Identification and Characterization of Novel Fc-Binding Heptapeptides from Experiments and Simulations

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products, therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 phage display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.

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Identification and Characterization of Novel Fc-Binding Heptapeptides from Experiments and Simulations

10.20944/preprints201806.0039.v1 ◽

2018 ◽

Author(s):

Xiaoquan Sun ◽

Justin Weaver ◽

Sumith Ranil Wickramasinghe ◽

Xianghong Qian

Keyword(s):

Hydrophobic Interactions ◽

Culture Media ◽

Protein A ◽

Md Simulations ◽

Production Costs ◽

Antibody Detection ◽

Small Peptides ◽

Affinity Ligands ◽

Binding Behavior ◽

Igg Isotypes

Purification of biologically-derived therapeutics is a major cost contributor to the production of this rapidly growing class of pharmaceuticals. Monoclonal antibodies comprise a large percentage of these products therefore new antibody purification tools are needed. Small peptides, as opposed to traditional antibody affinity ligands such as Protein A, may have advantages in stability and production costs. Multiple heptapeptides that demonstrate Fc binding behavior that have been identified from a combinatorial peptide library using M13 Phage Display are presented herein. Seven unique peptide sequences of diverse hydrophobicity and charge were identified. All seven peptides showed strong binding to the four major human IgG isotypes, human IgM, as well as binding to canine, rat, and mouse IgG. These seven peptides were also shown to bind human IgG4 from DMEM cell culture media with 5% FCS and 5 g/L ovalbumin present. These peptides may be useful as surface ligands for antibody detection and purification purposes. Molecular docking and classical molecular dynamics (MD) simulations were conducted to elucidate the mechanisms and energetics for the binding of these peptides to the Fc region. The binding site was found to be located between the two glycan chains inside the Fc fragment. Both hydrogen bonding and hydrophobic interactions were found to be crucial for the binding interactions. Excellent agreement for the binding strength was obtained between experimental results and simulations.

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Conformational Ensembles by NMR and MD Simulations in Model Heptapeptides with Select Tri-Peptide Motifs

International Journal of Molecular Sciences ◽

10.3390/ijms22031364 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1364

Author(s):

V. V. Krishnan ◽

Timothy Bentley ◽

Alina Xiong ◽

Kalyani Maitra

Keyword(s):

Principal Component ◽

Md Simulations ◽

Conformational Space ◽

Random Coil ◽

Radius Of Gyration ◽

Small Peptides ◽

Conformational Ensembles ◽

Solution State ◽

Peptide Motifs ◽

Explicit Solvent

Both nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations are routinely used in understanding the conformational space sampled by peptides in the solution state. To investigate the role of single-residue change in the ensemble of conformations sampled by a set of heptapeptides, AEVXEVG with X = L, F, A, or G, comprehensive NMR, and MD simulations were performed. The rationale for selecting the particular model peptides is based on the high variability in the occurrence of tri-peptide E*L between the transmembrane β-barrel (TMB) than in globular proteins. The ensemble of conformations sampled by E*L was compared between the three sets of ensembles derived from NMR spectroscopy, MD simulations with explicit solvent, and the random coil conformations. In addition to the estimation of global determinants such as the radius of gyration of a large sample of structures, the ensembles were analyzed using principal component analysis (PCA). In general, the results suggest that the -EVL- peptide indeed adopts a conformational preference that is distinctly different not only from a random distribution but also from other peptides studied here. The relatively straightforward approach presented herein could help understand the conformational preferences of small peptides in the solution state.

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Design of peptide affinity ligands for S-protein: a comparison of combinatorial and de novo design strategies

Molecular Diversity ◽

10.1007/s11030-013-9436-z ◽

2013 ◽

Vol 17 (2) ◽

pp. 357-369 ◽

Cited By ~ 10

Author(s):

Divya Chandra ◽

Christopher J. Morrison ◽

James Woo ◽

Steven Cramer ◽

Pankaj Karande

Keyword(s):

De Novo ◽

Protein A ◽

De Novo Design ◽

Design Strategies ◽

S Protein ◽

Affinity Ligands ◽

Peptide Affinity

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Exploration of Novel Xanthine Oxidase Inhibitors Based on 1,6-Dihydropyrimidine-5-Carboxylic Acids by an Integrated in Silico Study

International Journal of Molecular Sciences ◽

10.3390/ijms22158122 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8122

Author(s):

Na Zhai ◽

Chenchen Wang ◽

Fengshou Wu ◽

Liwei Xiong ◽

Xiaogang Luo ◽

...

Keyword(s):

Xanthine Oxidase ◽

Carboxylic Acids ◽

Hydrophobic Interactions ◽

Pharmacophore Model ◽

3D Qsar ◽

Md Simulations ◽

Pharmacophore Modeling ◽

Binding Modes ◽

Comfa Model ◽

Validation Parameters

Xanthine oxidase (XO) is an important target for the effective treatment of hyperuricemia-associated diseases. A series of novel 2-substituted 6-oxo-1,6-dihydropyrimidine-5-carboxylic acids (ODCs) as XO inhibitors (XOIs) with remarkable activities have been reported recently. To better understand the key pharmacological characteristics of these XOIs and explore more hit compounds, in the present study, the three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking, pharmacophore modeling, and molecular dynamics (MD) studies were performed on 46 ODCs. The constructed 3D-QSAR models exhibited reliable predictability with satisfactory validation parameters, including q2 = 0.897, R2 = 0.983, rpred2 = 0.948 in a CoMFA model, and q2 = 0.922, R2 = 0.990, rpred2 = 0.840 in a CoMSIA model. Docking and MD simulations further gave insights into the binding modes of these ODCs with the XO protein. The results indicated that key residues Glu802, Arg880, Asn768, Thr1010, Phe914, and Phe1009 could interact with ODCs by hydrogen bonds, π-π stackings, or hydrophobic interactions, which might be significant for the activity of these XOIs. Four potential hits were virtually screened out using the constructed pharmacophore model in combination with molecular dockings and ADME predictions. The four hits were also found to be relatively stable in the binding pocket by MD simulations. The results in this study might provide effective information for the design and development of novel XOIs.

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Making high-dimensional molecular distribution functions tractable through Belief Propagation on Factor Graphs

10.1101/2021.06.28.450193 ◽

2021 ◽

Author(s):

Zachary Smith ◽

Pratyush Tiwary

Keyword(s):

Belief Propagation ◽

Conformational Dynamics ◽

Joint Probability ◽

Md Simulations ◽

Distribution Functions ◽

Factor Graph ◽

High Dimensional ◽

Enhanced Sampling ◽

Small Peptides ◽

Approximate Probability

Molecular dynamics (MD) simulations provide a wealth of high-dimensional data at all-atom and femtosecond resolution but deciphering mechanistic information from this data is an ongoing challenge in physical chemistry and biophysics. Theoretically speaking, joint probabilities of the equilibrium distribution contain all thermodynamic information, but they prove increasingly difficult to compute and interpret as the dimensionality increases. Here, inspired by tools in probabilistic graphical modeling, we develop a factor graph trained through belief propagation that helps factorize the joint probability into an approximate tractable form that can be easily visualized and used. We validate the study through the analysis of the conformational dynamics of two small peptides with 5 and 9 residues. Our validations include testing the conditional dependency predictions through an intervention scheme inspired by Judea Pearl. Secondly we directly use the belief propagation based approximate probability distribution as a high-dimensional static bias for enhanced sampling, where we achieve spontaneous back-and-forth motion between metastable states that is up to 350 times faster than unbiased MD. We believe this work opens up useful ways to thinking about and dealing with high-dimensional molecular simulations.

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Residue-Level Contact Reveals Modular Domain Interactions of PICK1 Are Driven by Both Electrostatic and Hydrophobic Forces

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.616135 ◽

2021 ◽

Vol 7 ◽

Author(s):

Amy O. Stevens ◽

Yi He

Keyword(s):

Hydrophobic Interactions ◽

Pdz Domain ◽

Md Simulations ◽

Intramolecular Interactions ◽

Coarse Grained ◽

Scaffolding Protein ◽

Concave Surface ◽

Stable Complex ◽

Bar Domain ◽

C Terminus

PICK1 is a multi-domain scaffolding protein that is uniquely comprised of both a PDZ domain and a BAR domain. While previous experiments have shown that the PDZ domain and the linker positively regulate the BAR domain and the C-terminus negatively regulates the BAR domain, the details of internal regulation mechanisms are unknown. Molecular dynamics (MD) simulations have been proven to be a useful tool in revealing the intramolecular interactions at atomic-level resolution. PICK1 performs its biological functions in a dimeric form which is extremely computationally demanding to simulate with an all-atom force field. Here, we use coarse-grained MD simulations to expose the key residues and driving forces in the internal regulations of PICK1. While the PDZ and BAR domains do not form a stable complex, our simulations show the PDZ domain preferentially interacting with the concave surface of the BAR domain over other BAR domain regions. Furthermore, our simulations show that the short helix in the linker region can form interactions with the PDZ domain. Our results reveal that the surface of the βB-βC loop, βC strand, and αA-βD loop of the PDZ domain can form a group of hydrophobic interactions surrounding the linker helix. These interactions are driven by hydrophobic forces. In contrast, our simulations reveal a very dynamic C-terminus that most often resides on the convex surface of the BAR domain rather than the previously suspected concave surface. These interactions are driven by a combination of electrostatic and hydrophobic interactions.

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Investigation of alkaline effects on Protein A affinity ligands and resins using high resolution mass spectrometry

Journal of Chromatography B ◽

10.1016/j.jchromb.2020.122473 ◽

2021 ◽

Vol 1162 ◽

pp. 122473

Author(s):

Magnus Wetterhall ◽

Mats Ander ◽

Tomas Björkman ◽

Sravani Musunuri ◽

Ronnie Palmgren ◽

...

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

High Resolution Mass Spectrometry ◽

Protein A ◽

Affinity Ligands ◽

High Resolution Mass ◽

Resolution Mass

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Molecular docking and molecular dynamics simulations of fumarate hydratase and its mutant H235N complexed with pyromellitic acid and citrate

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720017500263 ◽

2017 ◽

Vol 15 (06) ◽

pp. 1750026 ◽

Cited By ~ 1

Author(s):

S. Subasri ◽

Santosh Kumar Chaudhary ◽

K. Sekar ◽

Manish Kesherwani ◽

D. Velmurugan

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

Model Building ◽

Hydrophobic Interactions ◽

Conformational Stability ◽

Fumarate Hydratase ◽

Md Simulations ◽

Major Effect ◽

Active Site Residues ◽

Pyromellitic Acid

Fumarase catalyzes the reversible, stereospecific hydration/dehydration of fumarate to L-malate during the Kreb’s cycle. In the crystal structure of the tetrameric fumarase, it was found that some of the active site residues S145, T147, N188 G364 and H235 had water-mediated hydrogen bonding interactions with pyromellitic acid and citrate which help to the protonation state for the conversion of fumarate to malate. When His 235 is mutated with Asn (H235N), water-mediated interactions were lost due to the shifting of active site water molecule by 0.7 Å away. Molecular dynamics (MD) simulations were also carried out by NAMD and analyzed using Assisted Model Building with Energy Refinement (AMBER) program to better understand the conformational stability and other aspects during the binding of pyromellitic acid and citrate with native and mutant FH. The role of hydrogen bonds and hydrophobic interactions was also analyzed. The present study confirms that the H235N mutation has a major effect on the catalytic activity of fumarase which is evident from the biochemical studies.

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Investigating the effect of Ser256 phosphorylation on gating of aquaporin-2: Molecular Dynamics study

10.1101/2021.01.18.427094 ◽

2021 ◽

Author(s):

Pragya Priyadarshini ◽

Balvinder Singh

Keyword(s):

Molecular Dynamics ◽

Water Permeability ◽

Water Movement ◽

Hydrophobic Interactions ◽

Md Simulations ◽

Post Translational Modification ◽

Functional Changes ◽

Gating Mechanism ◽

Intracellular Storage ◽

Dynamics Simulations

AbstractRegulation of water transport via aquaporins is crucial for osmoregulation and water homeostasis of an organism. This transport of water is regulated either by gating or trafficking wherein AQPs are transported from intracellular storage sites to plasma membrane. It has been proposed that water movement via AQP2 is regulated by post-translational modification. We aimed to explore the structural and functional changes occurring in AQP2 due to Ser256 phosphorylation. We have carried out molecular dynamics simulations to investigate molecular basis of effect of phosphorylation on water permeability of AQP2. MD simulations show that there are mild variations in the pore sizes of different monomers of the phosphorylated and unphosphorylated AQP2. Analysis of inter and intra-monomeric interactions such as hydrogen bond, electrostatic and hydrophobic interactions has been carried out. Structures of the phosphorylated AQP2 do not show any blocking of mouth of pore of the monomers during the course of MD simulations. Further, water permeability calculations do corroborate the above finding. This molecular dynamics study suggests that phosphorylation of C-terminal Ser-256 residue of AQP2 may not be directly responsible for gating mechanism.

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Key Interacting Residues Between RBD of SARS-CoV-2 and ACE2 Receptor: Combination of Molecular Dynamic Simulation and Density Functional Calculation

10.26434/chemrxiv.14582484.v1 ◽

2021 ◽

Author(s):

Bahaa Jawad ◽

Puja Adhikari ◽

Rudolf Podgornik ◽

Wai-Yim Ching

Keyword(s):

Density Functional ◽

Vaccine Development ◽

Hydrophobic Interactions ◽

Binding Free Energy ◽

Tight Binding ◽

Md Simulations ◽

Functional Theory ◽

Generalized Born ◽

The Density Functional Theory ◽

Key Residues

The spike protein of SARS-CoV-2 binds to ACE2 receptor via its receptor-binding domain (RBD), with the RBD-ACE2 complex presenting an essential molecular target for vaccine development to stall the virus infection proliferation. The computational analysis at molecular, amino acid (AA) and atomic levels have been performed systematically to identify the key interacting AAs in the formation of the RBD-ACE2 complex, including the MD simulations with molecular mechanics generalized Born surface area (MM-GBSA) method to predict binding free energy (BFE) and to determine the actual interacting AAs, as well as two ab initio quantum chemical protocols based on the density functional theory (DFT) implementation. Based on MD results, Q493, Y505, Q498, N501, T500, N487, Y449, F486, K417, Y489, F456, Y495, and L455 have been identified as hotspots in RBD, while those in ACE2 are K353, K31, D30, D355, H34, D38, Q24, T27, Y83, Y41, E35, and E37. Both the electrostatic and hydrophobic interactions are the main driving force to form the AA-AA binding pairs. We confirm that Q493, N501, F486, K417, and F456 in RBD are the key residues responsible for the tight binding of SARS-CoV-2 with ACE2 compared to SARS-CoV. The DFT results reveal that N487, Q493, Y449, T500, G496, G446 and G502 in RBD form pairs via specific hydrogen bonding with Q24, H34, E35, D38, Y41, Q42 and K353 in ACE2.

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