Dataset Information

Molecular models of human P-glycoprotein in two different catalytic states.

ABSTRACT: BACKGROUND: P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies. RESULTS: We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants. CONCLUSION: Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs), within the transmembrane domains (TMDs) or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission. Our models may characterize different steps in the catalytic cycle and may be important tools to understand the structure-function relationship of P-glycoprotein.

SUBMITTER: Becker JP

PROVIDER: S-EPMC2661087 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Molecular models of human P-glycoprotein in two different catalytic states.

Becker Jean-Paul JP Depret Grégoire G Van Bambeke Françoise F Tulkens Paul M PM Prévost Martine M

BMC structural biology 20090122

<h4>Background</h4>P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution struct ...[more]

PMID: 19159494

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Project description:ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+) with each NBD indicates that the coordination of ATP and Mg(2+) differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations.

Project description:The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle biopsy samples by removing invalid and low signal-high-variance probes (as for CDF GPL24047). The probes were then summarized into groups (probe-sets) reflecting either an ensembl full transcript identifier (FL-ENST, GPL24047) or just the probes targeting the 3' UTR or the 5' UTR of that particular ENST. Therefore, 3 different CDF were used to process the HTA 2.0 arrays in this study. Note that each CEL file was GC adjusted using APT while our custom CDF pipeline removes any probe that has >80% or <20% GC content (~50,000). The analysis was carried out only on the pairs of probe-sets i.e. FL-ENST vs 3'UTR or FL-ENST vs 5'UTR or 3'UTR vs 5'UTR. Dynamic muscle loading alters tissue phenotype reflecting altered metabolic and functional demands. In humans, heterogeneous adaptation to loading complicates identification of the underpinning molecular regulators. We present a within-person analysis strategy that reduced heterogeneity for changes in muscle mass by ~40% and employed a genome-wide transcriptome method that modeled each mRNA from coding exons and 3’/5’ untranslated (UTR) regions. Our strategy detected ~3-4 times more regulated genes than similarly sized studies, including substantial UTR-selective regulation that other methods would not detect. We discovered a core of 141 genes correlated to muscle growth validated from newly analyzed independent samples (n=100). Further validating these identified genes, via RNAi in primary muscle cells, we demonstrate that members of the core genes were regulators of protein synthesis e.g. Molecular Transducers of Physical Activity in Humans MoTrPAC. Employing proteome-constrained networks and pathway analysis revealed notable relationships with the molecular characteristics of human muscle aging and insulin sensitivity, as well as potential drug-therapies. https://doi.org/10.1016/j.celrep.2020.107980

Dataset Information

Molecular models of human P-glycoprotein in two different catalytic states.

Publications

Molecular models of human P-glycoprotein in two different catalytic states.

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