On the Role of Flexibility in Protein–Ligand Interactions: the Example of p53 Tetramerization Domain

The recognition of protein surfaces by designed ligands has become an attractive approach in drug discovery. However, the variable nature and irregular behavior of protein surfaces defy this new area of research. The easy to understand “lock‐and‐key” model is far from being the ideal paradigm in biomolecular interactions and, hence, any new finding on how proteins and ligands behave in recognition events paves a step of the way. Herein, we illustrate a clear example on how an increase in flexibility of both protein and ligand can result in an increase in the stability of the macromolecular complex. The biophysical study of the interaction between a designed flexible tetraguanidinium‐calix[4]arene and the tetramerization domain of protein p53 (p53TD) and its natural mutant p53TD‐R337H shows how the floppy mutant domain interacts more tightly with the ligand than the well‐packed wild‐type protein. Moreover, the flexible calixarene ligand interacts with higher affinity to both wild‐type and mutated protein domains than a conformationally rigid calixarene analog previously reported. These findings underscore the crucial role of flexibility in molecular recognition processes, for both small ligands and large biomolecular surfaces.     

The Thermodynamic Influence of Trapped Water Molecules on a Protein–Ligand Interaction

Water molecules doing time : Atomic‐resolution crystal structures of the PPIase domain of cyclophilin G, alone and in complex with cyclosporin A, and together with MD simulations and calorimetry, reveal how trapped water molecules influence the thermodynamic profile of a protein–ligand interaction.

     

Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

The allosteric modulation of G‐protein‐coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na⁺ ions bound to the inactive conformations at the conserved Asp^2.50. To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na⁺ binding to the allosteric site are limited. We performed hundreds‐of‐microsecond long simulations of 18 GPCRs and elucidated their Na⁺ binding mechanism. In class A GPCRs, the Na⁺ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na⁺ binding to the allosteric site, which reveal a conserved mechanism of Na⁺ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na⁺ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.     

On‐Bead Screens Sample Narrower Affinity Ranges of Protein–Ligand Interactions Compared to Equivalent Solution Assays

Conceptually, on‐bead screening is one of the most efficient high‐throughput screening (HTS) methods. One of its inherent advantages is that the solid support has a dual function: it serves as a synthesis platform and as a screening compartment. Compound purification, cleavage and storage and extensive liquid handling are not necessary in bead‐based HTS. Since the establishment of one‐bead one‐compound library synthesis, the properties of polymer beads in chemical reactions have been thoroughly investigated. However, the characterization of the kinetics and thermodynamics of protein–ligand interactions on the beads used for screening has received much less attention. Consequently, the majority of reported on‐bead screens are based on empirically derived procedures, independent of measured equilibrium constants and rate constants of protein binding to ligands on beads. More often than not, on‐bead screens reveal apparent high affinity binders through strong protein complexation on the matrix of the solid support. After decoding, resynthesis, and solution testing the primary hits turn out to be unexpectedly weak binders, or may even fall out of the detection limit of the solution assay. Only a quantitative comparison of on‐bead binding and solution binding events will allow systematically investigating affinity differences as function of protein and small molecule properties. This will open up routes for optimized bead materials, blocking conditions and other improved assay procedures. By making use of the unique features of our previously introduced confocal nanoscanning (CONA) method, we investigated the kinetic and thermodynamic properties of protein–ligand interactions on TentaGel beads, a popular solid support for on‐bead screening. The data obtained from these experiments allowed us to determine dissociation constants for the interaction of bead‐immobilized ligands with soluble proteins. Our results therefore provide, for the first time, a comparison of on‐bead versus solution binding thermodynamics. Our data indicate that affinity ranges found in on‐bead screening are indeed narrower compared to equivalent interactions in homogeneous solution. A thorough physico‐chemical understanding of the molecular recognition between proteins and surface bound ligands will further strengthen the role of on‐bead screening as an ultimately cost‐effective method in hit and lead finding.     

Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein–Ligand Interactions

Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.     

Flexibility and Rigidity of Proteins and Protein–Pigment Complexes

Proteins may be rigid or flexible to various degrees as required for optimal function. Flexibility of large parts of a protein, which rearrange or move, is particularly interesting and will be discussed in this article. We differentiate between several categories, although the boundaries between them are diffuse: flexibility of peptide segments, order–disorder transitions of spatially contiguous regions, and domain motions. The domains may be flexibly linked to allow rather unrestricted motions or the motions may be constrained to certain modes. The various categories of large-scale flexibility will be illustrated with the following examples: (1) Small protein proteinase inhibitors are rather rigid molecules which provide binding surfaces complementary to their cognate proteases but show also limited segmental flexibility and adaptation. (2) Large plasma proteinase inhibitors exhibit large conformational changes after interaction with proteases probably for regulatory purposes. (3) Pancreatic serine proteases employ a disorder–order transition of their activation domain as a means to regulate enzymic activity. (4) Immunoglobulins show rather unrestricted and also hinged domain motions in different parts of the molecule probably to allow binding to antigens in different arrangements. (5) Citrate synthase adopts open and closed forms by a hinged domain motion to bind substrates and release products and to perform the catalytic condensation reaction, respectively. (6) Riboflavin synthase, a bifunctional multienzyme complex, catalyzes two consecutive reactions by means of two subunits, α and β. The β-subunits form a shell, in which the α-subunits are enclosed. Diffusional motion of the catalytic intermediates is therefore restricted. In addition, rearrangement of the N-terminal segment occurs during the assembly of the β-subunit. In contrast, rigidity is dominant in the structures of the light-harvesting complexes and the photosynthetic reaction centers involved in photosynthetic light reactions. These are large protein–pigment complexes in which the proteins serve as matrices to hold the pigments in the appropriate conformation and relative arrangement. Since motion would contribute to deactivation of the photoexcited states of the pigments and diminish the efficiency of light-energy and electron transfer, the functional role of rigidity is easy to rationalize for these proteins.     

Rationally Designed Semisynthetic Natural Product Analogues for Stabilization of 14‐3‐3 Protein–Protein Interactions

The natural product family of fusicoccanes are stabilizers of 14‐3‐3 mediated protein–protein interactions (PPIs), some of which possess antitumor activity. In this study, the first use of molecular dynamics (MD) to rationally design PPI stabilizers with increased potency is presented. Synthesis of a focused library, with subsequent characterization by fluorescence polarization, mutational studies, and X‐ray crystallography confirmed the power of the MD‐based design approach, revealing the potential for an additional hydrogen bond with the 14‐3‐3 protein to lead to significantly increased potency. Additionally, these compounds exert their action in a cellular environment with increased potency. The newly found polar interaction could provide an anchoring point for new small‐molecule PPI stabilizers. These results facilitate the development of fusicoccanes towards drugs or tool compounds, as well as allowing the study of the fundamental principles behind PPI stabilization.     

Full-Atom Model of the Agonist LPS-Bound Toll-like Receptor 4 Dimer in a Membrane Environment

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) innate immunity system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We have combined ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies give functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular Toll/Interleukin-1 receptor domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS)₂ architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Kinetic Analysis and Structural Interpretation of Competitive Ligand Binding for NO Dioxygenation in Truncated Hemoglobin N

The conversion of nitric oxide (NO) into nitrate (NO₃^?) by dioxygenation protects cells from lethal NO. Starting from NO‐bound heme, the first step in converting NO into benign NO₃^? is the ligand exchange reaction FeNO+O₂→FeO₂+NO, which is still poorly understood at a molecular level. For wild‐type (WT) truncated hemoglobin N (trHbN) and its Y33A mutant, the calculated barriers for the exchange reaction differ by 1.5 kcal mol^?1, compared with 1.7 kcal mol^?1 from experiment. It is directly confirmed that the ligand exchange reaction is rate‐limiting in trHbN and that entropic contributions account for 75 % of the difference between the WT and the mutant. Residues Tyr 33, Phe 46, Val 80, His 81, and Gln 82 surrounding the active site are expected to control the reaction path. By comparison with electronic structure calculations, the transition state separating the two ligand‐bound states was assigned to a ²A state.     

Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding

Aberrant RNA–protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA–protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.     

Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3

The chemokine receptor CXCR3 is a G protein‐coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small‐molecule allosteric CXCR3‐agonists do not bind to the same allosteric binding pocket as 8‐azaquinazolinone‐based negative allosteric modulators. We have now performed molecular‐dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3‐binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a “shallow” and a second “deeper” pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.