David J. E. Callaway

A molecular model of Alzheimer amyloid beta-peptide fibril formation.

Polymerization of the amyloid beta (Aβ) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer’s disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Aβ-fragments containing the Aβ16–20sequence, previously shown essential for Aβ-Aβ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Aβ14–23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Aβ1–42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Aβ14–23 oligomers in an antiparallel β-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Aβ-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

Triviality pursuit: Can elementary scalar particles exist?

Abstract Great effort is presently being expended in the search for elementary scalar “Higgs” particles. These particles have yet to be observed. The primary justification for this search is the theoretically elegant Higgs-Kibble mechanism, in which the interactions of elemetary scalars are used to generate gauge boson masses in a quantum field theory. However, strong evidence suggests that at least a pure φ 4 scalar field theory is trivial or noninteracting. Should this triviality persist in more complicated systems such as the standard model of the weak interaction, the motivation for looking for Higgs particles would be seriously undermined. Alternatively, the presence of gauge and fermion fields can rescue a pure scalar theory from triviality. Phenomenological constraints (such as a bounded or even predictable Higgs mass) may then be implied. In this report the evidence for triviality in various field theories is reviewed, and the implications for high energy physics are discussed.

Ezrin Controls the Macromolecular Complexes Formed between an Adapter Protein Na+/H+ Exchanger Regulatory Factor and the Cystic Fibrosis Transmembrane Conductance Regulator

Na+/H+ exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)2·NHERF·ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR·NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton.

Protein kinase C phosphorylation disrupts Na+/H+ exchanger regulatory factor 1 autoinhibition and promotes cystic fibrosis transmembrane conductance regulator macromolecular assembly.

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Proteins MOVE! Protein dynamics and long-range allostery in cell signaling

An emerging point of view in protein chemistry is that proteins are not the static objects that are displayed in textbooks but are instead dynamic actors. Protein dynamics plays a fundamental role in many diseases, and spans a large hierarchy of timescales, from picoseconds to milliseconds or even longer. Nanoscale protein domain motion on length scales comparable to protein dimensions is key to understanding how signals are relayed through multiple protein-protein interactions. A canonical example is how the scaffolding proteins NHERF1 and ezrin work in coordination to assemble crucial membrane complexes. As membrane-cytoskeleton scaffolding proteins, these provide excellent prototypes for understanding how regulatory signals are relayed through protein-protein interactions between the membrane and the cytoskeleton. Here, we review recent progress in understanding the structure and dynamics of the interaction. We describe recent novel applications of neutron spin echo spectroscopy to reveal the dynamic propagation of allosteric signals by nanoscale protein motion, and present a guide to the future study of dynamics and its application to the cure of disease.

Non-triviality of gauge theories with elementary scalars and upper bounds on Higgs masses

Strong evidence supports the idea that pure φ4 field theory is trivial (non-interacting) in four dimensions. In the context of perturbation theory when gauge fields or fermions are present the combined theory may become non-trivial for a limited range of values of the renormalized coupling constants. Calculable upper bounds on the masses of elementary Higgs particles are implied by the restrictions on these coupling constants. Other constraints (on, e.g. the number of fermions in the theory) are also implied.

Ezrin induces long-range interdomain allostery in the scaffolding protein NHERF1

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains-PDZ1 and PDZ2-and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 A. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.

A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation

The mammalian Na+/H+ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.

Monte Carlo renormalization group study of φ4 field theory

A previously proposed general method for evaluating block renormalized coupling constants within the framework of the Monte Carlo renormalization group (MCRG) is applied to φ4 field theory. The flow diagrams, fixed points, and critical exponents are determined in two, three and four dimensions. Results in four dimensions are consistent with the idea that φ4 field theory is trivial (non-interacting) in the continuum limit. The possibility of using MCRG techniques to ascertain whether a general non-asymptotically free theory is trivial or not is also discussed.

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Background: The structure of activated ezrin is not known. Results: We have determined the conformation of activated ezrin upon binding to PIP2 and to F-actin. Conclusion: Activated ezrin forms more extensive contacts with F-actin than generally depicted. Significance: This study provides new insight into the mechanisms by which ezrin assembles signaling complexes at the membrane-cytoskeleton interface. Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP2. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP2 binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP2 binding. The study provides the first structural view of the activated ezrin bound to PIP2 and to F-actin.