ABSTRACT
The electrostatic properties of proteins arise from the number and distribution of polar and charged residues. Electrostatic interactions in proteins play a critical role in numerous processes such as molecular recognition, protein solubility, viscosity, and antibody developability. Thus, characterizing and quantifying electrostatic properties of a protein are prerequisites for understanding these processes. Here, we present PEP-Patch, a tool to visualize and quantify the electrostatic potential on the protein surface in terms of surface patches, denoting separated areas of the surface with a common physical property. We highlight its applicability to elucidate protease substrate specificity and antibody-antigen recognition and predict heparin column retention times of antibodies as an indicator of pharmacokinetics.
Subject(s)
Antibodies , Proteins , Static Electricity , Proteins/chemistry , Solubility , ViscosityABSTRACT
Hydration thermodynamics play a fundamental role in fields ranging from the pharmaceutical industry to environmental research. Numerous methods exist to predict solvation thermodynamics of compounds ranging from small molecules to large biomolecules. Arguably the most precise methods are those based on molecular dynamics (MD) simulations in explicit solvent. One theory that has seen increased use is inhomogeneous solvation theory (IST). However, while many applications require accurate description of salt-water mixtures, no implementation of IST is currently able to estimate solvation properties involving more than one solvent species. Here, we present an extension to grid inhomogeneous solvation theory (GIST) that can take salt contributions into account. At the example of carbazole in 1 M NaCl solution, we compute the solvation energy as well as first and second order entropies. While the effect of the first order ion entropy is small, both the water-water and water-ion entropies contribute strongly. We show that the water-ion entropies are efficiently approximated using the Kirkwood superposition approximation. However, this approach cannot be applied to the water-water entropy. Furthermore, we test the quantitative validity of our method by computing salting-out coefficients and comparing them to experimental data. We find a good correlation to experimental salting-out constants, while the absolute values are overpredicted due to the approximate second order entropy. Since ions are frequently used in MD, either to neutralize the system or as a part of the investigated process, our method greatly extends the applicability of GIST. The use-cases range from biopharmaceuticals, where many assays require high salt concentrations, to environmental research, where solubility in sea water is important to model the fate of organic substances.
Subject(s)
Gastrointestinal Stromal Tumors , Entropy , Humans , Solvents , Thermodynamics , WaterABSTRACT
FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.
Subject(s)
Enzyme Inhibitors/pharmacology , Hydrolases/antagonists & inhibitors , Enzyme Inhibitors/metabolism , Humans , Hydrolases/chemistry , Hydrolases/metabolism , Molecular Docking Simulation , Protein ConformationABSTRACT
The use of fragments to biophysically characterize a protein binding pocket and determine the strengths of certain interactions is a computationally and experimentally commonly applied approach. Almost all drug like molecules contain at least one aromatic moiety forming stacking interactions in the binding pocket. In computational drug design, the strength of stacking and the resulting optimization of the aromatic core or moiety is usually calculated using high level quantum mechanical approaches. However, as these calculations are performed in a vacuum, solvation properties are neglected. We close this gap by using Grid Inhomogeneous Solvation Theory (GIST) to describe the properties of individual heteroaromatics and complexes and thereby estimate the desolvation penalty. In our study, we investigated the solvation free energies of heteroaromatics frequently occurring in drug design projects in complex with truncated side chains of phenylalanine, tyrosine, and tryptophan. Furthermore, we investigated the properties of drug-fragments crystallized in a fragment-based lead optimization approach investigating PDE-10-A. We do not only find good correlation for the estimated desolvation penalty and the experimental binding free energy, but our calculations also allow us to predict prominent interaction sites. We highlight the importance of including the desolvation penalty of the respective heteroaromatics in stacked complexes to explain the gain or loss in affinity of potential lead compounds.
Subject(s)
Drug Design , Pharmaceutical Preparations , Protein Binding , Tyrosine , Binding Sites , Quantum Theory , ThermodynamicsABSTRACT
Hydration is one of the key players in the protein-ligand binding process. It not only influences the binding process per se, but also the drug's absorption, distribution, metabolism, and excretion properties. To gain insights into the hydration of aromatic cores, the solvation thermodynamics of 40 aromatic mono- and bicyclic systems, frequently occurring in medicinal chemistry, are investigated. Thermodynamics is analyzed with two different methods: grid inhomogeneous solvation theory (GIST) and thermodynamic integration (TI). Our results agree well with previously published experimental and computational solvation free energies. The influence of adding heteroatoms to aromatic systems and how the position of these heteroatoms impacts the compound's interactions with water is studied. The solvation free energies of these heteroaromatics are highly correlated to their gas phase interaction energies with benzene: compounds showing a high interaction energy also have a high solvation free energy value. Therefore, replacing a compound with one having a higher gas phase interaction energy might not result in the expected improvement in affinity. The desolvation costs counteract the higher stacking interactions, hence weakening or even inverting the expected gain in binding free energy.
Subject(s)
Drug Design , Heterocyclic Compounds/chemistry , Hydrocarbons, Aromatic/chemistry , Computational Chemistry , Humans , Molecular Structure , Thermodynamics , WaterABSTRACT
Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible 'lid'-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes.
Subject(s)
Amino Acids/metabolism , Carboxy-Lyases/metabolism , Hydrolases/metabolism , Mitochondrial Proteins/metabolism , Amino Acids/chemistry , Amino Acids/genetics , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Catalytic Domain , Crystallography, X-Ray , Humans , Hydrolases/chemistry , Hydrolases/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Models, Molecular , Mutation , Protein Conformation , Pyruvates/chemistry , Pyruvates/metabolism , Substrate SpecificityABSTRACT
Prokaryotic and eukaryotic fumarylacetoacetate hydrolase (FAH) superfamily members, sharing conserved regions that form the so-called FAH-domain, catalyze a remarkable variety of reactions. These enzymes are essential in the metabolic pathways to degrade aromatic compounds in prokaryotes and eukaryotes. It appears that prokaryotic FAH superfamily members evolved mainly to allow microbes to generate energy and useful metabolites from complex carbon sources. We review recent findings, indicating that both prokaryotic and eukaryotic members of the FAH superfamily also display oxaloacetate decarboxylase (ODx) activity. The identification of human FAH domain-containing protein 1 as mitochondrial ODx regulating mitochondrial function supports the new concept that, during evolution, eukaryotic FAH superfamily members have acquired important regulatory functions beyond catabolism of complex carbon sources. Molecular studies on the evolution and function of FAH superfamily members are expected to provide new mechanistic insights in their physiological roles.
Subject(s)
Hydrolases/metabolism , Microbiota , Mitochondria/enzymology , Amino Acid Sequence , Carboxy-Lyases/metabolism , Humans , Hydrolases/chemistry , Sequence Homology, Amino Acid , Tyrosine/metabolismABSTRACT
Urea derivatives are ubiquitously found in many chemical disciplines. N,N'-substituted ureas may show different conformational preferences depending on their substitution pattern. The high energetic barrier for isomerization of the cis and trans state poses additional challenges on computational simulation techniques aiming at a reproduction of the biological properties of urea derivatives. Herein, we investigate energetics of urea conformations and their interconversion using a broad spectrum of methodologies ranging from data mining, via quantum chemistry to molecular dynamics simulation and free energy calculations. We find that the inversion of urea conformations is inherently slow and beyond the time scale of typical simulation protocols. Therefore, extra care needs to be taken by computational chemists to work with appropriate model systems. We find that both knowledge-driven approaches as well as physics-based methods may guide molecular modelers towards accurate starting structures for expensive calculations to ensure that conformations of urea derivatives are modeled as adequately as possible.
Subject(s)
Drug Design , Molecular Conformation , Molecular Dynamics Simulation , Pharmaceutical Preparations/chemistry , Computer-Aided Design , Data Mining , Humans , Isomerism , Quantum Theory , ThermodynamicsABSTRACT
Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.
Subject(s)
Protein Folding , Water , Proteins , Molecular Dynamics Simulation , Molecular Conformation , Thermodynamics , Protein Conformation , Protein UnfoldingABSTRACT
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.
ABSTRACT
The use of volatile biomarkers in exhaled breath as predictors to individual drug response would advance the field of personalised medicine by providing direct information on enzyme activity. This would result in enormous benefits, both for patients and for the healthcare sector. Non-invasive breath tests would also gain a high acceptance by patients. Towards this goal, differences in metabolism resulting from extensive polymorphisms in a major group of drug-metabolizing enzymes, the cytochrome P450 (CYP) family, need to be determined and quantified. CYP2C9 is responsible for metabolising many crucial drugs (e.g., diclofenac) and food ingredients (e.g., limonene). In this paper, we provide a proof-of-concept study that illustrates thein vitrobioconversion of diclofenac in recombinant HEK293T cells overexpressing CYP2C9 to 4'-hydroxydiclofenac. Thisin vitroapproach is a necessary and important first step in the development of breath tests to determine and monitor metabolic processes in the human body. By focusing on the metabolic conversion of diclofenac, we have been able to establish a workflow using a cell-based system for CYP2C9 activity. Furthermore, we illustrate how the bioconversion of diclofenac is limited in the presence of limonene, which is another CYP2C9 metabolising substrate. We show that increasing limonene levels continuously reduce the production of 4'-hydroxydiclofenac. Michaelis-Menten kinetics were performed for the diclofenac 4'-hydroxylation with and without limonene, giving a kinetic constant of the reaction,KM, of 103µM and 94.1µM, respectively, and a maximum reaction rate,Vmax, of 46.8 pmol min-1106cells-1and 56.0 pmol min-1106cells-1with and without the inhibitor, respectively, suggesting a non-competitive or mixed inhibition type. The half-maximal inhibitory concentration value (IC50) for the inhibition of the formation of 4'-hydroxydiclofenace by limonene is determined to be 1413µM.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Diclofenac , Humans , Diclofenac/metabolism , Diclofenac/pharmacology , Cytochrome P-450 CYP2C9/genetics , Cytochrome P-450 CYP2C9/metabolism , Aryl Hydrocarbon Hydroxylases/metabolism , HEK293 Cells , Limonene , Precision Medicine , Workflow , Breath Tests , Cytochrome P-450 Enzyme System/metabolismABSTRACT
Antibodies and other new antibody-like formats have emerged as one of the most rapidly growing classes of biotherapeutic proteins. Understanding the structural features that drive antibody function and, consequently, their molecular recognition is critical for engineering antibodies. Here, we present the structural architecture of conventional IgG antibodies alongside other formats. We emphasize the importance of considering antibodies as conformational ensembles in solution instead of focusing on single-static structures because their functions and properties are strongly governed by their dynamic nature. Thus, in this review, we provide an overview of the unique structural and dynamic characteristics of antibodies with respect to their antigen recognition, biophysical properties, and effector functions. We highlight the numerous technical advances in antibody structure prediction and design, enabled by the vast number of experimentally determined high-quality structures recorded with cryo-EM, NMR, and X-ray crystallography. Lastly, we assess antibody and vaccine design strategies in the context of structure and dynamics.
ABSTRACT
Recycling IgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.
Subject(s)
Bacteriophages , Histidine , Histidine/metabolism , Antigens/metabolism , Immunoglobulin G/genetics , Hydrogen-Ion Concentration , Bacteriophages/metabolism , Peptide LibraryABSTRACT
Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations, we show that a single static X-ray structure is not sufficient to identify determinants of antibody-antigen recognition. Here, we investigate antibodies that undergo substantial conformational changes upon antigen binding and have been classified as difficult cases in an extensive benchmark for antibody-antigen docking. We present thermodynamics and transition kinetics of these conformational rearrangements and show that paratope states can be used to improve antibody-antigen docking. By using the unbound antibody X-ray structure as starting structure for molecular dynamics simulations, we retain a binding competent conformation substantially different to the unbound antibody X-ray structure. We also observe that the kinetically dominant antibody paratope conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that paratope states in solution can improve antibody-antigen docking and structure prediction.
Subject(s)
Antibodies , Antigens , Antibodies/metabolism , Antigens/chemistry , Binding Sites, Antibody , Protein Binding , Protein ConformationABSTRACT
[This corrects the article DOI: 10.3389/fmolb.2022.812750.].
ABSTRACT
As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.
ABSTRACT
Stacking interactions play a crucial role in drug design, as we can find aromatic cores or scaffolds in almost any available small molecule drug. To predict optimal binding geometries and enhance stacking interactions, usually high-level quantum mechanical calculations are performed. These calculations have two major drawbacks: they are very time consuming, and solvation can only be considered using implicit solvation. Therefore, most calculations are performed in vacuum. However, recent studies have revealed a direct correlation between the desolvation penalty, vacuum stacking interactions and binding affinity, making predictions even more difficult. To overcome the drawbacks of quantum mechanical calculations, in this study we use neural networks to perform fast geometry optimizations and molecular dynamics simulations of heteroaromatics stacked with toluene in vacuum and in explicit solvation. We show that the resulting energies in vacuum are in good agreement with high-level quantum mechanical calculations. Furthermore, we show that using explicit solvation substantially influences the favored orientations of heteroaromatic rings thereby emphasizing the necessity to include solvation properties starting from the earliest phases of drug design.
ABSTRACT
Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed of 4 different kappa light chains paired with 4 different heavy chains. In this study, we systematically show that different heavy and light chain pairings strongly influence the paratope, interdomain interaction patterns and the relative VH-VL interface orientations. We observe changes in conformational diversity and substantial population shifts of the complementarity determining region (CDR) loops, resulting in distinct dominant solution structures and differently favored canonical structures. Additionally, we identify conformational changes in the structural diversity of the CDR-H3 loop upon different heavy and light chain pairings, as well as upon changes in sequence and structure of the neighboring CDR loops, despite having an identical CDR-H3 loop amino acid sequence. These results can also be transferred to all CDR loops and to the relative VH-VL orientation, as certain paratope states favor distinct interface angle distributions. Furthermore, we directly compare the timescales of sidechain rearrangements with the well-described transition kinetics of conformational changes in the backbone of the CDR loops. We show that sidechain flexibilities are strongly affected by distinct heavy and light chain pairings and decipher germline-specific structural features co-determining stability. These findings reveal that all CDR loops are strongly correlated and that distinct heavy and light chain pairings can result in different paratope states in solution, defined by a characteristic combination of CDR loop conformations and VH-VL interface orientations. Thus, these results have broad implications in the field of antibody engineering, as they clearly show the importance of considering paired heavy and light chains to understand the antibody binding site, which is one of the key aspects in the design of therapeutics.
Subject(s)
Binding Sites, Antibody , Germ Cells/immunology , Molecular Dynamics Simulation , Complementarity Determining Regions/chemistry , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Light Chains/chemistry , Immunoglobulin Variable Region/chemistry , Protein ConformationABSTRACT
During the affinity maturation process the immune system produces antibodies with higher specificity and activity through various rounds of somatic hypermutations in response to an antigen. Elucidating the affinity maturation process is fundamental in understanding immunity and in the development of biotherapeutics. Therefore, we analyzed 10 pairs of antibody fragments differing in their specificity and in distinct stages of affinity maturation using metadynamics in combination with molecular dynamics (MD) simulations. We investigated differences in flexibility of the CDR-H3 loop and global changes in plasticity upon affinity maturation. Among all antibody pairs we observed a substantial rigidification in flexibility and plasticity reflected in a substantial decrease of conformational diversity. To visualize and characterize these findings we used Markov-states models to reconstruct the kinetics of CDR-H3 loop dynamics and for the first time provide a method to define and localize surface plasticity upon affinity maturation.
ABSTRACT
T-cell receptors are an important part in the adaptive immune system as they are responsible for detecting foreign proteins presented by the major histocompatibility complex (MHC). The affinity is predominantly determined by structure and sequence of the complementarity determining regions (CDRs), of which the CDR3 loops are responsible for peptide recognition. We present a kinetic classification of T-cell receptor CDR3 loops with different loop lengths into canonical and non-canonical solution structures. Using molecular dynamics simulations, we do not only sample available X-ray structures, but we also observe a substantially broader CDR3 loop ensemble with various distinct kinetic minima in solution. Our results strongly imply, that for given CDR3 loop sequences several canonical structures have to be considered to characterize the conformational diversity of these loops. Our suggested dominant solution structures could extend the repertoire of available canonical clusters by including kinetic minimum structures present in solution. Thus, the CDR3 loops need to be characterized as conformational ensembles in solution. Furthermore, the conformational changes of the CDR3 loops follow the paradigm of conformational selection, because the experimentally determined binding competent state is present within this ensemble of pre-existing conformations without the presence of the antigen. We also identify strong correlations between the CDR3 loops and include combined state descriptions. Additionally, we observe a strong dependency of the CDR3 loop conformations on the relative Vα-Vß interdomain orientations, revealing that certain CDR3 loop states favor specific interface orientations.