Dataset Information

Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

ABSTRACT: Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC₄), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC₄ binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC₄ binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC₄-binding site.

SUBMITTER: Szeri F

PROVIDER: S-EPMC8267652 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Protein receptor conformations, obtained from molecular dynamics (MD) simulations, have become a promising treatment of its explicit flexibility in molecular docking experiments applied to drug discovery and development. However, incorporating the entire ensemble of MD conformations in docking experiments to screen large candidate compound libraries is currently an unfeasible task. Clustering algorithms have been widely used as a means to reduce such ensembles to a manageable size. Most studies investigate different algorithms using pairwise Root-Mean Square Deviation (RMSD) values for all, or part of the MD conformations. Nevertheless, the RMSD only may not be the most appropriate gauge to cluster conformations when the target receptor has a plastic active site, since they are influenced by changes that occur on other parts of the structure. Hence, we have applied two partitioning methods (k-means and k-medoids) and four agglomerative hierarchical methods (Complete linkage, Ward's, Unweighted Pair Group Method and Weighted Pair Group Method) to analyze and compare the quality of partitions between a data set composed of properties from an enzyme receptor substrate-binding cavity and two data sets created using different RMSD approaches. Ensembles of representative MD conformations were generated by selecting a medoid of each group from all partitions analyzed. We investigated the performance of our new method for evaluating binding conformation of drug candidates to the InhA enzyme, which were performed by cross-docking experiments between a 20 ns MD trajectory and 20 different ligands. Statistical analyses showed that the novel ensemble, which is represented by only 0.48% of the MD conformations, was able to reproduce 75% of all dynamic behaviors within the binding cavity for the docking experiments performed. Moreover, this new approach not only outperforms the other two RMSD-clustering solutions, but it also shows to be a promising strategy to distill biologically relevant information from MD trajectories, especially for docking purposes.

Project description:ContextEgypt has a high prevalence of the hepatitis C virus (HCV) genotype 4a (GT-4a). Unfortunately, the high resistance it exhibited still was not given the deserved attention in the scientific community. There is currently no consensus on the NS5A binding site because the crystal structure of HCV NS5A has not been resolved. The prediction of the binding modes of direct-acting antivirals (DAA) with the NS5A is a point of controversy due to the fact that several research groups presented different interaction models to elucidate the NS5A binding site. Consequently, a 3D model of HCV NS5A GT-4a was constructed and evaluated using molecular dynamics (MD) simulations. The generated model implies an intriguing new orientation of the AH relative to domain I. Additionally, the probable binding modes of marketed NS5A inhibitors were explored. MD simulations validated the stability of the predicted protein-ligand complexes. The suggested model predicts that daclatasvir and similar drugs bind symmetrically to HCV NS5A GT-4a. This will allow for the development of new NS5A-directed drugs, which may result in reduced resistance and/or a wider range of effectiveness against HCV.MethodsThe 3D model of HCV NS5A GT-4a was constructed using the comparative modeling approach of the web-based application Robetta. Its stability was tested with 200-ns MD simulations using the Desmond package of Schrodinger. The OPLS2005 force field was assigned for minimization, and the RMSD, RMSF, and rGyr were tracked throughout the MD simulations. Fpocket was used to identify druggable protein pockets (cavities) over the simulation trajectories. The binding modes of marketed NS5A inhibitors were then generated and refined with the aid of docking predictions made by FRED and AutoDock Vina. The stability of these drugs in complex with GT-4a was investigated by using energetic and structural analyses over MD simulations. The Prime MM-GBSA (molecular mechanics/generalized Born surface area) method was used as a validation tool after the docking stage and for the averaged clusters after the MD simulation stage. We utilized PyMOL and VMD to visualize the data.

Project description:Despite designating catalytic roles of Asp299 and Arg131 during the transfer of gamma-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591-600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in K(m) (Ado) with a 1000-fold decrease in k(cat)/K(m), implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2'or 3'-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme-Ado complex, the binding energy (DeltaDeltaG(B)) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered K(m) (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.

Dataset Information

Mutagenic Analysis of the Putative ABCC6 Substrate-Binding Cavity Using a New Homology Model.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets