Biomolecular recognition between proteins follows complex mechanisms, the understanding of which can substantially advance drug discovery efforts. Here, we track each step of the binding process in ...atomistic detail with molecular dynamics simulations using trypsin and its inhibitor bovine pancreatic trypsin inhibitor (BPTI) as a model system. We use umbrella sampling to cover a range of unbinding pathways. Starting from these simulations, we subsequently seed classical simulations at different stages of the process and combine them to a Markov state model. We clearly identify three kinetically separated states (an unbound state, an encounter state, and the final complex) and describe the mechanisms that dominate the binding process. From our model, we propose the following sequence of events. The initial formation of the encounter complex is driven by long-range interactions because opposite charges in trypsin and BPTI draw them together. The encounter complex features the prealigned binding partners with binding sites still partially surrounded by solvation shells. Further approaching leads to desolvation and increases the importance of van der Waals interactions. The native binding pose is adopted by maximizing short-range interactions. Thereby side-chain rearrangements ensure optimal shape complementarity. In particular, BPTI’s P1 residue adapts to the S1 pocket and prime site residues reorient to optimize interactions. After the paradigm of conformation selection, binding-competent conformations of BPTI and trypsin are already present in the apo ensembles and their probabilities increase during this proposed two-step association process. This detailed characterization of the molecular forces driving the binding process includes numerous aspects that have been discussed as central to the binding of trypsin and BPTI and protein complex formation in general. In this study, we combine all these aspects into one comprehensive model of protein recognition. We thereby contribute to enhance our general understanding of this fundamental mechanism, which is particularly critical as the development of biopharmaceuticals continuously gains significance.
Molecular dynamics simulations are an invaluable tool to characterize the dynamic motions of proteins in atomistic detail. However, the accuracy of models derived from simulations inevitably relies ...on the quality of the underlying force field. Here, we present an evaluation of current non-polarizable and polarizable force fields (AMBER ff14SB, CHARMM 36m, GROMOS 54A7, and Drude 2013) based on the long-standing biophysical challenge of protein folding. We quantify the thermodynamics and kinetics of the β-hairpin formation using Markov state models of the fast-folding mini-protein CLN025. Furthermore, we study the (partial) folding dynamics of two more complex systems, a villin headpiece variant and a WW domain. Surprisingly, the polarizable force field in our set, Drude 2013, consistently leads to destabilization of the native state, regardless of the secondary structure element present. All non-polarizable force fields, on the other hand, stably characterize the native state ensembles in most cases even when starting from a partially unfolded conformation. Focusing on CLN025, we find that the conformational space captured with AMBER ff14SB and CHARMM 36m is comparable, but the ensembles from CHARMM 36m simulations are clearly shifted toward disordered conformations. While the AMBER ff14SB ensemble overstabilizes the native fold, CHARMM 36m and GROMOS 54A7 ensembles both agree remarkably well with experimental state populations. In addition, GROMOS 54A7 also reproduces experimental folding times most accurately. Our results further indicate an over-stabilization of helical structures with AMBER ff14SB. Nevertheless, the presented investigations strongly imply that reliable (un)folding dynamics of small proteins can be captured in feasible computational time with current additive force fields.
Abstract
To date, more than 263 million people have been infected with SARS-CoV-2 during the COVID-19 pandemic. In many countries, the global spread occurred in multiple pandemic waves characterized ...by the emergence of new SARS-CoV-2 variants. Here we report a sequence and structural-bioinformatics analysis to estimate the effects of amino acid substitutions on the affinity of the SARS-CoV-2 spike receptor binding domain (RBD) to the human receptor hACE2. This is done through qualitative electrostatics and hydrophobicity analysis as well as molecular dynamics simulations used to develop a high-precision empirical scoring function (ESF) closely related to the linear interaction energy method and calibrated on a large set of experimental binding energies. For the latest variant of concern (VOC), B.1.1.529 Omicron, our Halo difference point cloud studies reveal the largest impact on the RBD binding interface compared to all other VOC. Moreover, according to our ESF model, Omicron achieves a much higher ACE2 binding affinity than the wild type and, in particular, the highest among all VOCs except Alpha and thus requires special attention and monitoring.
The relation of surface polarity and conformational preferences is decisive for cell permeability and thus bioavailability of macrocyclic drugs. Here, we employ grid inhomogeneous solvation theory ...(GIST) to calculate solvation free energies for a series of six macrocycles in water and chloroform as a measure of passive membrane permeability. We perform accelerated molecular dynamics simulations to capture a diverse structural ensemble in water and chloroform, allowing for a direct profiling of solvent-dependent conformational preferences. Subsequent GIST calculations facilitate a quantitative measure of solvent preference in the form of a transfer free energy, calculated from the ensemble-averaged solvation free energies in water and chloroform. Hence, the proposed method considers how the conformational diversity of macrocycles in polar and apolar solvents translates into transfer free energies. Following this strategy, we find a striking correlation of 0.92 between experimentally determined cell permeabilities and calculated transfer free energies. For the studied model systems, we find that the transfer free energy exceeds the purely water-based solvation free energies as a reliable estimate of cell permeability and that conformational sampling is imperative for a physically meaningful model. We thus recommend this purely physics-based approach as a computational tool to assess cell permeabilities of macrocyclic drug candidates.
We present an approach to assess antibody CDR-H3 loops according to their dynamic properties using molecular dynamics simulations. We selected six antibodies in three pairs differing substantially in ...their individual promiscuity respectively specificity. For two pairs of antibodies crystal structures are available in different states of maturation and used as starting structures for the analyses. For a third pair we chose two antibody CDR sequences obtained from a synthetic library and predicted the respective structures. For all three pairs of antibodies we performed metadynamics simulations to overcome the limitations in conformational sampling imposed by high energy barriers. Additionally, we used classic molecular dynamics simulations to describe nano- to microsecond flexibility and to estimate up to millisecond kinetics of captured conformational transitions. The methodology represents the antibodies as conformational ensembles and allows comprehensive analysis of structural diversity, thermodynamics of conformations and kinetics of structural transitions. Referring to the concept of conformational selection we investigated the link between promiscuity and flexibility of the antibodies' binding interfaces. The obtained detailed characterization of the binding interface clearly indicates a link between structural flexibility and binding promiscuity for this set of antibodies.
Here, we demonstrate a method to capture local dynamics on a time scale 3 orders of magnitude beyond state-of-the-art simulation approaches. We apply accelerated molecular dynamics simulations for ...conformational sampling and extract reweighted backbone dihedral distributions. Local dynamics are characterized by torsional probabilities, resulting in residue-wise dihedral entropies. Our approach is successfully validated for three different protein systems of increasing size: alanine dipeptide, bovine pancreatic trypsin inhibitor (BPTI), and the major birch pollen allergen Bet v 1a. We demonstrate excellent agreement of flexibility profiles with both large-scale computer simulations and NMR experiments. Thus, our method provides efficient access to local protein dynamics on extended time scales of high biological relevance.
Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of ...protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.
Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family ...comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.
Despite their structural similarity, members of the chymotrypsin family of serine proteases exhibit striking differences in their substrate preferences. A high correlation between differences in knowledge‐based substrate preferences, focusing only on electrostatics, and in physics‐based electrostatic molecular interaction fields was found. Additionally, we suggest that electrostatic interactions and shape complementarity are orthogonal aspects of substrate recognition.
The equilibrium between active E and inactive E* forms of thrombin is assumed to be governed by the allosteric binding of a Na
ion. Here we use molecular dynamics simulations and Markov state models ...to sample transitions between active and inactive states. With these calculations we are able to compare thermodynamic and kinetic properties depending on the presence of Na
. For the first time, we directly observe sodium-induced conformational changes in long-timescale computer simulations. Thereby, we are able to explain the resulting change in activity. We observe a stabilization of the active form in presence of Na
and a shift towards the inactive form in Na
-free simulations. We identify key structural features to quantify and monitor this conformational shift. These include the accessibility of the S1 pocket and the reorientation of W215, of R221a and of the Na
loop. The structural characteristics exhibit dynamics at various timescales: Conformational changes in the Na
binding loop constitute the slowest observed movement. Depending on its orientation, it induces conformational shifts in the nearby substrate binding site. Only after this shift, residue W215 is able to move freely, allowing thrombin to adopt a binding-competent conformation.
Reliable information on partition coefficients plays a key role in drug development, as solubility decisively affects bioavailability. In a physicochemical context, the partition coefficient of a ...solute between two different solvents can be described as a function of solvation free energies. Hence, substantial scientific efforts have been made toward accurate predictions of solvation free energies in various solvents. The grid inhomogeneous solvation theory (GIST) facilitates the calculation of solvation free energies. In this study, we introduce an extended version of the GIST algorithm, which enables the calculation for chloroform in addition to water. Furthermore, GIST allows localization of enthalpic and entropic contributions. We test our approach by calculating partition coefficients between water and chloroform for a set of eight small molecules. We report a Pearson correlation coefficient of 0.96 between experimentally determined and calculated partition coefficients. The capability to reliably predict partition coefficients between water and chloroform and the possibility to localize their contributions allow the optimization of a compound’s partition coefficient. Therefore, we presume that this methodology will be of great benefit for the efficient development of pharmaceuticals.
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