Conflicting Interfacial Electrostatic Interactions as a Design Principle to Modulate Long-Range Interdomain Communication.

The extent and molecular basis of interdomain communication in multidomain proteins, central to understanding allostery and function, is an open question. One simple evolutionary strategy could involve the selection of either conflicting or favorable electrostatic interactions across the interface of two closely spaced domains to tune the magnitude of interdomain connectivity. Here, we study a bilobed domain FF34 from the eukaryotic p190A RhoGAP protein to explore one such design principle that mediates interdomain communication. We find that while the individual structural units in wild-type FF34 are marginally coupled, they exhibit distinct intrinsic stabilities and low cooperativity, manifesting as slow folding. The FF3-FF4 interface harbors a frustrated network of highly conserved electrostatic interactions-a charge troika-that promotes the population of multiple, decoupled, and non-native structural modes on a rugged native landscape. Perturbing this network via a charge-reversal mutation not only enhances stability and cooperativity but also dampens the fluctuations globally and speeds up the folding rate by at least an order of magnitude. Our work highlights how a conserved but nonoptimal network of interfacial electrostatic interactions shapes the native ensemble of a bilobed protein, a feature that could be exploited in designing molecular systems with long-range connectivity and enhanced cooperativity.

Ensemble origins and distance-dependence of long-range mutational effects in proteins.

Mutational effects in globular proteins exhibit an exponential-like decreasing dependence on distance from the mutated site, suggestive of long-range modulation of structural-thermodynamic features. Here, we extract the physical origins of this pattern by employing a statistical-mechanical model to construct conformational ensembles of three archetypal proteins. Through large-scale in silico alanine-scanning mutagenesis, we show that inter-residue differential coupling free energies, which are characteristic ensemble thermodynamic properties, follow a similar exponential distance dependence with the effects felt until ∼15-20 Å from the mutated site. From the perspective of an ensemble-averaged structure, this feature arises via long-range reorganization of the interaction network on mutations which is more significant for charged residues compared to hydrophobic residues. Our work highlights how subtle alterations in the microscopic distribution of states manifest as a macroscopic distance dependence, the physical origins of mutation-induced dynamic allostery, and the necessity to consider the global intra-protein interaction network to understand mutational outcomes.

Structural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble.

The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural-energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical-mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge-charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a "gatekeeper", modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge-charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains.

A hierarchy of coupling free energies underlie the thermodynamic and functional architecture of protein structures.

Protein sequences and structures evolve by satisfying varied physical and biochemical constraints. This multi-level selection is enabled not just by the patterning of amino acids on the sequence, but also via coupling between residues in the native structure. Here, we employ an energetically detailed statistical mechanical model with millions of microstates to extract such long-range structural correlations, i.e. thermodynamic coupling free energies, from a diverse family of protein structures. We find that despite the intricate and anisotropic distribution of coupling patterns, the majority of residues (>70%) are only marginally coupled contributing to functional motions and catalysis. Physical origins of 'sectors', determinants of native ensemble heterogeneity in extant, ancient and designed proteins, and the basis for allostery emerge naturally from coupling free energies. The statistical framework highlights how evolutionary selection and optimization occur at the level of global interaction network for a given protein fold impacting folding, function, and allosteric outputs.

Structural basis of the pleiotropic and specific phenotypic consequences of missense mutations in the multifunctional NAD(P)H:quinone oxidoreductase 1 and their pharmacological rescue.

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.

Tannic acid prevents macrophage-induced pro-fibrotic response in lung epithelial cells via suppressing TLR4-mediated macrophage polarization.

BACKGROUND: Polarized macrophages induce fibrosis through multiple mechanisms, including a process termed epithelial-to-mesenchymal transition (EMT). Mesenchymal cells contribute to the excessive accumulation of fibrous connective tissues, leading to organ failure. This study was aimed to investigate the effect of tannic acid (TA), a natural dietary polyphenol on M1 macrophage-induced EMT and its underlying mechanisms. MATERIALS: First, we induced M1 polarization in macrophage cell lines (RAW 264.7 and THP-1). Then, the conditioned-medium (CM) from these polarized macrophages was used to induce EMT in the human adenocarcinomic alveolar epithelial (A549) cells. We also analysed the role of TA on macrophage polarization. RESULTS: We found that TA pre-treated CM did not induce EMT in epithelial cells. Further, TA pre-treated CM showed diminished activation of MAPK in epithelial cells. Subsequently, TA was shown to inhibit LPS-induced M1 polarization in macrophages by directly targeting toll-like receptor 4 (TLR4), thereby repressing LPS binding to TLR4/MD2 complex and subsequent signal transduction. CONCLUSION: It was concluded that TA prevented M1 macrophage-induced EMT by suppressing the macrophage polarization possibly through inhibiting the formation of LPS-TLR4/MD2 complex and blockage of subsequent downstream signal activation. Further, our findings may provide beneficial information to develop new therapeutic strategies against chronic inflammatory diseases.