Francesca Massi

Simulation Study of the Structure and Dynamics of the Alzheimer’s Amyloid Peptide Congener in Solution

The amyloid Abeta(10-35)-NH2 peptide is simulated in an aqueous environment on the nanosecond time scale. One focus of the study is on the validation of the computational model through a direct comparison of simulated statistical averages with experimental observations of the peptides structure and dynamics. These measures include (1) nuclear magnetic resonance spectroscopy-derived amide bond order parameters and temperature-dependent H(alpha) proton chemical shifts, (2) the peptides radius of gyration and end-to-end distance, (3) the rates of peptide self-diffusion in water, and (4) the peptides hydrodynamic radius as measured by quasielastic light scattering experiments. A second focus of the study is the identification of key intrapeptide interactions that stabilize the central structural motif of the peptide. Particular attention is paid to the structure and fluctuation of the central LVFFA hydrophobic cluster (17-21) region and the VGSN turn (24-27) region. There is a strong correlation between preservation of the structure of these elements and interactions between the cluster and turn regions in imposing structure on the peptide monomer. The specific role of these interactions in relation to proposed mechanisms of amyloidosis is discussed.

Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1ρ relaxation experiments

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on‐ and off‐resonance rotating‐frame 15N R1ρ relaxation experiments to have a kinetic exchange rate constant of 25,000 sec−1 at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N‐cap hydrogen bonds of the α‐helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H‐15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin‐dependent protein degradation pathway.

Energy landscape theory for Alzheimer's amyloid ?-peptide fibril elongation

Recent experiments on the kinetics of deposition and fibril elongation of the Alzheimers β‐amyloid peptide on preexisting fibrils are analyzed. A mechanism is developed based on the dock‐and‐lock scheme recently proposed by Maggio and coworkers to organize their experimental observations of the kinetics of deposition of β‐peptide on preexisting amyloid fibrils and deposits. Our mechanism includes channels for (1) a one‐step prion‐like direct deposition on fibrils of activated monomeric peptide in solution, and (2) a two‐step deposition of unactivated peptide on fibrils and subsequent reorganization of the peptide–fibril complex. In this way, the mechanism and implied “energy landscape” unify a number of schemes proposed to describe the process of fibril elongation. This β‐amyloid landscape mechanism (βALM) is found to be in good agreement with existing experimental data. A number of experimental tests of the mechanism are proposed. The mechanism leads to a clear definition of overall equilibrium or rate constants in terms of the energetics of the elementary underlying processes. Analysis of existing experimental data suggests that fibril elongation occurs through a two‐step mechanism of nonspecific peptide absorption and reorganization. The mechanism predicts a turnover in the rate of fibril elongation as a function of temperature and denaturant concentration. Proteins 2001;42:217–229.

Charge states rather than propensity for β‐structure determine enhanced fibrillogenesis in wild‐type Alzheimer's β‐amyloid peptide compared to E22Q Dutch mutant

The activity of the Alzheimers amyloid β‐peptide is a sensitive function of the peptides sequence. Increased fibril elongation rate of the E22Q Dutch mutant of the Alzheimers amyloid β‐peptide relative to that of the wild‐type peptide has been observed. The increased activity has been attributed to a larger propensity for the formation of β structure in the monomeric E22Q mutant peptide in solution relative to the WT peptide. That hypothesis is tested using four nanosecond timescale simulations of the WT and Dutch mutant forms of the Aβ(10–35)‐peptide in aqueous solution. The simulation results indicate that the propensity for formation of β‐structure is no greater in the E22Q mutant peptide than in the WT peptide. A significant measure of “flickering” of helical structure in the central hydrophobic cluster region of both the WT and mutant peptides is observed. The simulation results argue against the hypothesis that the Dutch mutation leads to a higher probability of formation of β‐structure in the monomeric peptide in aqueous solution. We propose that the greater stability of the solvated WT peptide relative to the E22Q mutant peptide leads to decreased fibril elongation rate in the former. Stability difference is due to the differing charge state of the two peptides. The other proposal leads to the prediction that the fibril elongation rates for the WT and the mutant E22Q should be similar under acid conditions.

Structural and dynamical analysis of the hydration of the Alzheimer's β‐amyloid peptide

An analysis of the water molecules in the first solvation shell obtained from the molecular dynamics simulation of the amyloid β(10‐35)NH2 peptide and the amyloid β(10‐35)NH2E22Q “Dutch” mutant peptide is presented. The structure, energetics, and dynamics of water in the hydration shell have been investigated using a variety of measures, including the hydrogen bond network, the water residence times for all the peptide residues, the diffusion constant, experimentally determined HN amide proton exchange, and the transition probabilities for water to move from one residue to another or into the bulk. The results of the study indicate that: (1) the water molecules at the peptide‐solvent interface are organized in an ordered structure similar for the two peptide systems but different from that of the bulk, (2) the peptide structure inhibits diffusion perpendicular to the peptide surface by a factor of 3 to 5 relative to diffusion parallel to the peptide surface, which is comparable to diffusion of bulk water, (3) water in the first solvation shell shows dynamical relaxation on fast (1–2 ps) and slow (10–40 ps) time scales, (4) a novel solvent relaxation master equation is shown to capture the details of the fast relaxation of water in the peptides first solvation shell, (5) the interaction between the peptide and the solvent is stronger in the wild type than in the E22Q mutant peptide, in agreement with earlier results obtained from computer simulations [Massi, F.; Straub, J. E. Biophys J 2001, 81, 697] correlated with the observed enhanced activity of the E22Q mutant peptide.

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease

ABSTRACT Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state. DOI: http://dx.doi.org/10.7554/eLife.02848.001

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα.

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a >100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.

A Conserved Three-Nucleotide Core Motif Defines Musashi RNA-Binding Specificity

Background: The Musashi family of RNA-binding proteins promotes progenitor cell proliferation. Results: The majority of Musashi sequence specificity comes from a UAG motif. Sequences outside of UAG make minor contributions to affinity. Conclusion: UAG forms the core Musashi recognition element. Significance: Delineating the sequences that contribute to binding is critical to understanding RNA target selection. Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.

Accurate Estimates of Free Energy Changes in Charge Mutations.

The ability to determine the effect of charge changes on the free energy is necessary for fundamental studies of the electrostatic contribution to protein binding and stability. Currently, calculations of differences in free energy for charge mutations carried out in systems with periodic boundary conditions must include an approximate self-energy correction that can be on the same order of magnitude as the calculated free energy change. Here, a new method for accurately calculating the free energy change associated with any alchemical mutation, regardless of charge, is presented. In this method, paired mutations of opposite charge exactly cancel the self-energy term because of its quadratic charge dependence. Since the self-energy term implicitly cancels within the method, a correction never needs to be applied, and the statistical uncertainty associated is thereby removed. An implementation procedure is described and applied to the free energy of ionic hydration and a charged amino acid mutation.