Intrinsic determinants of prion protein neurotoxicity in Drosophila: from sequence to (dys)function.

Prion diseases are fatal brain disorders characterized by deposition of insoluble isoforms of the prion protein (PrP). The normal and pathogenic structures of PrP are relatively well known after decades of studies. Yet our current understanding of the intrinsic determinants regulating PrP misfolding are largely missing. A 3D subdomain of PrP comprising the ß2-α2 loop and helix 3 contains high sequence and structural variability among animals and has been proposed as a key domain regulating PrP misfolding. We combined in vivo work in Drosophila with molecular dynamics (MD) simulations, which provide additional insight to assess the impact of candidate substitutions in PrP from conformational dynamics. MD simulations revealed that in human PrP WT the ß2-α2 loop explores multiple ß-turn conformations, whereas the Y225A (rabbit PrP-like) substitution strongly favors a 310-turn conformation, a short right-handed helix. This shift in conformational diversity correlates with lower neurotoxicity in flies. We have identified additional conformational features and candidate amino acids regulating the high toxicity of human PrP and propose a new strategy for testing candidate modifiers first in MD simulations followed by functional experiments in flies. In this review we expand on these new results to provide additional insight into the structural and functional biology of PrP through the prism of the conformational dynamics of a 3D domain in the C-terminus. We propose that the conformational dynamics of this domain is a sensitive measure of the propensity of PrP to misfold and cause toxicity. This provides renewed opportunities to identify the intrinsic determinants of PrP misfolding through the contribution of key amino acids to different conformational states by MD simulations followed by experimental validation in transgenic flies.

Y225A induces long-range conformational changes in human prion protein that are protective in Drosophila.

Prion protein (PrP) misfolding is the key trigger in the devastating prion diseases. Yet the sequence and structural determinants of PrP conformation and toxicity are not known in detail. Here, we describe the impact of replacing Y225 in human PrP with A225 from rabbit PrP, an animal highly resistant to prion diseases. We first examined human PrP-Y225A by molecular dynamics simulations. We next introduced human PrP in Drosophila and compared the toxicity of human PrP-WT and Y225A in the eye and in brain neurons. Y225A stabilizes the ß2-α2 loop into a 310-helix from six different conformations identified in WT and lowers hydrophobic exposure. Transgenic flies expressing PrP-Y225A exhibit less toxicity in the eye and in brain neurons and less accumulation of insoluble PrP. Overall, we determined that Y225A lowers toxicity in Drosophila assays by promoting a structured loop conformation that increases the stability of the globular domain. These findings are significant because they shed light on the key role of distal α-helix 3 on the dynamics of the loop and the entire globular domain.

Insight From Animals Resistant to Prion Diseases: Deciphering the Genotype - Morphotype - Phenotype Code for the Prion Protein.

Prion diseases are a group of neurodegenerative diseases endemic in humans and several ruminants caused by the misfolding of native prion protein (PrP) into pathological conformations. Experimental work and the mad-cow epidemic of the 1980s exposed a wide spectrum of animal susceptibility to prion diseases, including a few highly resistant animals: horses, rabbits, pigs, and dogs/canids. The variable susceptibility to disease offers a unique opportunity to uncover the mechanisms governing PrP misfolding, neurotoxicity, and transmission. Previous work indicates that PrP-intrinsic differences (sequence) are the main contributors to disease susceptibility. Several residues have been cited as critical for encoding PrP conformational stability in prion-resistant animals, including D/E159 in dog, S167 in horse, and S174 in rabbit and pig PrP (all according to human numbering). These amino acids alter PrP properties in a variety of assays, but we still do not clearly understand the structural correlates of PrP toxicity. Additional insight can be extracted from comparative structural studies, followed by molecular dynamics simulations of selected mutations, and testing in manipulable animal models. Our working hypothesis is that protective amino acids generate more compact and stable structures in a C-terminal subdomain of the PrP globular domain. We will explore this idea in this review and identify subdomains within the globular domain that may hold the key to unravel how conformational stability and disease susceptibility are encoded in PrP.

FRET Analysis of Ionic Strength Sensors in the Hofmeister Series of Salt Solutions Using Fluorescence Lifetime Measurements.

Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor-acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor-acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.

Mechanical Unfolding of Spectrin Repeats Induces Water-Molecule Ordering.

Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin's spectrin repeat 1 and related the changes in the protein's structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein's small protrusions in structurally ordered waters that are characteristic of the so-called "small-molecule regime," which allows water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.

Dynamics of Dystrophin's Actin-Binding Domain.

We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Tuning Sulfur Oxidation States on Thioether-Bridged Peptide Macrocycles for Modulation of Protein Interactions.

Thioethers, sulfoxides, and sulfonium ions, despite diverse physicochemical properties, all engage in noncovalent interactions with proteins. Thioether-containing macrocycles are also attracting attention as protein-protein interaction (PPI) inhibitors. Here, we used a model PPI between α-helical mixed lineage leukemia (MLL) protein and kinase-inducible domain interacting (KIX) domain to evaluate oxidation effects on sulfurcontaining macrocycle structure, stability, and protein affinity. Desolvation effects from various polarity states were evaluated computationally and experimentally at the side chain, amino acid, and peptide level. Sulfur-containing side chains spanned polarity ranges between all-hydrocarbon and lactam bridges for modulating solubility, cellular uptake, and affinity. Helical propensity studies showed that, although oxidized sulfur-containing side chains could be tolerated, conformational effects were sequence-dependent. In some cases, proteolytic stability, binding capacity with KIX, and increased helicity were obtained as first steps toward developing PPI inhibitors.

Oxidation increases the strength of the methionine-aromatic interaction.

Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we found that oxidation increased the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction, by 0.5-1.4 kcal/mol. We found that non-hydrogen-bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups were enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR spectroscopy experiments on model peptides demonstrated that oxidation of methionine stabilized the interaction by 0.5-0.6 kcal/mol. We confirmed the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on (i) calmodulin structure and dynamics, and (ii) lymphotoxin-α binding toTNFR1. Thus, the methionine-aromatic motif was a determinant of protein structural and functional sensitivity to oxidative stress.

Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation.

The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to ß-adrenergic stimulation. We found that the single deletion of arginine in phospholamban's recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations and motions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a well-tuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca(2+) signaling and normal cardiac function.

Synchronous opening and closing motions are essential for cAMP-dependent protein kinase A signaling.

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and closing of the active cleft without changing the enzyme's structure, rendering it catalytically inefficient. Since the opening and closing motions govern the rate-determining product release, we conclude that optimal and coherent conformational fluctuations are necessary for efficient turnover of protein kinases.

Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution.

Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

NMR mapping of protein conformational landscapes using coordinated behavior of chemical shifts upon ligand binding.

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the linear trajectories of NMR chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues' response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein's approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins.

Role of conformational entropy in the activity and regulation of the catalytic subunit of protein kinase A.

Protein kinase A (PKA) is the archetypical phosphokinase, sharing a catalytic core with the entire protein kinase superfamily. In eukaryotes, the ubiquitous location of PKA makes it one of the most important cellular signaling molecules, involved in a myriad of events. The catalytic subunit of PKA (PKA-C) is one of the most studied enzymes and was the first kinase to be crystallized; however, the effects of ligand binding, post-translational modifications and mutations on the activity of the kinase have been difficult to understand with only structural data. Here, we review our latest NMR studies on PKA-C, the results of which underscore the role of fast and slow conformational dynamics in the activation and inhibition of the kinase.

Concerted hydrogen atom and electron transfer mechanism for catalysis by lysine-specific demethylase.

We calculate the free energy profile for the postulated hydride transfer reaction mechanism for the catalysis of lysine demethylation by lysine-specific demethylase LSD1. The potential energy surface is obtained by using combined electrostatically embedded multiconfiguration molecular mechanics (EE-MCMM) and single-configuration molecular mechanics (MM). We employ a constant valence bond coupling term to obtain analytical energies and gradients of the EE-MCMM subsystem, which contains 45 quantum mechanics (QM) atoms and which is parametrized with density functional calculations employing specific reaction parameters obtained by matching high-level wave function calculations. In the MM region, we employ the Amber ff03 and TIP3P force fields. The free energy of activation at 300 K is calculated by molecular dynamics (MD) umbrella sampling on a system with 102,090 atoms as the maximum of the free energy profile along the reaction coordinate as obtained by the weighted histogram analysis method with 17 umbrella sampling windows. This yields a free energy of activation of only 10 kcal/mol, showing that the previously postulated direct hydride transfer reaction mechanism is plausible, although we find that it is better interpreted as a concerted transfer of a hydrogen atom and an electron.

Connecting protein conformational dynamics with catalytic function as illustrated in dihydrofolate reductase.

Combined quantum mechanics/molecular mechanics molecular dynamics simulations reveal that the M20 loop conformational dynamics of dihydrofolate reductase (DHFR) is severely restricted at the transition state of the hydride transfer as a result of the M42W/G121V double mutation. Consequently, the double-mutant enzyme has a reduced entropy of activation, i.e., increased entropic barrier, and altered temperature dependence of kinetic isotope effects in comparison with those of wild-type DHFR. Interestingly, in both wild-type DHFR and the double mutant, the average donor-acceptor distances are essentially the same in the Michaelis complex state (~3.5 Å) and the transition state (2.7 Å). It was found that an additional hydrogen bond is formed to stabilize the M20 loop in the closed conformation in the M42W/G121V double mutant. The computational results reflect a similar aim designed to knock out precisely the dynamic flexibility of the M20 loop in a different double mutant, N23PP/S148A.

The Third Dimension of a More O'Ferrall-Jencks Diagram for Hydrogen Atom Transfer in the Isoelectronic Hydrogen Exchange Reactions of (PhX)(2)H(•) with X = O, NH, and CH(2).

A critical element in theoretical characterization of the mechanism of proton-coupled electron transfer (PCET) reactions, including hydrogen atom transfer (HAT), is the formulation of the electron and proton localized diabatic states, based on which a More O'Ferrall-Jencks diagram can be represented to determine the step-wise and concerted nature of the reaction. Although the More O'Ferrall-Jencks diabatic states have often been used empirically to develop theoretical models for PCET reactions, the potential energy surfaces for these states have never been determined directly based on first principles calculations using electronic structure theory. The difficulty is due to a lack of practical method to constrain electron and proton localized diabatic states in wave function or density functional theory calculations. Employing a multistate density functional theory (MSDFT), in which the electron and proton localized diabatic configurations are constructed through block-localization of Kohn-Sham orbitals, we show that distinction between concerted proton-electron transfer (CPET) and HAT, which are not distinguishable experimentally from phenomenological kinetic data, can be made by examining the third dimension of a More O'Ferrall-Jencks diagram that includes both the ground and excited state potential surfaces. In addition, we formulate a pair of effective two-state valence bond models to represent the CPET and HAT mechanisms. We found that the lower energy of the CPET and HAT effective diabatic states at the intersection point can be used as an energetic criterion to distinguish the two mechanisms. In the isoelectronic series of hydrogen exchange reaction in (PhX)(2)H(•), where X = O, NH, and CH(2), there is a continuous transition from a CPET mechanism for the phenoxy radical-phenol pair to a HAT process for benzyl radical and toluene, while the reaction between PhNH(2) and PhNH(•) has a mechanism intermediate of CPET and HAT. The electronically nonadiabatic nature of the CPET mechanism in the phenol system can be attributed to the overlap interactions between the ground and excited state surfaces, resulting in roughly orthogonal minimum energy paths on the adiabatic ground and excited state potential energy surfaces. On the other hand, the minimum energy path on the adiabatic ground state for the HAT mechanism coincides with that on the excited state, producing a large electronic coupling that separates the two surfaces by more than 120 kcal/mol.

Conformational equilibrium of N-myristoylated cAMP-dependent protein kinase A by molecular dynamics simulations.

The catalytic subunit of protein kinase A (PKA-C) is subject to several post- or cotranslational modifications that regulate its activity both spatially and temporally. Among those, N-myristoylation increases the kinase affinity for membranes and might also be implicated in substrate recognition and allosteric regulation. Here, we investigated the effects of N-myristoylation on the structure, dynamics, and conformational equilibrium of PKA-C using atomistic molecular dynamics simulations. We found that the myristoyl group inserts into the hydrophobic pocket and leads to a tighter packing of the A-helix against the core of the enzyme. As a result, the conformational dynamics of the A-helix are reduced and its motions are more coupled with the active site. Our simulations suggest that cation-π interactions among W30, R190, and R93 are responsible for coupling these motions. Two major conformations of the myristoylated N-terminus are the most populated: a long loop (LL conformation), similar to Protein Data Bank (PDB) entry 1CMK , and a helix-turn-helix structure (HTH conformation), similar to PDB entry 4DFX , which shows stronger coupling between the conformational dynamics observed at the A-helix and active site. The HTH conformation is stabilized by S10 phosphorylation of the kinase via ionic interactions between the protonated amine of K7 and the phosphate group on S10, further enhancing the dynamic coupling to the active site. These results support a role of N-myristoylation in the allosteric regulation of PKA-C.

The methionine-aromatic motif plays a unique role in stabilizing protein structure.

Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1-1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.