been used in protein and phospholipid bilayers simulations to improve conformational sampling. Molecular simulation input generation via CHARMM-GUI now supports HMR for diverse simulation programs. In addition, considering ambiguous pH at the bacterial outer membrane surface, different protonation states, either -2e or -1e, of phosphate groups in lipopolysaccharides (LPS) are also supported in CHARMM-GUI . To examine the robustness of HMR and the influence of protonation states of phosphate groups on LPS bilayer properties, eight different LPS-type all-atom systems with two phosphate protonation states are modeled and simulated utilizing both OpenMM 2-fs (standard) and 4-fs (HMR) schemes. Consistency in the conformational space sampled by standard and HMR simulations shows the reliability of HMR even in LPS, one of the most complex biomolecules. For systems with different protonation states, similar conformations are sampled with a PO or PO group, but different phosphate protonation states make slight impacts on lipid packing and conformational properties of LPS acyl chains. Systems with PO have a slightly smaller area per lipid and thus slightly more ordered lipid A acyl chains compared to those with PO, due to more electrostatic repulsion between PO even with neutralizing Ca ions. HMR and different protonation states of phosphates of LPS available in CHARMM-GUI are expected to be useful for further investigations of biological systems of diverse origin.
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http://dx.doi.org/10.1021/acs.jcim.0c01360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902386PMC
February 2021

Ligand-Binding-Site Refinement to Generate Reliable Holo Protein Structure Conformations from Apo Structures.

J Chem Inf Model 2021 01 18;61(1):535-546. Epub 2020 Dec 18.

Departments of Biological Sciences, Chemistry, Bioengineering, and Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

The first important step in a structure-based virtual screening is the judicious selection of a receptor protein. In cases where the holo protein receptor structure is unavailable, significant reduction in virtual screening performance has been reported. In this work, we present a robust method to generate reliable holo protein structure conformations from apo structures using molecular dynamics (MD) simulation with restraints derived from holo structure binding-site templates. We perform benchmark tests on two different datasets: 40 structures from a directory of useful decoy-enhanced (DUD-E) and 84 structures from the Gunasekaran dataset. Our results show successful refinement of apo binding-site structures toward holo conformations in 82% of the test cases. In addition, virtual screening performance of 40 DUD-E structures is significantly improved using our MD-refined structures as receptors with an average enrichment factor (EF), an EF value of 6.2 compared to apo structures with 3.5. Docking of native ligands to the refined structures shows an average ligand root mean square deviation (RMSD) of 1.97 Å (DUD-E dataset and Gunasekaran dataset) relative to ligands in the holo crystal structures, which is comparable to the self-docking (i.e., docking of the native ligand back to its crystal structure receptor) average, 1.34 Å (DUD-E dataset) and 1.36 Å (Gunasekaran dataset). On the other hand, docking to the apo structures yields an average ligand RMSD of 3.65 Å (DUD-E) and 2.90 Å (Gunasekaran). These results indicate that our method is robust and can be useful to improve virtual screening performance of apo structures.
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http://dx.doi.org/10.1021/acs.jcim.0c01354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856192PMC
January 2021

CHARMM-GUI Free Energy Calculator for Absolute and Relative Ligand Solvation and Binding Free Energy Simulations.

J Chem Theory Comput 2020 Nov 28;16(11):7207-7218. Epub 2020 Oct 28.

Department of Biological Sciences, Chemistry, Bioengineering, and Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Alchemical free energy simulations have long been utilized to predict free energy changes for binding affinity and solubility of small molecules. However, while the theoretical foundation of these methods is well established, seamlessly handling many of the practical aspects regarding the preparation of the different thermodynamic end states of complex molecular systems and the numerous processing scripts often remains a burden for successful applications. In this work, we present CHARMM-GUI (http://www.charmm-gui.org/input/fec) that provides various alchemical free energy perturbation molecular dynamics (FEP/MD) systems with input and post-processing scripts for NAMD and GENESIS. Four submodules are available: (for absolute ligand binding FEP/MD), (for relative ligand binding FEP/MD), (for absolute ligand solvation FEP/MD), and (for relative ligand solvation FEP/MD). Each module is designed to build multiple systems of a set of selected ligands at once for high-throughput FEP/MD simulations. The capability of is illustrated by absolute and relative solvation FEP/MD of a set of ligands and absolute and relative binding FEP/MD of a set of ligands for T4-lysozyme in solution and the adenosine A receptor in a membrane. The calculated free energy values are overall consistent with the experimental and published free energy results (within ∼1 kcal/mol). We hope that is useful to carry out high-throughput FEP/MD simulations in the field of biomolecular sciences and drug discovery.
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http://dx.doi.org/10.1021/acs.jctc.0c00884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658063PMC
November 2020

Experimentally Guided Computational Methods Yield Highly Accurate Insights into Transmembrane Interactions within the T Cell Receptor Complex.

J Phys Chem B 2020 11 8;124(46):10303-10310. Epub 2020 Oct 8.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

Understanding how molecular interactions within the plasma membrane govern assembly, clustering, and conformational changes in single-pass transmembrane (TM) receptors has long presented substantial experimental challenges. Our previous work on activating immune receptors has combined direct biochemical and biophysical characterizations with both independent and experimentally restrained computational methods to provide novel insights into the key TM interactions underpinning assembly and stability of complex, multisubunit receptor systems. The recently published cryo-EM structure of the intact T cell receptor (TCR)-CD3 complex provides a unique opportunity to test the models and predictions arising from these studies, and we find that they are accurate, which we attribute to robust simulation environments and careful consideration of limitations related to studying TM interactions in isolation from additional receptor domains. With this in mind, we revisit results in other immune receptors and look forward to how similar methods may be applied to understand receptors for which little or no structural information is currently available.
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http://dx.doi.org/10.1021/acs.jpcb.0c06403DOI Listing
November 2020

Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 with Heparan Sulfates in a Membrane.

Glycobiology 2021 Jun;31(5):593-602

Department of Biological Sciences, Lehigh University, Bethlehem, PA, 18015, United States.

Glypican-1 and its heparan sulfate (HS) chains play important roles in modulating many biological processes including growth factor signaling. Glypican-1 is bound to a membrane surface via a glycosylphosphatidylinositol (GPI)-anchor. In this study, we used all-atom molecular modeling and simulation to explore the structure, dynamics, and interactions of GPI-anchored glypican-1, three HS chains, membranes, and ions. The folded glypican-1 core structure is stable, but has substantial degrees of freedom in terms of movement and orientation with respect to the membrane due to the long unstructured C-terminal region linking the core to the GPI-anchor. With unique structural features depending on the extent of sulfation, high flexibility of HS chains can promote multi-site interactions with surrounding molecules near and above the membrane. This study is a first step toward all-atom molecular modeling and simulation of the glycocalyx, as well as its modulation of interactions between growth factors and their receptors.
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http://dx.doi.org/10.1093/glycob/cwaa092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176774PMC
June 2021

Conformational States of the Cytoprotective Protein Bcl-xL.

Biophys J 2020 10 20;119(7):1324-1334. Epub 2020 Aug 20.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California. Electronic address:

Bcl-xL is a major inhibitor of apoptosis, a fundamental homeostatic process of programmed cell death that is highly conserved across evolution. Because it plays prominent roles in cancer, Bcl-xL is a major target for anticancer therapy and for studies aimed at understanding its structure and activity. Although Bcl-xL is active primarily at intracellular membranes, most studies have focused on soluble forms of the protein lacking both the membrane-anchoring C-terminal tail and the intrinsically disordered loop, and this has resulted in a fragmented view of the protein's biological activity. Here, we describe the conformation of full-length Bcl-xL. Using NMR spectroscopy, molecular dynamics simulations, and isothermal titration calorimetry, we show how the three structural elements affect the protein's structure, dynamics, and ligand-binding activity in both its soluble and membrane-anchored states. The combined data provide information about the molecular basis for the protein's functionality and a view of its complex molecular mechanisms.
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http://dx.doi.org/10.1016/j.bpj.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567986PMC
October 2020

Biomechanical Characterization of SARS-CoV-2 Spike RBD and Human ACE2 Protein-Protein Interaction.

bioRxiv 2020 Jul 31. Epub 2020 Jul 31.

The current COVID-19 pandemic has already had a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use its surface spike (S) protein's receptor binding domain (RBD) to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS epidemic. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.
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http://dx.doi.org/10.1101/2020.07.31.230730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402033PMC
July 2020

CHARMM-GUI supports the Amber force fields.

J Chem Phys 2020 Jul;153(3):035103

Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

As part of our ongoing efforts to support diverse force fields and simulation programs in CHARMM-GUI, this work presents the development of FF-Converter to prepare Amber simulation inputs with various Amber force fields within the current CHARMM-GUI workflow. The currently supported Amber force fields are ff14SB/ff19SB (protein), Bsc1 (DNA), OL3 (RNA), GLYCAM06 (carbohydrate), Lipid17 (lipid), GAFF/GAFF2 (small molecule), TIP3P/TIP4P-EW/OPC (water), and 12-6-4 ions, and more will be added if necessary. The robustness and usefulness of this new CHARMM-GUI extension are demonstrated by two exemplary systems: a protein/N-glycan/ligand/membrane system and a protein/DNA/RNA system. Currently, CHARMM-GUI supports the Amber force fields only for the Amber program, but we will expand the FF-Converter functionality to support other simulation programs that support the Amber force fields.
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http://dx.doi.org/10.1063/5.0012280DOI Listing
July 2020

Calcium and hydroxyapatite binding site of human vitronectin provides insights to abnormal deposit formation.

Proc Natl Acad Sci U S A 2020 08 22;117(31):18504-18510. Epub 2020 Jul 22.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037;

The human blood protein vitronectin (Vn) is a major component of the abnormal deposits associated with age-related macular degeneration, Alzheimer's disease, and many other age-related disorders. Its accumulation with lipids and hydroxyapatite (HAP) has been demonstrated, but the precise mechanism for deposit formation remains unknown. Using a combination of solution and solid-state NMR experiments, cosedimentation assays, differential scanning fluorimetry (DSF), and binding energy calculations, we demonstrate that Vn is capable of binding both soluble ionic calcium and crystalline HAP, with high affinity and chemical specificity. Calcium ions bind preferentially at an external site, at the top of the hemopexin-like (HX) domain, with a group of four Asp carboxylate groups. The same external site is also implicated in HAP binding. Moreover, Vn acquires thermal stability upon association with either calcium ions or crystalline HAP. The data point to a mechanism whereby Vn plays an active role in orchestrating calcified deposit formation. They provide a platform for understanding the pathogenesis of macular degeneration and other related degenerative disorders, and the normal functions of Vn, especially those related to bone resorption.
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http://dx.doi.org/10.1073/pnas.2007699117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414086PMC
August 2020

A systematic analysis of protein-carbohydrate interactions in the Protein Data Bank.

Glycobiology 2021 Feb;31(2):126-136

Departments of Biological Sciences, Chemistry, Bioengineering, and Computer Sciences and Engineering, Lehigh University, Bethlehem, PA 18015, USA.

Protein-carbohydrate interactions underlie essential biological processes. Elucidating the mechanism of protein-carbohydrate recognition is a prerequisite for modeling and optimizing protein-carbohydrate interactions, which will help in discovery of carbohydrate-derived therapeutics. In this work, we present a survey of a curated database consisting of 6,402 protein-carbohydrate complexes in the Protein Data Bank (PDB). We performed an all-against-all comparison of a subset of nonredundant binding sites, and the result indicates that the interaction pattern similarity is not completely relevant to the binding site structural similarity. Investigation of both binding site and ligand promiscuities reveals that the geometry of chemical feature points is more important than local backbone structure in determining protein-carbohydrate interactions. A further analysis on the frequency and geometry of atomic interactions shows that carbohydrate functional groups are not equally involved in binding interactions. Finally, we discuss the usefulness of protein-carbohydrate complexes in the PDB with acknowledgement that the carbohydrates in many structures are incomplete.
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http://dx.doi.org/10.1093/glycob/cwaa062DOI Listing
February 2021

Developing a Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein Model in a Viral Membrane.

J Phys Chem B 2020 08 6;124(33):7128-7137. Epub 2020 Jul 6.

Departments of Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

This technical study describes all-atom modeling and simulation of a fully glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB: 6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19) so that researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.
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http://dx.doi.org/10.1021/acs.jpcb.0c04553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341691PMC
August 2020

Modeling and Simulation of a Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane.

bioRxiv 2020 May 21. Epub 2020 May 21.

Departments of Computer Science and Engineering, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

This technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.
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http://dx.doi.org/10.1101/2020.05.20.103325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263518PMC
May 2020

Modeling and Simulation of Bacterial Outer Membranes with Lipopolysaccharides and Enterobacterial Common Antigen.

J Phys Chem B 2020 07 30;124(28):5948-5956. Epub 2020 Jun 30.

Department of Biological Sciences, Department of Chemistry, and Department of Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Enterobacterial common antigen (ECA) is a surface glycolipid shared by all members of the family. In addition to lipopolysaccharides (LPS), ECA is an important component in the outer membrane (OM) of Gram-negative bacteria, making the OM an effective, selective barrier against the permeation of toxic molecules. Previous modeling and simulation studies represented OMs exclusively with LPS in the outer leaflet. In this work, various ECA molecules were first modeled and incorporated into symmetric bilayers with LPS in different ratios, and all-atom molecular dynamics simulations were conducted to investigate the properties of the mixed bilayers mimicking OM outer leaflets. Dynamic and flexible conformational ensembles are sampled for each ECA/LPS system. Incorporation of ECA (an LPS core-linked form) and ECA (a phosphatidylglycerol-linked form) affects lipid packing and ECA/LPS distributions on the bilayer surface. Hydrophobic thickness and chain order parameter analyses indicate that incorporation of ECA makes the acyl chains of LPS more flexible and disordered and thus increases the area per lipid of LPS. The calculated area per lipid of each ECA/LPS provides a good estimate for building more realistic OMs with different ratios of ECA/LPS, which will be useful in order to characterize their interactions with outer membrane proteins in more realistic OMs.
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http://dx.doi.org/10.1021/acs.jpcb.0c03353DOI Listing
July 2020

Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival.

Mol Microbiol 2020 09 25;114(3):510-520. Epub 2020 Jun 25.

Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS) enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.
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http://dx.doi.org/10.1111/mmi.14530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594906PMC
September 2020

Cooperativity in Proteasome Core Particle Maturation.

iScience 2020 May 22;23(5):101090. Epub 2020 Apr 22.

Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA; Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, 338 Ackert Hall, Manhattan, KS 66506, USA. Electronic address:

Proteasomes are multi-subunit protease complexes found in all domains of life. The maturation of the core particle (CP), which harbors the active sites, involves dimerization of two half CPs (HPs) and an autocatalytic cleavage that removes β propeptides. How these steps are regulated remains poorly understood. Here, we used the Rhodococcus erythropolis CP to dissect this process in vitro. Our data show that propeptides regulate the dimerization of HPs through flexible loops we identified. Furthermore, N-terminal truncations of the propeptides accelerated HP dimerization and decelerated CP auto-activation. We identified cooperativity in autocatalysis and found that the propeptide can be partially cleaved by adjacent active sites, potentially aiding an otherwise strictly autocatalytic mechanism. We propose that cross-processing during bacterial CP maturation is the underlying mechanism leading to the observed cooperativity of activation. Our work suggests that the bacterial β propeptide plays an unexpected and complex role in regulating dimerization and autocatalytic activation.
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http://dx.doi.org/10.1016/j.isci.2020.101090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210456PMC
May 2020

Improving Protein-Ligand Docking Results with High-Throughput Molecular Dynamics Simulations.

J Chem Inf Model 2020 04 10;60(4):2189-2198. Epub 2020 Apr 10.

Departments of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Structure-based virtual screening relies on classical scoring functions that often fail to reliably discriminate binders from nonbinders. In this work, we present a high-throughput protein-ligand complex molecular dynamics (MD) simulation that uses the output from AutoDock Vina to improve docking results in distinguishing active from decoy ligands in a directory of useful decoy-enhanced (DUD-E) dataset. MD trajectories are processed by evaluating ligand-binding stability using root-mean-square deviations. We select 56 protein targets (of 7 different protein classes) and 560 ligands (280 actives, 280 decoys) and show 22% improvement in ROC AUC (area under the curve, receiver operating characteristics curve), from an initial value of 0.68 (AutoDock Vina) to a final value of 0.83. The MD simulation demonstrates a robust performance across all seven different protein classes. In addition, some predicted ligand-binding modes are moderately refined during MD simulations. These results systematically validate the reliability of a physics-based approach to evaluate protein-ligand binding interactions.
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http://dx.doi.org/10.1021/acs.jcim.0c00057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534544PMC
April 2020

Broadening Activity of Polymyxin by Quaternary Ammonium Grafting.

ACS Infect Dis 2020 06 3;6(6):1427-1435. Epub 2020 Apr 3.

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Bacterial pathogens continue to impose a tremendous health burden across the globe. Here, we describe a novel series of polymyxin-based agents grafted with membrane-active quaternary ammonium warheads to combine two important classes of Gram-negative antimicrobial scaffolds. The goal was to deliver a targeted quaternary ammonium warhead onto the surface of bacterial pathogens using the outer membrane homing properties of polymyxin. The most potent agents resulted in new scaffolds that retained the ability to target Gram-negative bacteria and had limited toxicity toward mammalian cells. We showed, using a molecular dynamics approach, that the new agents retained their ability to engage in specific interactions with lipopolysaccharide molecules. Significantly, the combination of quaternary ammonium and polymyxin widens the activity to the pathogen . Our results serve as an example of how two membrane-active agents can be combined to produce a class of novel scaffolds with potent biological activity.
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http://dx.doi.org/10.1021/acsinfecdis.0c00037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293573PMC
June 2020

Dynamics and Interactions of GPI-Linked lynx1 Protein with/without Nicotinic Acetylcholine Receptor in Membrane Bilayers.

J Phys Chem B 2020 05 9;124(20):4017-4025. Epub 2020 Apr 9.

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, United States.

Nicotinic acetylcholine receptors (nAChRs) participate in diverse biological processes, such as mood, learning, and addiction. Glycosylphosphatidylinositol-linked lynx1 is an allosteric modulator of nAChR function, including shifts in agonist sensitivity, reduced desensitization, and slower recovery from desensitization. This modulation is thought to be achieved by lynx1's interaction with nAChR subunits, particularly at the α4:α4 interface. In this study, we used molecular modeling and simulation to study the structure, dynamics, and interactions of lynx1 when bound to nAChRs, as well as unbound, monomeric lynx1 in membranes. Though lynx1 structures are similar in both states, its dynamics is more restricted in the bound state than in the unbound one. When bound, interactions between lynx1 and nAChR are observed to be maintained throughout the simulations. Of particular note, lynx1 demonstrates prolonged interactions with the receptor C-loop in one of the nAChR α4 subunits, a region important for agonist binding and possibly the transition between open/closed states. During interactions with lynx1, an α4 C-loop tends to be restricted in either a closed or open state, whereas the C-loop state transitions are more evident when lynx1 is unbound. Interestingly, the conformational change of the C-loop is stochastic, suggesting that lynx1 can influence nAChR (critical for its multimodal action), for instance, by shifting its agonist sensitivity and recovery from desensitization.
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http://dx.doi.org/10.1021/acs.jpcb.0c00159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820712PMC
May 2020

Molecular Simulations of Gram-Negative Bacterial Membranes Come of Age.

Annu Rev Phys Chem 2020 04 18;71:171-188. Epub 2020 Feb 18.

School of Chemistry, University of Southampton, Southampton S017 1BJ, United Kingdom; email:

Gram-negative bacteria are protected by a multicompartmental molecular architecture known as the cell envelope that contains two membranes and a thin cell wall. As the cell envelope controls influx and efflux of molecular species, in recent years both experimental and computational studies of such architectures have seen a resurgence due to the implications for antibiotic development. In this article we review recent progress in molecular simulations of bacterial membranes. We show that enormous progress has been made in terms of the lipidic and protein compositions of bacterial systems. The simulations have moved away from the traditional setup of one protein surrounded by a large patch of the same lipid type toward a more bio-logically representative viewpoint. Simulations with multiple cell envelope components are also emerging. We review some of the key method developments that have facilitated recent progress, discuss some current limitations, and offer a perspective on future directions.
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http://dx.doi.org/10.1146/annurev-physchem-103019-033434DOI Listing
April 2020