The thermodynamics of secondary p53 binding sites on MDM2 and MDMX were evaluated using p53 peptides containing residues 16-29, 17-35, and 1-73. All the peptides had large, negative heat capacity (ΔCp), consistent with the burial of p53 residues F19, W23, and L26 in the primary binding sites of MDM2 and MDMX. MDMX has a higher affinity and more negative ΔCp than MDM2 for p5317-35, which is due to MDMX stabilization and not additional interactions with the secondary binding site. ΔCp measurements show binding to the secondary site is inhibited by the disordered tails of MDM2 for WT p53 but not a more helical mutant where proline 27 is changed to alanine. This result is supported by all-atom molecular dynamics simulations showing that p53 residues 30-35 turn away from the disordered tails of MDM2 in P27A17-35 and make direct contact with this region in p5317-35. Molecular dynamics simulations also suggest that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site. ΔCp measurements also show that tighter binding of the P27A mutant to MDM2 and MDMX is due to increased helicity, which reduces the energetic penalty associated with coupled folding and binding. Our results will facilitate the design of selective p53 inhibitors for MDM2 and MDMX.

The first- and second-generation clinically used HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) are key components of antiretroviral therapy (ART), which work by blocking the integration step in the HIV-1 replication cycle that is catalyzed by a nucleoprotein assembly called an intasome. However, resistance to even the latest clinically used INSTIs is beginning to emerge. Developmental third-generation INSTIs, based on naphthyridine scaffolds, are promising candidates to combat drug-resistant viral variants. Among these novel INSTIs, compound 4f exhibits two distinct conformations when binding with intasomes from HIV-1 and the closely related prototype foamy virus (PFV) despite the high structural similarity of their INSTI binding pockets. The molecular mechanism and the key active site residues responsible for these differing binding modes in closely related intasomes remain elusive. To unravel the molecular determinants governing the two distinct binding modes, we applied a novel molecular dynamics-based free energy method that utilizes alchemical pathways to overcome the sampling challenges associated with transitioning between the two bound conformations of ligand 4f within the crowded environments of the INSTI binding pockets in these intasomes. The calculated conformational free energies successfully recapitulate the experimentally observed binding mode preferences in the two viral intasomes. Analysis of the simulated structures suggests that the observed binding mode preferences are caused by amino acid residue differences in both the front and the central catalytic sub-pocket of the INSTI binding site in HIV-1 and PFV. Additional free energy calculations on mutants of HIV-1 and PFV revealed that while both sub-pockets contribute to binding mode selection, the central sub-pocket plays a more important role. These results highlight the importance of both side chain and solvent reorganization, as well as the conformational entropy in determining the ligand binding mode, and will help inform the development of more effective INSTIs for combatting drug-resistant viral variants.

In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAP\'s entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.

As proposed here, β-turns play an essential role in protein self-assembly. This compact, four-residue motif affects protein conformation dramatically by reversing the overall chain direction. Turns are the \"hinges\" in globular proteins. This new proposal broadens a previous hypothesis that globular proteins solve the folding problem in part by filtering conformers with unsatisfied backbone hydrogen bonds, thereby preorganizing the folding population. Recapitulating that hypothesis: unsatisfied conformers would be dramatically destabilizing, shifting the U(nfolded) ⇌ N(ative) equilibrium far to the left. If even a single backbone polar group is satisfied by solvent when unfolded but buried and unsatisfied when folded, that energy penalty alone, approximately +5 kcal/mol, would rival almost the entire free energy of protein stabilization at room temperature. Consequently, globular proteins are built on scaffolds of hydrogen-bonded α-helices and/or strands of β-sheet, motifs that can be extended indefinitely, with intra-segment hydrogen bond partners for their backbone polar groups and without steric clash. Scaffolds foster a protein-wide hydrogen-bonded network, and, of thermodynamic necessity, they self-assemble cooperatively. Unlike elements of repetitive secondary structure, α-helices and β-sheet, a four-residue β-turn has only a single hydrogen bond (from i + 3 → i), not a cooperatively formed assembly of hydrogen bonds. As such, turns can form autonomously and are poised to initiate assembly of scaffold elements by bringing them together in an orientation and registration that promotes cooperative \"zipping\". The overall effect of this self-assembly mechanism is to induce substantial preorganization in the thermodynamically accessible folding population and, concomitantly, to reduce the folding entropy.

Natural taste/flavor enhancers are essential ingredients that could potentially address condiments overconsumption. For the first time, we report that hyaluronic acid (HA) could modulate taste perception, governed by the dynamic interactions among taste compounds, mucin, and HA. Various conformations of HA impact taste perception. The high molecular weight (Mw) of 1090 kDa HA inhibits the sense of taste due to its increased viscosity, which hinders the penetration of Na+ into the mucin layer. HA with low and medium Mw (100 kDa, 400 kDa) could enhance taste perception. Isothermal titration calorimetry analysis confirms the stronger binding between mucin and HA. The intensity of their interaction increases as the Mw of HA increases from 8 kDa to 400 kDa. Quartz crystal microbalance with dissipation characterization further indicates that the rigid conformation of 100 kDa HA facilitates the binding of Na+ with taste receptors, thereby enhancing taste perception. The flexible conformation of 400 kDa HA may conceal the taste receptor cells, reducing taste enhancement. Our work advances the understanding of conformational entropy of natural mucoadhesion and mucopenetration polymers, which lays the foundation for their potential use as taste enhancers.

The d-GlcNAc moiety in sialyl Lewisx (sLex, 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a 1H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups.

The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5\'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity-a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

β-glucosidases play a pivotal role in second-generation biofuel (2G-biofuel) production. For this application, thermostable enzymes are essential due to the denaturing conditions on the bioreactors. Random amino acid substitutions have originated new thermostable β-glucosidases, but without a clear understanding of their molecular mechanisms. Here, we probe by different molecular dynamics simulation approaches with distinct force fields and submitting the results to various computational analyses, the molecular bases of the thermostabilization of the Paenibacillus polymyxa GH1 β-glucosidase by two-point mutations E96K (TR1) and M416I (TR2). Equilibrium molecular dynamic simulations (eMD) at different temperatures, principal component analysis (PCA), virtual docking, metadynamics (MetaDy), accelerated molecular dynamics (aMD), Poisson-Boltzmann surface analysis, grid inhomogeneous solvation theory and colony method estimation of conformational entropy allow to converge to the idea that the stabilization carried by both substitutions depend on different contributions of three classic mechanisms: (i) electrostatic surface stabilization; (ii) efficient isolation of the hydrophobic core from the solvent, with energetic advantages at the solvation cap; (iii) higher distribution of the protein dynamics at the mobile active site loops than at the protein core, with functional and entropic advantages. Mechanisms i and ii predominate for TR1, while in TR2, mechanism iii is dominant. Loop A integrity and loops A, C, D, and E dynamics play critical roles in such mechanisms. Comparison of the dynamic and topological changes observed between the thermostable mutants and the wildtype protein with amino acid co-evolutive networks and thermostabilizing hotspots from the literature allow inferring that the mechanisms here recovered can be related to the thermostability obtained by different substitutions along the whole family GH1. We hope the results and insights discussed here can be helpful for future rational approaches to the engineering of optimized β-glucosidases for 2G-biofuel production for industry, biotechnology, and science.