Molecular models of the open and closed states of the whole human CFTR protein

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (ΔF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.     

An approach to molecular dynamics of chemical reaction

Zusammenfassung Der bei Ablauf einer chemischen Reaktion stattfindende Elementarvorgang wird mit Hilfe des Diffusionsmodells untersucht. Der hierbei für die Geschwindigkeitskonstante gewonnene Ausdruck [Gl. (17)] stimmt in dem Grenzfalle, dass der Reibungskoeffizient verschwindet, mit dem Ergebnis nach der Eyringschen Übergangsmethode und im Grenzfalle grosser Reibungskoeffizienten mit der Darstellung Gleichung (24) für den Smoluchowskischen Diffusionsfluss im Lagekoordinatenraume überein.

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The author takes great pleasure in acknowledging the encouragement of Prof.K. Oomori.     

Molecular ontogenesis and molecular genetics in the crosses of toads

Riassunto È stata studiata la comparsa di antigeni nel corso dello sviluppo di due specie di rospi:Bufo bufo eB. viridis e dei loro incroci reciproci. Alcuni antigeni al loro primo apparire precipitano solo con l'antisiero specie-specifico e, solo dopo lo stadio di bottone caudale, sono precipitati anche dall'antisiero dell'altra specie; questo viene interpretato quale una ontogenesi molecolare. Negli incroci, le molecole con gruppi reattivi per i due antisieri sono più frequenti nell'incrocio vitale (B. bufo ×B. viridis ), mentre l'incrocio letale (B. viridis ×B. bufo ) mostra alcuni antigeni separati che precipitano ognuno con un antisiero.

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This investigation was supported by a grant from the Consiglio Nazionale delle Richerche.     

Role of CFTR in epithelial physiology

Salt and fluid absorption and secretion are two processes that are fundamental to epithelial function and whole body fluid homeostasis, and as such are tightly regulated in epithelial tissues. The CFTR anion channel plays a major role in regulating both secretion and absorption in a diverse range of epithelial tissues, including the airways, the GI and reproductive tracts, sweat and salivary glands. It is not surprising then that defects in CFTR function are linked to disease, including life-threatening secretory diarrhoeas, such as cholera, as well as the inherited disease, cystic fibrosis (CF), one of the most common life-limiting genetic diseases in Caucasian populations. More recently, CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease (COPD), and the hyper-responsiveness in asthma, underscoring its fundamental role in whole body health and disease. CFTR regulates many mechanisms in epithelial physiology, such as maintaining epithelial surface hydration and regulating luminal pH. Indeed, recent studies have identified luminal pH as an important arbiter of epithelial barrier function and innate defence, particularly in the airways and GI tract. In this chapter, we will illustrate the different operational roles of CFTR in epithelial function by describing its characteristics in three different tissues: the airways, the pancreas, and the sweat gland.     

The gating of the CFTR channel

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed in the apical membrane of epithelia. Mutations in the CFTR gene are the cause of cystsic fibrosis. CFTR is the only ABC-protein that constitutes an ion channel pore forming subunit. CFTR gating is regulated in complex manner as phosphorylation is mandatory for channel activity and gating is directly regulated by binding of ATP to specific intracellular sites on the CFTR protein. This review covers our current understanding on the gating mechanism in CFTR and illustrates the relevance of alteration of these mechanisms in the onset of cystic fibrosis.     

Architecture and functional properties of the CFTR channel pore

The main function of the cystic fibrosis transmembrane conductance regulator (CFTR) is as an ion channel for the movement of small anions across epithelial cell membranes. As an ion channel, CFTR must form a continuous pathway across the cell membrane—referred to as the channel pore—for the rapid electrodiffusional movement of ions. This review summarizes our current understanding of the architecture of the channel pore, as defined by electrophysiological analysis and molecular modeling studies. This includes consideration of the characteristic functional properties of the pore, definition of the overall shape of the entire extent of the pore, and discussion of how the molecular structure of distinct regions of the pore might control different facets of pore function. Comparisons are drawn with closely related proteins that are not ion channels, and also with structurally unrelated proteins with anion channel function. A simple model of pore function is also described.     

New paradigms of CFTR chloride channel regulation

The Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel controls salt and water transport across epithelial tissues. Alterations in the activity of this ion channel lead to two major human diseases: cystic fibrosis (low CFTR activity) and secretory diarrhea (excessive CFTR activity). The goal of this article is to review recent developments in our understanding of two aspects of CFTR biology: (i) interactions between CFTR domains (intramolecular interactions) that control the gating of this epithelial chloride channel and (ii) interactions between CFTR and other proteins (intermolecular interactions) that couple the activity of this ion channel to additional cellular processes in epithelial cells (e.g. membrane traffic). Clarifying the nature of these interactions may lead to the development of novel strategies for treating diseases that involve the CFTR chloride channel. Received 12 October 1999; accepted 31 December 1999     

Biochemical,biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.     

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

Since their initial discovery, 30 years ago, antimicrobial peptides (AMPs) have been intensely investigated as a possible solution to the increasing problem of drug-resistant bacteria. The interaction of antimicrobial peptides with the cellular membrane of bacteria is the key step of their mechanism of action. Fluorescence spectroscopy can provide several structural details on peptide–membrane systems, such as partition free energy, aggregation state, peptide position and orientation in the bilayer, and the effects of the peptides on the membrane order. However, these “low-resolution” structural data are hardly sufficient to define the structural requirements for the pore formation process. Molecular dynamics simulations, on the other hand, provide atomic-level information on the structure and dynamics of the peptide–membrane system, but they need to be validated experimentally. In this review we summarize the information that can be obtained by both approaches, highlighting their versatility and complementarity, suggesting that their synergistic application could lead to a new level of insight into the mechanism of membrane destabilization by AMPs.     

Combining theoretical and experimental data to decipher CFTR 3D structures and functions

Cryo-electron microscopy (cryo-EM) has recently provided invaluable experimental data about the full-length cystic fibrosis transmembrane conductance regulator (CFTR) 3D structure. However, this experimental information deals with inactive states of the channel, either in an apo, quiescent conformation, in which nucleotide-binding domains (NBDs) are widely separated or in an ATP-bound, yet closed conformation. Here, we show that 3D structure models of the open and closed forms of the channel, now further supported by metadynamics simulations and by comparison with the cryo-EM data, could be used to gain some insights into critical features of the conformational transition toward active CFTR forms. These critical elements lie within membrane-spanning domains but also within NBD1 and the N-terminal extension, in which conformational plasticity is predicted to occur to help the interaction with filamin, one of the CFTR cellular partners.     

The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically ‘rescued’ F508del CFTR displays instability at the cell’s surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.     

A SAXS-based ensemble model of the native and phosphorylated regulatory domain of the CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is an anion channel activated by protein kinase A phosphorylation. The regulatory domain (RD) of CFTR has multiple phosphorylation sites, and is responsible for channel activation. This domain is intrinsically disordered, rendering the structural analysis a difficult task, as high-resolution techniques are barely applicable. In this work, we obtained a biophysical characterization of the native and phosphorylated RD in solution by employing complementary structural methods. The native RD has a gyration radius of 3.25 nm, and a maximum molecular dimension of 11.4 nm, larger than expected for a globular protein of the same molecular mass. Phosphorylation causes compaction of the structure, yielding a significant reduction of the gyration radius, to 2.92 nm, and on the maximum molecular dimension to 10.2 nm. Using an ensemble optimization method, we were able to generate a low-resolution, three-dimensional model of the native and the phosphorylated RD based on small-angle X-ray scattering data. We have obtained the first experiment-based model of the CFTR regulatory domain, which will be useful to understand the molecular mechanisms of normal and pathological CFTR functioning.     

Molecular weight of human alpha2-H-ferroglobulin subunits. Comparison with molecular weight of ferritin subunits]

alpha2 H globulin, a glycoferroprotein, was first demonstrated in the sera of patients with malignant diseases. This protein was isolated from cancerous human liver, and compared with ferritin, a ferroprotein showing some identical properties (presence of iron, high molecular weight, common antigenic determinants). However, physicochemical differences were observed between these two proteins. The study of protein dissociation was performed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction by mercaptoethanol. A similar molecular weight of 19 000 is obtained for subunits of these two proteins. This value agrees well with the results obtained by other authors for ferritin.     

Transfer and templates in scientific modelling

The notion of template has been advocated by Paul Humphreys and others as an illuminating unit of analysis in the philosophy of scientific modelling. Templates are supposed to have the dual functions of representing target systems and of facilitating quantitative manipulation. A resulting worry is that wide-ranging cross-disciplinary use of templates might compromise their representational function and reduce them to mere formalisms. In this paper, we argue that templates are valuable units of analysis in reconstructing cross-disciplinary modelling. Central to our discussion are the ways in which Lotka-Volterra models are used to analyse processes of technology diffusion. We illuminate both the similarities and differences between contributions to this case of cross-disciplinary modelling by reconstructing them as transfer of a template, without reducing the template to a mere formalism or a computational model. This requires differentiating the interpretation of templates from that of the models based on them. This differentiation allows us to claim that the LV models of technology diffusion that we review are the result of template transfer - conformist in some contributions, creative in others.     

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.     

Conformational change of the extracellular parts of the CFTR protein during channel gating

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^? channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd²⁺ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.     

Robust modelling of ARCH models

The autoregressive conditional heteroscedastic (ARCH) model and its extensions have been widely used in modelling changing variances in financial time series. Since the asset return distributions frequently display tails heavier than normal distributions, it is worth while studying robust ARCH modelling without a specific distribution assumption. In this paper, rather than modelling the conditional variance, we study ARCH modelling for the conditional scale. We examine the L₁‐estimation of ARCH models and derive the limiting distributions of the estimators. A robust standardized absolute residual autocorrelation based on least absolute deviation estimation is proposed. Then a robust portmanteau statistic is constructed to test the adequacy of the model, especially the specification of the conditional scale. We obtain their asymptotic distributions under mild conditions. Examples show that the suggested L₁‐norm estimators and the goodness‐of‐fit test are robust against error distributions and are accurate for moderate sample sizes. This paper provides a useful tool in modelling conditional heteroscedastic time series data. Copyright © 2001 John Wiley & Sons, Ltd.