Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region.

Digging into the 3D Structure Predictions of AlphaFold2 with Low Confidence: Disorder and Beyond.

AlphaFold2 (AF2) has created a breakthrough in biology by providing three-dimensional structure models for whole-proteome sequences, with unprecedented levels of accuracy. In addition, the AF2 pLDDT score, related to the model confidence, has been shown to provide a good measure of residue-wise disorder. Here, we combined AF2 predictions with pyHCA, a tool we previously developed to identify foldable segments and estimate their order/disorder ratio, from a single protein sequence.

Pharmacological chaperones improve intra-domain stability and inter-domain assembly via distinct binding sites to rescue misfolded CFTR.

Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target.

New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation.

C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein.

Functional and Pharmacological Characterization of the Rare CFTR Mutation W361R.

Understanding the functional consequence of rare cystic fibrosis (CF) mutations is mandatory for the adoption of precision therapeutic approaches for CF. Here we studied the effect of the very rare CF mutation, W361R, on CFTR processing and function. We applied western blot, patch clamp and pharmacological modulators of CFTR to study the maturation and ion transport properties of pEGFP-WT and mutant CFTR constructs, W361R, F508del and L69H-CFTR, expressed in HEK293 cells.

Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity.

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC close to 1 μM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III.

A topology-based investigation of protein interaction sites using Hydrophobic Cluster Analysis.

Hydrophobic clusters, as defined by Hydrophobic Cluster Analysis (HCA), are conditioned binary patterns, made of hydrophobic and non-hydrophobic positions, whose limits fit well those of regular secondary structures. They were proved to be useful for predicting secondary structures in proteins from the only information of a single amino acid sequence and have permitted to assess, in a comprehensive way, the leading role of binary patterns in secondary structure preference towards a particular state. Here, we considered the available experimental 3D structures of protein globular domains to enlarge our previously reported hydrophobic cluster database (HCDB), almost doubling the number of hydrophobic cluster species (each species being defined by a unique binary pattern) that represent the most frequent structural bricks encountered within protein globular domains.

Order in Disorder as Observed by the "Hydrophobic Cluster Analysis" of Protein Sequences.

Hydrophobic cluster analysis (HCA) is an original approach for protein sequence analysis, which provides access to the foldable repertoire of the protein universe, including yet unannotated protein segments ("dark proteome"). Foldable segments correspond to ordered regions, as well as to intrinsically disordered regions (IDRs) undergoing disorder to order transitions. In this review, how HCA can be used to give insight into this last category of foldable segments is illustrated, with examples matching known 3D structures.

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.

Cis variants identified in F508del complex alleles modulate CFTR channel rescue by small molecules.

Molecules correcting the trafficking (correctors) and gating defects (potentiators) of the cystic fibrosis causing mutation c.1521_1523delCTT (p.Phe508del) begin to be a useful treatment for CF patients bearing p.

The implications of CFTR structural studies for cystic fibrosis drug development.

Development of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators, targeting the root cause of cystic fibrosis (CF), represents a challenge in the era of personalized medicine, as CFTR mutations lead to a variety of phenotypes, which likely require different, specific treatments. CF drug development is also complicated by the need to preserve the right balance between stability and flexibility, required for optimal function of the CFTR protein. In this review, we highlight how structural data can be exploited in this context to understand the molecular mechanisms of disease-associated mutations, to characterize the mechanisms of action of known modulators and to rationalize the search for novel, specific compounds.

Molecular modelling and molecular dynamics of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations.

The respective roles of polar/nonpolar binary patterns and amino acid composition in protein regular secondary structures explored exhaustively using hydrophobic cluster analysis.

Several studies have highlighted the leading role of the sequence periodicity of polar and nonpolar amino acids (binary patterns) in the formation of regular secondary structures (RSS). However, these were based on the analysis of only a few simple cases, with no direct mean to correlate binary patterns with the limits of RSS. Here, HCA-derived hydrophobic clusters (HC) which are conditioned binary patterns whose positions fit well those of RSS, were considered.

Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.

In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today.

Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues.

Ferroportin (SLC40A1) is the only known iron exporter in mammals and is considered a key coordinator of the iron balance between intracellular and systemic iron homeostasis. However, the structural organization of ferroportin in the lipid bilayer remains controversial and very little is known about the mechanism underlying iron egress. In the present study, we have developed an approach based on comparative modeling, which has led to the construction of a model of the three-dimensional (3D) structure of ferroportin by homology to the crystal structure of a Major Facilitator Superfamily member (EmrD).

CFTR: effect of ICL2 and ICL4 amino acids in close spatial proximity on the current properties of the channel.

Background: CFTR is the only ABC transporter functioning as a chloride (Cl(-)) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains.

Methods: Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording.

The PWAPA cassette: Intimate association of a PHD-like finger and a winged-helix domain in proteins included in histone-modifying complexes.

Polycomb complexes function as enforcers of epigenetically repressed state, balanced by an antagonist state mediated by Trithorax. Using sensitive methods of sequence analysis, we show here that Polycomb-like proteins (PCLs) contain a tandem of intimately associated domains, which we named PWAPA and which is also present in ASH2L, a member of the Trithorax group. Polycomb-like proteins and ASH2L belong to the PCR2 and MLL histone methyltransferase complexes, respectively.

Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF.

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor.

C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand.

LOTUS, a new domain associated with small RNA pathways in the germline.

We describe here LOTUS, a hitherto uncharacterized small globular domain, which was identified using sensitive sequence profile analysis. The LOTUS domain is found in germline-specific proteins that are present in the nuage/polar granules of germ cells. TDRD5 and TDRD7, two mammalian members of the germline Tudor group, possess three copies of the LOTUS domain in their extreme N-termini.

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.

A concanavalin A-like lectin domain in the CHS1/LYST protein, shared by members of the BEACH family.

CHS1/LYST, the causative protein of the Chediak-Higashi syndrome (CHS), belongs to the BEACH (named after BEige And Chediak-Higashi) family, which includes various large proteins sharing the same C-terminal domain architecture [a PH (Pleckstrin homology)-BEACH domain followed by WD repeats). Members of the BEACH family are generally defined as vesicle-trafficking regulatory proteins, but their functions remain to be determined at the molecular level. Here, using a panel of sensitive methods of sequence analysis, we show that the N-terminal regions of BEACH proteins contain an as yet not described domain, which shares striking similarities with clostridial neurotoxins and defines a novel family within the concanavalin A (ConA)-like lectin superfamily.

The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo.

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4.