Conformational selection of allergen-antibody complexes—surface plasticity of paratopes and epitopes

Abstract Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.

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Antibodies exhibit multiple paratope states influencing VH–VL domain orientations

Communications Biology ◽

10.1038/s42003-020-01319-z ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Monica L. Fernández-Quintero ◽

Nancy D. Pomarici ◽

Barbara A. Math ◽

Katharina B. Kroell ◽

Franz Waibl ◽

...

Keyword(s):

Molecular Dynamics ◽

Structural Information ◽

Conformational Transitions ◽

Antigen Binding ◽

Antigen Binding Site ◽

Solution Structures ◽

Conformational Rearrangements ◽

Complementarity Determining Regions ◽

Dynamics Simulations

Abstract In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as complementarity determining regions (CDRs) shaping the antigen-binding site, the paratope. Whereas it was assumed that certain sequences can only adopt a limited set of backbone conformations, in this study we present a kinetic classification of several paratope states in solution. Using molecular dynamics simulations in combination with experimental structural information we capture the involved conformational transitions between different canonical clusters and additional dominant solution structures occurring in the micro-to-millisecond timescale. Furthermore, we observe a strong correlation of CDR loop movements. Another important aspect when characterizing different paratope states is the relative VH/VL orientation and the influence of the distinct CDR loop states on the VH/VL interface. Conformational rearrangements of the CDR loops do not only have an effect on the relative VH/VL orientations, but also influence in some cases the elbow-angle dynamics and shift the respective distributions. Thus, our results show that antibodies exist as several interconverting paratope states, each contributing to the antibody’s properties.

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A β-Alanine Catabolism Pathway Containing a Highly Promiscuous ω-Transaminase in the 12-Aminododecanate-Degrading Pseudomonas sp. Strain AAC

Applied and Environmental Microbiology ◽

10.1128/aem.00665-16 ◽

2016 ◽

Vol 82 (13) ◽

pp. 3846-3856 ◽

Cited By ~ 13

Author(s):

Matthew Wilding ◽

Thomas S. Peat ◽

Janet Newman ◽

Colin Scott

Keyword(s):

Conformational Changes ◽

Active Site ◽

Building Block ◽

Protein A ◽

Physiological Role ◽

Substantial Contribution ◽

Content Type ◽

Nylon 12 ◽

Dynamics Simulations

ABSTRACTWe previously isolated the transaminase KES23458 fromPseudomonassp. strain AAC as a promising biocatalyst for the production of 12-aminododecanoic acid, a constituent building block of nylon-12. Here, we report the subsequent characterization of this transaminase. It exhibits activity with a broad substrate range which includes α-, β-, and ω-amino acids, as well as α,ω-diamines and a number of other industrially relevant compounds. It is therefore a prospective candidate for the biosynthesis of a range of polyamide monomers. The crystal structure of KES23458 revealed that the protein forms a dimer containing a large active site pocket and unusual phosphorylated histidine residues. To infer the physiological role of the transaminase, we expressed, purified, and characterized a dehydrogenase from the same operon, KES23460. Unlike the transaminase, the dehydrogenase was shown to be quite selective, catalyzing the oxidation of malonic acid semialdehyde, formed from β-alanine transamination via KES23458. In keeping with previous reports, the dehydrogenase was shown to catalyze both a coenzyme A (CoA)-dependent reaction to form acetyl-CoA and a significantly slower CoA-independent reaction to form acetate. These findings support the original functional assignment of KES23458 as a β-alanine transaminase. However, a seemingly well-adapted active site and promiscuity toward unnatural compounds, such as 12-aminododecanoic acid, suggest that this enzyme could perform multiple functions forPseudomonassp. strain AAC.IMPORTANCEWe describe the characterization of an industrially relevant transaminase able to metabolize 12-aminododecanoic acid, a constituent building block of the widely used polymer nylon-12, and we report the biochemical and structural characterization of the transaminase protein. A physiological role for this highly promiscuous enzyme is proposed based on the characterization of a related gene from the host organism. Molecular dynamics simulations were carried out to compare the conformational changes in the transaminase protein to better understand the determinants of specificity in the protein. This study makes a substantial contribution that is of interest to the broad biotechnology and enzymology communities, providing insights into the catalytic activity of an industrially relevant biocatalyst as well as the biological function of this operon.

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The answer lies in the energy: how simple atomistic molecular dynamics simulations may hold the key to epitope prediction on the fully glycosylated SARS-CoV-2 spike protein

10.1101/2020.07.22.214254 ◽

2020 ◽

Cited By ~ 1

Author(s):

Stefano Serapian ◽

Filippo Marchetti ◽

Alice Triveri ◽

Giulia Morra ◽

Massimiliano Meli ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Structural Information ◽

Epitope Prediction ◽

Spike Protein ◽

Cell Infection ◽

Monosaccharide Residue ◽

Decomposition Approach ◽

S Protein ◽

Dynamics Simulations

AbstractBetacoronavirus SARS-CoV-2 is posing a major threat to human health and its diffusion around the world is having dire socioeconomical consequences. Thanks to the scientific community’s unprecedented efforts, the atomic structure of several viral proteins has been promptly resolved. As the crucial mediator of host cell infection, the heavily glycosylated trimeric viral Spike protein (S) has been attracting the most attention and is at the center of efforts to develop antivirals, vaccines, and diagnostic solutions.Herein, we use an energy-decomposition approach to identify antigenic domains and antibody binding sites on the fully glycosylated S protein. Crucially, all that is required by our method are unbiased atomistic molecular dynamics simulations; no prior knowledge of binding properties or ad hoc combinations of parameters/measures extracted from simulations is needed. Our method simply exploits the analysis of energy interactions between all intra-protomer aminoacid and monosaccharide residue pairs, and cross-compares them with structural information (i.e., residueresidue proximity), identifying potential immunogenic regions as those groups of spatially contiguous residues with poor energetic coupling to the rest of the protein.Our results are validated by several experimentally confirmed structures of the S protein in complex with anti- or nanobodies. We identify poorly coupled sub-domains: on the one hand this indicates their role in hosting (several) epitopes, and on the other hand indicates their involvement in large functional conformational transitions. Finally, we detect two distinct behaviors of the glycan shield: glycans with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes; those with weaker coupling could themselves be poised for antibody recognition. Predicted Immunoreactive regions can be used to develop optimized antigens (recombinant subdomains, synthetic (glyco)peptidomimetics) for therapeutic applications.

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Time-resolved footprinting for the study of the structural dynamics of DNA–protein interactions

Biochemical Society Transactions ◽

10.1042/bst0360745 ◽

2008 ◽

Vol 36 (4) ◽

pp. 745-748 ◽

Cited By ~ 3

Author(s):

Bianca Sclavi

Keyword(s):

Rna Polymerase ◽

Conformational Changes ◽

Protein Interactions ◽

Structural Information ◽

Double Helix ◽

High Temporal Resolution ◽

Kinetic Properties ◽

Time Resolved ◽

Sequence Recognition

Transcription is often regulated at the level of initiation by the presence of transcription factors or nucleoid proteins or by changing concentrations of metabolites. These can influence the kinetic properties and/or structures of the intermediate RNA polymerase–DNA complexes in the pathway. Time-resolved footprinting techniques combine the high temporal resolution of a stopped-flow apparatus with the specific structural information obtained by the probing agent. Combined with a careful quantitative analysis of the evolution of the signals, this approach allows for the identification and kinetic and structural characterization of the intermediates in the pathway of DNA sequence recognition by a protein, such as a transcription factor or RNA polymerase. The combination of different probing agents is especially powerful in revealing different aspects of the conformational changes taking place at the protein–DNA interface. For example, hydroxyl radical footprinting, owing to their small size, provides a map of the solvent-accessible surface of the DNA backbone at a single nucleotide resolution; modification of the bases using potassium permanganate can reveal the accessibility of the bases when the double helix is distorted or melted; cross-linking experiments report on the formation of specific amino acid–DNA contacts, and DNase I footprinting results in a strong signal-to-noise ratio from DNA protection at the binding site and hypersensitivity at curved or kinked DNA sites. Recent developments in protein footprinting allow for the direct characterization of conformational changes of the proteins in the complex.

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Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa291 ◽

2020 ◽

Vol 75 (11) ◽

pp. 3209-3217 ◽

Cited By ~ 3

Author(s):

Jorge Arca-Suárez ◽

Juan Carlos Vázquez-Ucha ◽

Pablo Arturo Fraile-Ribot ◽

Emilio Lence ◽

Gabriel Cabot ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Conformational Changes ◽

Catalytic Efficiency ◽

The Novel ◽

Development Of Resistance ◽

New Variant ◽

Ic50 Values ◽

Dynamics Simulations

Abstract Background Pseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. Objectives To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. Methods Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. Results WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. Conclusions We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa.

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Preparation and characterization of porous TiO2-SiO2 mixed oxide

MRS Proceedings ◽

10.1557/proc-848-ff3.25 ◽

2004 ◽

Vol 848 ◽

Author(s):

Ang Thiam Peng ◽

Zhong Ziyi ◽

James Highfield

Keyword(s):

Electron Microscopy ◽

Mixed Oxide ◽

Mixed Oxides ◽

Structural Information ◽

High Surface ◽

X Ray Diffraction ◽

Scanning Calorimetry ◽

Surface Areas ◽

Transmission Electron

ABSTRACTA study on the comparison of porous TiO2-SiO2 mixed oxides synthesized variously via the “amine directed” method is reported. The amine capping groups were octylamine, dodecylamine, octyldecylamine, aniline, and isobutylamine. The mixed oxide is characterized by x-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), infrared spectroscopy (IR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and Brunauer-Emmett-Teller analysis (BET). While XRD, SEM and TEM provide mainly (bulk) structural information on the mixed oxide preparations, BET (N2 physisorption) probes into their surface area and texture. IR evidence suggests that intimate chemical mixing of both oxides has occurred, while BET measurements show that high surface areas are attainable and that the mixed oxide is more thermally stable than pure (control) samples of TiO2.

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Surprisingly Fast Interface and Elbow Angle Dynamics of Antigen-Binding Fragments

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2020.609088 ◽

2020 ◽

Vol 7 ◽

Author(s):

Monica L. Fernández-Quintero ◽

Katharina B. Kroell ◽

Martin C. Heiss ◽

Johannes R. Loeffler ◽

Patrick K. Quoika ◽

...

Keyword(s):

Light Chain ◽

Variable Region ◽

Affinity Maturation ◽

Antigen Binding ◽

Antigen Specificity ◽

Antigen Binding Site ◽

Elbow Angle ◽

Distinct Distribution ◽

Complementarity Determining Region

Fab consist of a heavy and light chain and can be subdivided into a variable (VH and VL) and a constant region (CH1 and CL). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable loops, shaping the antigen binding site, the paratope. Apart from the CDR loops, both the elbow angle and the relative interdomain orientations of the VH–VL and the CH1–CL domains influence the shape of the paratope. Thus, characterization of the interface and elbow angle dynamics is essential to antigen specificity. We studied nine antigen-binding fragments (Fab) to investigate the influence of affinity maturation, antibody humanization, and different light-chain types on the interface and elbow angle dynamics. While the CDR loops reveal conformational transitions in the micro-to-millisecond timescale, both the interface and elbow angle dynamics occur on the low nanosecond timescale. Upon affinity maturation, we observe a substantial rigidification of the VH and VL interdomain and elbow-angle flexibility, reflected in a narrower and more distinct distribution. Antibody humanization describes the process of grafting non-human CDR loops onto a representative human framework. As the antibody framework changes upon humanization, we investigated if both the interface and the elbow angle distributions are changed or shifted. The results clearly showed a substantial shift in the relative VH–VL distributions upon antibody humanization, indicating that different frameworks favor distinct interface orientations. Additionally, the interface and elbow angle dynamics of five antibody fragments with different light-chain types are included, because of their strong differences in elbow angles. For these five examples, we clearly see a high variability and flexibility in both interface and elbow angle dynamics, highlighting the fact that Fab interface orientations and elbow angles interconvert between each other in the low nanosecond timescale. Understanding how the relative interdomain orientations and the elbow angle influence antigen specificity, affinity, and stability has broad implications in the field of antibody modeling and engineering.

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Purification and characterization of GAD65-specific monoclonal autoantibodies

F1000Research ◽

10.12688/f1000research.6467.1 ◽

2015 ◽

Vol 4 ◽

pp. 135 ◽

Cited By ~ 2

Author(s):

Wei Jiang ◽

Henriette Macmillan ◽

Anne-Marie Madec ◽

Elizabeth D. Mellins

Keyword(s):

Molecular Mechanisms ◽

Critical Role ◽

Antigen Binding ◽

Healthy Individuals ◽

Β Cells ◽

Purification And Characterization ◽

Antigen Binding Site ◽

Patients At Risk

Autoantibodies against antigens expressed by insulin-producing β cells are circulating in both healthy individuals and patients at risk of developing Type 1 diabetes. Recent studies suggest that another set of antibodies (anti-idiotypic antibodies) exists in this antibody/antigen interacting network to regulate auto-reactive responses. Anti-idiotypic antibodies may block the antigen-binding site of autoantibodies or inhibit autoantibody expression and secretion. The equilibrium between autoantibodies and anti-idiotypic antibodies plays a critical role in mediating or preventing autoimmunity. Herein, using GAD65/anti-GAD65 autoantibodies as a model system, we aimed at establishing reliable approaches for purification of highly pure autoantibodies for the downstream investigation of molecular mechanisms underlying such a network.

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Excited-state distortions control the reactivities and regioselectivities of photochemical 4π-electrocyclizations of fluorobenzenes

10.26434/chemrxiv-2021-6csxm ◽

2021 ◽

Author(s):

Jingbai Li ◽

Steven Lopez

Keyword(s):

Excited State ◽

Structural Information ◽

Computational Cost ◽

Conical Intersections ◽

Surface Hopping ◽

Plane Analysis ◽

Nonadiabatic Molecular Dynamics ◽

Jahn Teller ◽

Dynamics Simulations

The photochemistry of benzene is complex and non-selective because numerous mechanistic pathways are accessible in the ground- and excited-states. Fluorination is a known strategy to increase the chemoselectivities for Dewar-benzenes via 4π-disrotatory electrocyclization. However, the origin of the chemo- and regioselectivities of fluorobenzenes remains unexplained because of experimental limitations in resolving the excited-state structures on ultrafast timescales. The computational cost of multiconfigurational nonadiabatic molecular dynamics simulations is also generally prohibitive. We now provide high-fidelity structural information and reaction outcome predictions with machine-learning-accelerated photodynamics simulations of a series of fluorobenzenes, C6F6-nHn, n=0–3 to study their S1→S0 decay in 4 ns. We trained neural networks with XMS-CASPT2(6,7)/aug-cc-pVDZ calculations, which reproduced the S1 absorption features with mean absolute errors of 0.04 eV (< 2 nm). The predicted S1 excited-state lifetimes for C6F4H2, C6F6, C6F5H, and C6F3H3 are 64, 40, 18, and 8 ps, respectively. The trend is in excellent agreement with the experimental lifetimes. Our calculations show that the pseudo Jahn-Teller distortions create the S1 minimum region that prolongs the excited-state lifetime of fluorobenzenes. The pseudo Jahn-Teller distortions reduce when fluorination decreases. Characterization of the surface hopping structures suggests that the S1 relaxation first involves a cis-trans isomerization of a 𝜋C-C-bond in the benzene ring, promoted by the pseudo-Jahn-Teller distortions. A branching plane analysis revealed that the conical intersections favoring 4π-electrocyclization are less energetically accessible through the S1 relaxation; lower-energy conical intersections resemble the reactant and favor reversion.

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Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

10.1101/2021.12.22.473911 ◽

2021 ◽

Author(s):

Mohanraj Gopalswamy ◽

Tobias Kroeger ◽

David Bickel ◽

Benedikt Frieg ◽

Shahina Akter ◽

...

Keyword(s):

Small Molecule ◽

Conformational Changes ◽

Chromosomal Translocation ◽

Small Molecule Inhibitor ◽

Molecule Inhibitor ◽

Immature Myeloid Cells ◽

Dynamics Simulations ◽

Tetramer Stability

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 +/- 1.28 micromolar. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

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