The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-ß4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-CF100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-ß4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-ß4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid ß42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

Accurate and Efficient SAXS/SANS Implementation Including Solvation Layer Effects Suitable for Molecular Simulations.

Small-angle X-ray and neutron scattering (SAXS/SANS) provide valuable insights into the structure and dynamics of biomolecules in solution, complementing a wide range of structural techniques, including molecular dynamics simulations. As contrast-based methods, they are sensitive not only to structural properties but also to solvent-solute interactions. Their use in molecular dynamics simulations requires a forward model that should be as fast and accurate as possible. In this work, we demonstrate the feasibility of calculating SAXS and SANS intensities using a coarse-grained representation consisting of one bead per amino acid and three beads per nucleic acid, with form factors that can be corrected on the fly to account for solvation effects at no additional computational cost. By coupling this forward model with molecular dynamics simulations restrained with SAS data, it is possible to determine conformational ensembles or refine the structure and dynamics of proteins and nucleic acids in agreement with the experimental results. To assess the robustness of this approach, we applied it to gelsolin, for which we acquired SAXS data on its closed state, and to a UP1-microRNA complex, for which we used previously collected measurements. Our hybrid-resolution small-angle scattering (hySAS) implementation, being distributed in PLUMED, can be used with atomistic and coarse-grained simulations using diverse restraining strategies.

The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-ß4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-CF100A toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-ß4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-ß4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.

hERG stereoselective modulation by mexiletine-derived ureas: Molecular docking study, synthesis, and biological evaluation.

Long QT syndrome (LQTS) is a disorder of cardiac electrophysiology resulting in life-threatening arrhythmias; nowadays, only a few drugs are available for the management of LQTS. Focusing our attention on LQT2, one of the most common subtypes of LQTS caused by mutations in the human ether-à-go-go-related gene (hERG), in the present work, the stereoselectivity of the recently discovered mexiletine-derived urea 8 was investigated on the hERG potassium channel. According to preliminary in silico predictions, in vitro studies revealed a stereoselective behavior, with the meso form showing the greatest hERG opening activity. In addition, functional studies on guinea pig isolated left atria, aorta, and ileum demonstrated that 8 does not present any cardiac or intestinal liability in our ex vivo studies. Due to its overall profile, (R,S)-8 paves the way for the design and development of a new series of compounds potentially useful in the treatment of both congenital and drug-induced forms of LQTS.

Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery.

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

A Small Molecule Stabilizes the Disordered Native State of the Alzheimer's Aß Peptide.

The stabilization of native states of proteins is a powerful drug discovery strategy. It is still unclear, however, whether this approach can be applied to intrinsically disordered proteins. Here, we report a small molecule that stabilizes the native state of the Aß42 peptide, an intrinsically disordered protein fragment associated with Alzheimer's disease. We show that this stabilization takes place by a disordered binding mechanism, in which both the small molecule and the Aß42 peptide remain disordered. This disordered binding mechanism involves enthalpically favorable local π-stacking interactions coupled with entropically advantageous global effects. These results indicate that small molecules can stabilize disordered proteins in their native states through transient non-specific interactions that provide enthalpic gain while simultaneously increasing the conformational entropy of the proteins.

Multi-eGO: An in silico lens to look into protein aggregation kinetics at atomic resolution.

Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 associated diseases that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the structural characterization of the transient oligomers formed during the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long time scales, typical of aggregation processes, have limited the use of computational methods to date. To surmount these limitations, we here present multi-eGO, an atomistic, hybrid structure-based model which, leveraging the knowledge of monomers conformational dynamics and of fibril structures, efficiently captures the essential structural and kinetics aspects of protein aggregation. Multi-eGO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments carried out on an amyloidogenic peptide from Transthyretin, a protein responsible for one of the most common cardiac amyloidoses. Multi-eGO simulations allow the formation of primary nuclei in a sea of transient lower-order oligomers to be observed over time and at atomic resolution, following their growth and the subsequent secondary nucleation events, until the maturation of multiple fibrils is achieved. Multi-eGO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases.

Disordered Regions Flanking the Binding Interface Modulate Affinity between CBP and NCOA.

Recognition motifs that mediate protein-protein interactions are usually embedded within longer intrinsically disordered regions. While binding interfaces involving the recognition motif in such interactions are well studied, less is known about the role of disordered regions flanking the motifs. The interaction between the transcriptional co-activators NCOA3 (ACTR) and CBP is mediated by coupled binding and folding of the two domains CID and NCBD. Here, we used circular dichroism and kinetics to directly quantify the contribution of the adjacent flanking regions of CID to its interaction with NCBD. Using N- and C-terminal combinatorial variants we found that the flanking regions promote binding in an additive fashion while retaining a large degree of disorder in the complex. Experiments at different ionic strengths demonstrated that the increase in affinity is not mediated by electrostatic interactions from the flanking regions. Instead, site-directed mutagenesis and molecular dynamics simulations suggest that binding is promoted by short-lived non-specific hydrophobic contacts between the flanking regions and NCBD. Our findings are consistent with highly frustrated interactions outside of the canonical binding interface resulting in a slightly energetically favorable fuzzy binding. Modulation of affinity via flanking regions could represent a general mechanism for functional regulation by intrinsically disordered protein regions.

l- to d-Amino Acid Substitution in the Immunodominant LCMV-Derived Epitope gp33 Highlights the Sensitivity of the TCR Recognition Mechanism for the MHC/Peptide Structure and Dynamics.

Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8+ T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wild-type peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2Db, predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2Db/gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2Db in complex with APLs, in which position 6 was subjected to an l- to d-amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the l- to d-modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.

Inactivation of the Pyrimidine Biosynthesis pyrD Gene Negatively Affects Biofilm Formation and Virulence Determinants in the Crohn's Disease-Associated Adherent Invasive Escherichia coli LF82 Strain.

In Crohn's disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers.

High Conformational Flexibility of the E2F1/DP1/DNA Complex.

The E2F1 transcription factor is a master regulator of cell-cycle progression whose uncontrolled activation contributes to tumor cells growth. E2F1 binds DNA as a heterodimer with DP partners, resulting in a multi-domain quaternary-structure complex composed of DNA binding domains, a coiled coil domain and a marked box domain separated by short linkers. Building on the 3D knowledge of the single domains of E2F and DPs, we characterized the structure and dynamics of the complete E2F1/DP1/DNA complex by a combination of small-angle X-ray scattering and molecular dynamics simulations. It shows an asymmetric contribution of the dynamics of the two proteins. Namely, the coiled-coil domain leans toward the DP1 side of the complex; the DP1 loop between α2 and α3 of the DBD partially populates a helical structure leaning far from the DNA and in the same direction of the coiled-coil domain; and the N-terminal disordered region of DP1, rich in basic residues, contributes to DNA binding stabilization. Intriguingly, tumor mutations in the flexible regions of the complex suggest that perturbation of protein dynamics could affect protein function in a context-dependent way. Our data suggest fundamental contributions of DP proteins in distinct aspects of E2F biology.

How to Determine Accurate Conformational Ensembles by Metadynamics Metainference: A Chignolin Study Case.

The reliability and usefulness of molecular dynamics simulations of equilibrium processes rests on their statistical precision and their capability to generate conformational ensembles in agreement with available experimental knowledge. Metadynamics Metainference (M&M), coupling molecular dynamics with the enhanced sampling ability of Metadynamics and with the ability to integrate experimental information of Metainference, can in principle achieve both goals. Here we show that three different Metadynamics setups provide converged estimate of the populations of the three-states populated by a model peptide. Errors are estimated correctly by block averaging, but higher precision is obtained by performing independent replicates. One effect of Metadynamics is that of dramatically decreasing the number of effective frames resulting from the simulations and this is relevant for M&M where the number of replicas should be large enough to capture the conformational heterogeneity behind the experimental data. Our simulations allow also us to propose that monitoring the relative error associated with conformational averaging can help to determine the minimum number of replicas to be simulated in the context of M&M simulations. Altogether our data provides useful indication on how to generate sound conformational ensemble in agreement with experimental data.

Refinement of α-Synuclein Ensembles Against SAXS Data: Comparison of Force Fields and Methods.

The inherent flexibility of intrinsically disordered proteins (IDPs) makes it difficult to interpret experimental data using structural models. On the other hand, molecular dynamics simulations of IDPs often suffer from force-field inaccuracies, and long simulation times or enhanced sampling methods are needed to obtain converged ensembles. Here, we apply metainference and Bayesian/Maximum Entropy reweighting approaches to integrate prior knowledge of the system with experimental data, while also dealing with various sources of errors and the inherent conformational heterogeneity of IDPs. We have measured new SAXS data on the protein α-synuclein, and integrate this with simulations performed using different force fields. We find that if the force field gives rise to ensembles that are much more compact than what is implied by the SAXS data it is difficult to recover a reasonable ensemble. On the other hand, we show that when the simulated ensemble is reasonable, we can obtain an ensemble that is consistent with the SAXS data, but also with NMR diffusion and paramagnetic relaxation enhancement data.

Converging experimental and computational views of the knotting mechanism of a small knotted protein.

MJ0366 from Methanocaldococcus jannaschii is the smallest topologically knotted protein known to date. 92 residues in length, MJ0366 ties a trefoil (31) knot by threading its C-terminal helix through a buttonhole formed by the remainder of the secondary structure elements. By generating a library of point mutations at positions pertinent to the knot formation, we systematically evaluated the contributions of individual residues to the folding stability and kinetics of MJ0366. The experimental Φ-values were used as restraints to computationally generate an ensemble of conformations that correspond to the transition state of MJ0366, which revealed several nonnative contacts. The importance of these nonnative contacts in stabilizing the transition state of MJ0366 was confirmed by a second round of mutagenesis, which also established the pivotal role of F15 in stapling the network of hydrophobic interactions around the threading C-terminal helix. Our converging experimental and computational results show that, despite the small size, the transition state of MJ0366 is formed at a very late stage of the folding reaction coordinate, following a polarized pathway. Eventually, the formation of extensive native contacts, as well as a number of nonnative ones, leads to the threading of the C-terminal helix that defines the topological knot.

The co-existence of cold activity and thermal stability in an Antarctic GH42 ß-galactosidase relies on its hexameric quaternary arrangement.

To survive in cold environments, psychrophilic organisms produce enzymes endowed with high specific activity at low temperature. The structure of these enzymes is usually flexible and mostly thermolabile. In this work, we investigate the structural basis of cold adaptation of a GH42 ß-galactosidase from the psychrophilic Marinomonas ef1. This enzyme couples cold activity with astonishing robustness for a psychrophilic protein, for it retains 23% of its highest activity at 5 °C and it is stable for several days at 37 °C and even 50 °C. Phylogenetic analyses indicate a close relationship with thermophilic ß-galactosidases, suggesting that the present-day enzyme evolved from a thermostable scaffold modeled by environmental selective pressure. The crystallographic structure reveals the overall similarity with GH42 enzymes, along with a hexameric arrangement (dimer of trimers) not found in psychrophilic, mesophilic, and thermophilic homologues. In the quaternary structure, protomers form a large central cavity, whose accessibility to the substrate is promoted by the dynamic behavior of surface loops, even at low temperature. A peculiar cooperative behavior of the enzyme is likely related to the increase of the internal cavity permeability triggered by heating. Overall, our results highlight a novel strategy of enzyme cold adaptation, based on the oligomerization state of the enzyme, which effectively challenges the paradigm of cold activity coupled with intrinsic thermolability. DATABASE: Structural data are available in the Protein Data Bank database under the accession number 6Y2K.

A kinetic ensemble of the Alzheimer's Aß peptide.

The conformational and thermodynamic properties of disordered proteins are commonly described in terms of structural ensembles and free energy landscapes. To provide information on the transition rates between the different states populated by these proteins, it would be desirable to generalize this description to kinetic ensembles. Approaches based on the theory of stochastic processes can be particularly suitable for this purpose. Here, we develop a Markov state model and apply it to determine a kinetic ensemble of Aß42, a disordered peptide associated with Alzheimer's disease. Through the Google Compute Engine, we generated 315-µs all-atom molecular dynamics trajectories. Using a probabilistic-based definition of conformational states in a neural network approach, we found that Aß42 is characterized by inter-state transitions on the microsecond timescale, exhibiting only fully unfolded or short-lived, partially folded states. Our results illustrate how kinetic ensembles provide effective information about the structure, thermodynamics and kinetics of disordered proteins.

Small-molecule sequestration of amyloid-ß as a drug discovery strategy for Alzheimer's disease.

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that modifies the properties of their monomeric states. Here, we identify a small molecule (10074-G5) capable of binding and sequestering the intrinsically disordered amyloid-ß (Aß) peptide in its monomeric, soluble state. Our analysis reveals that this compound interacts with Aß and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterize this interaction using biophysical experiments and integrative structural ensemble determination methods. We observe that this molecule increases the conformational entropy of monomeric Aß while decreasing its hydrophobic surface area. We also show that it rescues a Caenorhabditis elegans model of Aß-associated toxicity, consistent with the mechanism of action identified from the in silico and in vitro studies. These results illustrate the strategy of stabilizing the monomeric states of disordered proteins with small molecules to alter their behavior for therapeutic purposes.

Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.