Synthesis and Biological Evaluation of Pyrazole-Pyrimidones as a New Class of Correctors of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis (CF) is caused by the functional expression defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Despite the recent success in CFTR modulator development, the available correctors only partially restore the F508del-CFTR channel function, and several rare CF mutations show resistance to available drugs. We previously identified compound 4172 that synergistically rescued the F508del-CFTR folding defect in combination with the existing corrector drugs VX-809 and VX-661. Here, novel CFTR correctors were designed by applying a classical medicinal chemistry approach on the 4172 scaffold. Molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted to propose a plausible binding site and design more potent and effective analogs. We identified three optimized compounds, which, in combination with VX-809 and the investigational corrector 3151, increased the plasma membrane density and function of F508del-CFTR and other rare CFTR mutants resistant to the currently approved therapies.

Analysis of AlphaMissense data in different protein groups and structural context.

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of missense variants. Since heterogenous performance on different types of proteins can be expected, we assessed the efficacy of AlphaMissense across several protein groups (e.g. soluble, transmembrane, and mitochondrial proteins) and regions (e.g. intramembrane, membrane interacting, and high confidence AlphaFold segments) using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on disordered datasets and ClinVar data related to the CFTR ABC protein. However, a superior performance was shown when benchmarked against the high quality CFTR2 database. Our results with CFTR emphasizes AlphaMissense's potential in pinpointing functional hot spots, with its performance likely surpassing benchmarks calculated from ClinVar and ProteinGym datasets.

PtdIns4P is required for the autophagosomal recruitment of STX17 (syntaxin 17) to promote lysosomal fusion.

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion.Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P2: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.

Plasma membrane Ca2+ pump isoform 4 function in cell migration and cancer metastasis.

The Ca2+ ion is a universal second messenger involved in many vital physiological functions including cell migration and development. To fulfil these tasks the cytosolic Ca2+ concentration is tightly controlled, and this involves an intricate functional balance between a variety of channels and pumps of the Ca2+ signalling machinery. Among these proteins, plasma membrane Ca2+ ATPases (PMCAs) represent the major high-affinity Ca2+ extrusion systems in the cell membrane that are effective in maintaining free Ca2+ concentration at exceedingly low cytosolic levels, which is essential for normal cell function. An imbalance in Ca2+ signalling can have pathogenic consequences including cancer and metastasis. Recent studies have highlighted the role of PMCAs in cancer progression and have shown that a particular variant, PMCA4b, is downregulated in certain cancer types, causing delayed attenuation of the Ca2+ signal. It has also been shown that loss of PMCA4b leads to increased migration and metastasis of melanoma and gastric cancer cells. In contrast, an increased PMCA4 expression has been reported in pancreatic ductal adenocarcinoma that coincided with increased cell migration and shorter patient survival, suggesting distinct roles of PMCA4b in various tumour types and/or different stages of tumour development. The recently discovered interaction of PMCAs with basigin, an extracellular matrix metalloproteinase inducer, may provide further insights into our understanding of the specific roles of PMCA4b in tumour progression and cancer metastasis.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.

Expression, Function and Trafficking of the Human ABCG2 Multidrug Transporter Containing Mutations in an Unstructured Cytoplasmic Loop.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354-367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357-360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.

Synergistic Inhibitory Effect of Quercetin and Cyanidin-3O-Sophoroside on ABCB1.

The human ABCB1 (P-glycoprotein, Pgp) protein is an active exporter expressed in the plasma membrane of cells forming biological barriers. In accordance with its broad substrate spectrum and tissue expression pattern, it affects the pharmacokinetics of numerous chemotherapeutic drugs and it is involved in unwanted drug-drug interactions leading to side effects or toxicities. When expressed in tumor tissues, it contributes to the development of chemotherapy resistance in malignancies. Therefore, the understanding of the molecular details of the ligand-ABCB1 interactions is of crucial importance. In a previous study, we found that quercetin (QUR) hampers both the transport and ATPase activity of ABCB1, while cyandin-3O-sophroside (C3S) stimulates the ATPase activity and causes only a weak inhibition of substrate transport. In the current study, when QUR and C3S were applied together, both a stronger ATPase inhibition and a robust decrease in substrate transport were observed, supporting their synergistic ABCB1 inhibitory effect. Similar to cyclosporine A, a potent ABCB1 inhibitor, co-treatment with QUR and C3S shifted the conformational equilibrium to the "inward-facing" conformer of ABCB1, as it was detected by the conformation-selective UIC2 mAb. To gain deeper insight into the molecular details of ligand-ABCB1 interactions, molecular docking experiments and MD simulations were also carried out. Our in silico studies support that QUR and C3S can bind simultaneously to ABCB1. The most favourable ligand-ABCB1 interaction is obtained when C3S binds to the central substrate binding site and QUR occupies the "access tunnel". Our results also highlight that the strong ABCB1 inhibitory effect of the combined treatment with QUR and C3S may be exploited in chemotherapy protocols for the treatment of multidrug-resistant tumors or for improving drug delivery through pharmacological barriers.

Association between Hyperglycemia and Medication-Related Osteonecrosis of the Jaw (MRONJ).

BACKGROUND: Medication-related osteonecrosis of the jaw (MRONJ) is a type of jawbone necrosis caused by the use of drugs for some types of cancer and osteoporosis. The current study aimed to evaluate the associations between hyperglycemia and the development of medication-related osteonecrosis of the jaw. METHODS: Our research group investigated data collected between 1 January 2019 and 31 December 2020. A total of 260 patients were selected from the Inpatient Care Unit, Department of Oromaxillofacial Surgery and Stomatology, Semmelweis University. Fasting glucose data were used and included in the study. RESULTS: Approximately 40% of the necrosis group and 21% of the control group presented with hyperglycemia. There was a significant association between hyperglycemia and MRONJ (p < 0.05, p = 0.003). Vascular anomaly and immune dysfunction caused by hyperglycemia can lead to necrosis after tooth extraction. Necrosis is more common in the mandible (75.0%) and in the case of parenteral antiresorptive treatment (intravenous Zoledronate and subcutaneous Denosumab). Hyperglycemia is a more relevant risk factor than bad oral habits (26.7%). CONCLUSIONS: Ischemia is a complication of abnormal glucose levels, a possible risk factor for necrosis development. Hence, uncontrolled or poorly regulated plasma glucose levels can significantly increase the risk of jawbone necrosis after invasive dental or oral surgical interventions.

The Reentry Helix Is Potentially Involved in Cholesterol Sensing of the ABCG1 Transporter Protein.

ABCG1 has been proposed to play a role in HDL-dependent cellular sterol regulation; however, details of the interaction between the transporter and its potential sterol substrates have not been revealed. In the present work, we explored the effect of numerous sterol compounds on the two isoforms of ABCG1 and ABCG4 and made efforts to identify the molecular motifs in ABCG1 that are involved in the interaction with cholesterol. The functional readouts used include ABCG1-mediated ATPase activity and ABCG1-induced apoptosis. We found that both ABCG1 isoforms and ABCG4 interact with several sterol compounds; however, they have selective sensitivities to sterols. Mutational analysis of potential cholesterol-interacting motifs in ABCG1 revealed altered ABCG1 functions when F571, L626, or Y586 were mutated. L430A and Y660A substitutions had no functional consequence, whereas Y655A completely abolished the ABCG1-mediated functions. Detailed structural analysis of ABCG1 demonstrated that the mutations modulating ABCG1 functions are positioned either in the so-called reentry helix (G-loop/TM5b,c) (Y586) or in its close proximity (F571 and L626). Cholesterol molecules resolved in the structure of ABCG1 are also located close to Y586. Based on the experimental observations and structural considerations, we propose an essential role for the reentry helix in cholesterol sensing in ABCG1.

Comprehensive Collection and Prediction of ABC Transmembrane Protein Structures in the AI Era of Structural Biology.

The number of unique transmembrane (TM) protein structures doubled in the last four years, which can be attributed to the revolution of cryo-electron microscopy. In addition, AlphaFold2 (AF2) also provided a large number of predicted structures with high quality. However, if a specific protein family is the subject of a study, collecting the structures of the family members is highly challenging in spite of existing general and protein domain-specific databases. Here, we demonstrate this and assess the applicability and usability of automatic collection and presentation of protein structures via the ABC protein superfamily. Our pipeline identifies and classifies transmembrane ABC protein structures using the PFAM search and also aims to determine their conformational states based on special geometric measures, conftors. Since the AlphaFold database contains structure predictions only for single polypeptide chains, we performed AF2-Multimer predictions for human ABC half transporters functioning as dimers. Our AF2 predictions warn of possibly ambiguous interpretation of some biochemical data regarding interaction partners and call for further experiments and experimental structure determination. We made our predicted ABC protein structures available through a web application, and we joined the 3D-Beacons Network to reach the broader scientific community through platforms such as PDBe-KB.

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the ß-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

User Experience and Sustainability of 3D Printing in Dentistry.

BACKGROUND: 3D printing is a rapidly developing technology in the healthcare industry and in dentistry. Its application clearly shows that this area of digital dentistry has potential for everyday usage across all fields, including prosthodontics, orthodontics, maxillofacial surgery, and oral implantology. However, despite gaining ground, there is a lack of information about how specialists (dentists and dental technicians) use additive technology. Our research group aimed to investigate the impact of social media on additive manufacturing technology among dental specialists and their everyday usage of 3D printing. METHODS: This paper investigated specialists' everyday usage of 3D printers via an online survey (Google Forms). The survey questions aimed to discover the number of 3D printers used, the accessibility of the devices, the annual cost, and the design programs. Since specialists tend to build online communities on social media, we circulated our study questionnaire using our profiles on LinkedIn, Facebook, and Instagram platforms during our research. RESULTS: A total of 120 responses were received from 20 countries, with the most significant numbers being from Hungary 23.7% (n = 27), the United States 18.4% (n = 21), and the United Kingdom 7.9% (n = 9). Most of the participants were dentists (n = 68) or dental technicians (n = 29), but some CAD/CAM specialists (n = 23) also completed our survey. The participants had an average of 3.8 years (±0.7) of experience in the 3D printing field, and owned a total of 405 printing devices (3.6 on average/person). CONCLUSIONS: The impact of social media on this research field is growing increasingly. Hence, we support specialists in joining virtual communities on professional platforms. This article intended to provide a practical overview, feedback, and direction for dentists interested in 3D printing technology. From our survey, we can conclude that additive technology is broadening dental applications and the services that we can provide for our patients.

Oral Health Knowledge and Habits of People With Type 1 and Type 2 Diabetes.

OBJECTIVES: This study aimed to collect information about oral health knowledge and the habits of people living with diabetes (PwD), primarily type 1 diabetes, using the newly developed World Health Organisation Oral Health Questionnaire for Adults (Annex 7). MATERIALS AND METHODS: Comparable and reliable questionnaires, comprising 23 questions for PwD, were sent to diabetes social media groups, mailing lists, and associations. The survey explored the relationships amongst demographic factors, age, dental education, eating habits, and other factors. RESULTS: The 23-question survey was answered by 307 individuals from 60 different countries. Alcohol and tobacco use, dental anxiety, and bad habits were often reported. Of the participants, 61.2% (n = 188) had at least 1 drink during the past 30 days. Of the participants, 22.8% (n = 70) were smokers. In total, 80.8% (n = 248) of the participants consumed biscuits, 76.2% (n = 234) consumed sweets, and 63.2% (n = 194) consumed soft drinks regularly. A total of 26.4% (n = 81) of the participants reported being afraid of dental treatment. Of the participants, 48.5% (n = 149) reported dry mouth and other oral complications. The frequency of visits to the dentist was satisfactory. A total of 71.3% (n = 219) of the participants reported visiting a dentist during the past 12 months. CONCLUSIONS: There is a need for proper oral health education for PwD. Trained diabetes advocates could be core messengers. However, interdisciplinary cooperation is mandatory for both education and the clinical aspect of diabetes care. For example, diabetes nurses need to be educated with the help of dentists or oral hygienists.

The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations.

Atomic-level structural insight on the human ABCG2 membrane protein, a pharmacologically important transporter, has been recently revealed by several key papers. In spite of the wealth of structural data, the pathway of transmembrane movement for the large variety of structurally different ABCG2 substrates and the physiological lipid regulation of the transporter has not been elucidated. The complex molecular dynamics simulations presented here may provide a breakthrough in understanding the steps of the substrate transport process and its regulation by cholesterol. Our analysis revealed drug binding cavities other than the central binding site and delineated a putative dynamic transport pathway for substrates with variable structures. We found that membrane cholesterol accelerated drug transport by promoting the closure of cytoplasmic protein regions. Since ABCG2 is present in all major biological barriers and drug-metabolizing organs, influences the pharmacokinetics of numerous clinically applied drugs, and plays a key role in uric acid extrusion, this information may significantly promote a reliable prediction of clinically important substrate characteristics and drug-drug interactions.

A twist in the ABC: regulation of ABC transporter trafficking and transport by FK506-binding proteins.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds.

The ABCG2/BCRP transporter and its variants - from structure to pathology.

The ABCG2 protein has a key role in the transport of a wide range of structurally dissimilar endo- and xenobiotics in the human body, especially in the tissue barriers and the metabolizing or secreting organs. The human ABCG2 gene harbors a high number of polymorphisms and mutations, which may significantly modulate its expression and function. Recent high-resolution structural data, complemented with molecular dynamic simulations, may significantly help to understand intramolecular movements and substrate handling, as well as the effects of mutations on the membrane transporter function of ABCG2. As reviewed here, structural alterations may result not only in direct alterations in drug binding and transporter activity, but also in improper folding or problems in the carefully regulated process of trafficking, including vesicular transport, endocytosis, recycling, and degradation. Here, we also review the clinical importance of altered ABCG2 expression and function in general drug metabolism, cancer multidrug resistance, and impaired uric acid excretion, leading to gout.

Discovering the chloride pathway in the CFTR channel.

Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.

Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism.

The human ABCG2 is an important plasma membrane multidrug transporter, involved in uric acid secretion, modulation of absorption of drugs, and in drug resistance of cancer cells. Variants of the ABCG2 transporter, affecting cellular processing and trafficking, have been shown to cause gout and increased drug toxicity. In this paper, we overview the key cellular pathways involved in the processing and trafficking of large membrane proteins, focusing on ABC transporters. We discuss the information available for disease-causing polymorphic variants and selected mutations of ABCG2, causing increased degradation and impaired travelling of the transporter to the plasma membrane. In addition, we provide a detailed in silico analysis of an as yet unrecognized loop region of the ABCG2 protein, in which a recently discovered mutation may actually promote ABCG2 membrane expression. We suggest that post-translational modifications in this unstructured loop at the cytoplasmic surface of the protein may have special influence on ABCG2 processing and trafficking.

Quantitative comparison of ABC membrane protein type I exporter structures in a standardized way.

An increasing number of ABC membrane protein structures are determined by cryo-electron microscopy and X-ray crystallography, consequently identifying differences between their conformations has become an arising issue. Therefore, we propose to define standardized measures for ABC Type I exporter structure characterization. We set conformational vectors, conftors, which describe the relative orientation of domains and can highlight structural differences. In addition, continuum electrostatics calculations were performed to characterize the energetics of membrane insertion illuminating functionally crucial regions. In summary, the proposed metrics contribute to deeper understanding of ABC membrane proteins' structural features, structure validation, and analysis of movements observed in a molecular dynamics trajectory. Moreover, the concept of standardized metrics can be applied not only to ABC membrane protein structures (http://conftors.hegelab.org).