Motif Of Nucleotide Binding Domain Research Articles

Rescue of Rare CFTR Trafficking Mutants Highlights a Structural Location-Dependent Pattern for Correction.

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Currently, more than 2100 variants have been identified in the gene, with a large number being very rare. The approval of modulators that act on mutant CFTR protein, correcting its molecular defect and thus alleviating the burden of the disease, revolutionized the field of CF. However, these drugs do not apply to all patients with CF, especially those with rare mutations-for which there is a lack of knowledge on the molecular mechanisms of the disease and the response to modulators. In this work, we evaluated the impact of several rare putative class II mutations on the expression, processing, and response of CFTR to modulators. Novel cell models consisting of bronchial epithelial cell lines expressing CFTR with 14 rare variants were created. The variants studied are localized at Transmembrane Domain 1 (TMD1) or very close to the signature motif of Nucleotide Binding Domain 1 (NBD1). Our data show that all mutations analyzed significantly decrease CFTR processing and while TMD1 mutations respond to modulators, those localized in NBD1 do not. Molecular modeling calculations confirm that the mutations in NBD1 induce greater destabilization of CFTR structure than those in TMD1. Furthermore, the structural proximity of TMD1 mutants to the reported binding site of CFTR modulators such as VX-809 and VX-661, make them more efficient in stabilizing the CFTR mutants analyzed. Overall, our data suggest a pattern for mutation location and impact in response to modulators that correlates with the global effect of the mutations on CFTR structure.

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast.

Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast Saccharomyces cerevisiae but also in the pathogenic yeast Candida albicans We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of S. cerevisiae, Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in C. albicans, Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.

Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration.

ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.

Comparative analysis of ABCB1 reveals novel structural and functional conservation between monocots and dicots.

Phytohormone auxin plays a critical role in modulating plant architecture by creating a gradient regulated via its transporters such as ATP-binding cassette (ABC) B1. Except for Arabidopsis and maize, where it was shown to interrupt auxin transport, ABCB1's presence, structure and function in crop species is not known. Here we describe the structural and putative functional organization of ABCB1 among monocots relative to that of dicots. Identified from various plant species following specific and stringent criteria, ZmABCB1's “true” orthologs sequence identity ranged from 56–90% at the DNA and 75–91% at the predicted amino acid (aa) level. Relative to ZmABCB1, the size of genomic copies ranged from −27 to +1.5% and aa from −7.7 to +0.6%. With the average gene size being similar (5.8 kb in monocots and 5.7 kb in dicots), dicots have about triple the number of introns with an average size of 194 bp (total 1743 bp) compared to 556 bp (total 1667 bp) in monocots. The intron-exon junctions across species were however conserved. N-termini of the predicted proteins were highly variable: in monocots due to mismatches and small deletions of 1–13 aa compared to large, species-specific deletions of up to 77 aa in dicots. The species-, family- and group- specific conserved motifs were identified in the N-terminus and linker region of protein, possibly responsible for the specific functions. The near-identical conserved motifs of Nucleotide Binding Domains (NBDs) in two halves of the protein showed subtle aa changes possibly favoring ATP binding to the N-terminus. Predicted 3-D protein structures showed remarkable similarity with each other and for the residues involved in auxin binding.

Abstract 898: Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1).

Abstract P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) efflux transporter implicated in the development of multidrug resistance in cancer cells. The catalytic cycle of P-gp is still not fully understood. Both the C431 and C1074 residues in the Walker A motifs of nucleotide-binding domains (NBDs) are highly conserved in P-gp from different species and these residues are excellent reporter sites to study the interaction between the two NBDs during the catalytic cycle. Using homo-bifunctional crosslinkers, disulfide crosslinking of the 431C and 1074C residues in a cysless background can be observed in the presence of copper phenanthroline (CuP) and M17M, which have spacer arm lengths of 0 Å and 25 Å, respectively. However, crosslinking with M17M was prevented in the ADP-vanadate-trapped (closed) conformation, indicating that the distance between 431C and 1074C or the accessibility of these residues changes upon ATP hydrolysis. In addition, the accessibility of the residues, separately or together (431C/1074C) in both the apo and closed conformations was determined with fluorescein-5-maleimide (FM) and methanethio-sulfonate derivatives of rhodamine (MTS-rhodamine) and verapamil (MTS-verapamil), which exhibit different physical properties. The crosslinking of cysteine residues with thiol-reactive compounds is observed both in apo (open) and ADP-vanadate-trapped (closed) conformations, though at a reduced level in the latter case, indicating the flexible nature of the cysteines in Walker A domains. Consistent with these findings, the docking of FM in a homology model of human P-gp ATP sites shows that the sulfur atoms of 431C or 1074C can be oriented either facing the ATP pocket or facing away from the pocket. A different degree of labeling was observed between the two residues; for 1074C having 1.4- fold and 2-fold higher labeling with FM compared to 431C in the apo and closed conformations respectively, suggesting that accessibility of 1074C is greater than that of 431C in both conformations. In the presence of the transport substrate cyclosporine A (50 μM), crosslinking is still observed with CuP and M17M, suggesting that the binding of substrates to the drug-binding site in the transmembrane domains does not change the accessibility of the cysteines in the NBDs. In summary, the cysteines in the Walker A motifs of human P-gp are flexible and differentially accessible in the apo and closed conformations. In addition, the NBDs of human P-gp are closer together (20-25 Å) than the NBDs observed in the X-ray structure of mouse P-gp (36 Å). Citation Format: Hong-May Sim, Jaya Bhatnagar, Samantha A. Green, Adam Gonzalez, Eduardo E. Chufan, Suresh V. Ambudkar. Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 898. doi:10.1158/1538-7445.AM2013-898

Divergent signature motifs of nucleotide binding domains of ABC multidrug transporter, CaCdr1p of pathogenic Candida albicans, are functionally asymmetric and noninterchangeable

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

Conserved Asp327 of Walker B Motif in the N-Terminal Nucleotide Binding Domain (NBD-1) of Cdr1p ofCandida albicansHas Acquired a New Role in ATP Hydrolysis

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.

Phosphorylation of Candida glabrata ATP-binding Cassette Transporter Cdr1p Regulates Drug Efflux Activity and ATPase Stability

Fungal ATP-binding cassette transporter regulation was investigated using Candida glabrata Cdr1p and Pdh1p expressed in Saccharomyces cerevisiae. Rephosphorylation of Pdh1p and Cdr1p was protein kinase A inhibitor-sensitive but responded differentially to Tpk isoforms, stressors, and glucose concentration. Cdr1p Ser(307), which borders the nucleotide binding domain 1 ABC signature motif, and Ser(484), near the membrane, were dephosphorylated on glucose depletion and independently rephosphorylated during glucose exposure or under stress. The S484A enzyme retained half the wild type ATPase activity without affecting azole resistance, but the S307A enzyme was unstable to plasma membrane isolation. Studies of pump function suggested conformational interaction between Ser(484) and Ser(307). An S307A/S484A double mutant, which failed to efflux the Cdr1p substrate rhodamine 6G, had a fluconazole susceptibility 4-fold greater than the Cdr1p expressing strain, twice that of the S307A mutant, but 64-fold less than the control null strain. Stable intragenic suppressors indicative of homodimer nucleotide binding domain 1-nucleotide binding domain 1 interactions partially restored rhodamine 6G pumping and increased fluconazole and rhodamine 6G resistance in the S307A/S484A mutant. Nucleotide binding domain 1 of Cdr1p is a sensor of important physiological stimuli.

Crystal structure of human L-isoaspartyl-O-methyl-transferase with S-adenosyl homocysteine at 1.6-A resolution and modeling of an isoaspartyl-containing peptide at the active site.

Spontaneous formation of isoaspartyl residues (isoAsp) disrupts the structure and function of many normal proteins. Protein isoaspartyl methyltransferase (PIMT) reverts many isoAsp residues to aspartate as a protein repair process. We have determined the crystal structure of human protein isoaspartyl methyltransferase (HPIMT) complexed with adenosyl homocysteine (AdoHcy) to 1.6-A resolution. The core structure has a nucleotide binding domain motif, which is structurally homologous with the N-terminal domain of the bacterial Thermotoga maritima PIMT. Highly conserved residues in PIMTs among different phyla are placed at positions critical to AdoHcy binding and orienting the isoAsp residue substrate for methylation. The AdoHcy is completely enclosed within the HPIMT and a conformational change must occur to allow exchange with adenosyl methionine (AdoMet). An ordered sequential enzyme mechanism is supported because C-terminal residues involved with AdoHcy binding also form the isoAsp peptide binding site, and a change of conformation to allow AdoHcy to escape would preclude peptide binding. Modeling experiments indicated isoAsp groups observed in some known protein crystal structures could bind to the HPIMT active site.