Prediction Of Binding Affinities Of Peptides To TAP And Molecular Simulation Studies

The transporter associated with antigen processing (TAP) belongs to theATP-binding cassette (ABC) transporter superfamily, which plays a vital role inhuman adaptive immune defense. Driven by ATP hydrolysis, TAP translocatesantigenic peptides from the cytosol into the ER (endoplasmic reticulum) lumenwhere the antigenic peptides are loaded onto the HLA class I molecules. Recently,numerous studies show that TAP is closely related with various diseases such asautoimmune diseases (typeⅠdiabetes), viral infections, and different malignancies.Therefore, the understanding of the detailed structure and mechanism of TAP will be ofgreat importance in exploring cellular immunity, viruses escape mechanism, relatedimmunological diseases, tumor prevention and cure, and so on. Despite the rapidadvances in the study of TAP, many fundamental questions linked to ATP binding andhydrolysis and its relation to the transport cycle remain unanswered due to the absenceof a high-resolution three-dimensional crystal structure.Today, with the rapid development of computer technology, molecular simulationhave been an important research method in biology science, such as Quantitativestructural-activity relationship (QSAR), molecular dynamics (MD), homology modeling,molecular docking and so on. In this paper, these methods were used to study thebinding affinities of peptides to TAP and selectivity, coupling relationship between ATPhydrolysis and the conformational change of NBD as well as the structure-functionrelationship of TAP. The main results are summarized as follows:①VHSE (Principal component score vector of hydrophobic, steric, and electronicproperties), a novel set of descriptors based on the physical and chemicalcharacteristics of20nature amino acids, was used to characterize613nonamer peptidesof known affinity to TAP. Then, support vector regression (SVR) was adopted toestablish prediction models of TAP binding affinity of peptide. An optimal SVM modelwith linear kernel was obtained, of which R~2, Q~2and R~2extwere0.7386,0.7270and0.6057, respectively. Results show that electronic, steric, and hydrophobic properties ofthe amino acid sites are closely related to TAP binding affinity and substrate specificity,especially for electrical property. The amino acids at P1, P2, P7and P9of antigenicpeptides have the most impact on TAP binding affinity, while those at P3, P4, P5andP6have less impact and amino acid at P8has no impact. According to prediction results of single point mutated antigenic peptides by the optimal linear SVM model togetherwith the contribution weights of selected variables, substrate specificity of TAP wassummarized.②Molecular dynamics simulations are performed to investigate the couplingrelationship between ATP hydrolysis and the conformational change of NBD. Startingwith the ATP-bound, closed homodimer of TAP1-NBD, four simulation systems withall possible combinations of nucleotides bound to the two nucleotide binding sites areconstructed as follows: ATP/ATP, ATP/ADP, ADP/ADP and NO ATP, and simulatedwith equilibrium molecular dynamics for80ns each. The results show that significantchanges in the monitored distances between the monomers after ATP hydrolysis, eventhough the apparent dimer opening was not observed in any of the four80nssimulations. The major conformational changes are localized at specific regions of theprotein,namely, involving segments2030and90160. The results suggest that theclosed form of the NBD dimer can only be maintained with two bound ATP molecules;in other words, hydrolysis of one ATP can lead to the change of the dimer interface ofthe NBD dimer. Furthermore, only in the ADP/ADP-bound dimer, the relative rotationof the α helical subdomain results in the distances increased in each active site, probablysufficient or close to sufficient to allow nucleotide exchange. Additionally, the openingis an immediate effect of ATP hydrolysis rather than the dissociation of hydrolysisproducts. These results support a switch model for the mechnism of ATP hydrolysis incontrast to the constant contact model.③Based on a protein-protein BLAST search and a multiple sequence alignmentwith known structures of full-length, four homology models of TAP were presentedbuilt on the crystal structures of P-glycoprotein (P-gp), TM0287/0288, and Sav1866.The models represent the transporter in inward-and outward-facing conformations,which could represent initial and final states of the transport cycle, respectively. Theassessment of the TAP homology models was carried out by means of Procheck,What_CHECK, and ERRAT and so on. The values obtained from these analyses suggestthe models are comparable with with the crystal. The conserved regions in theER-facing loops with a role in the opening and closing of the cavity were described. Theconserved π-stacking interactions in the cytosolic part of the TMDs were also identified.Further, combined with the experimental data, structural insights into the role ofresidues involved in peptide binding were taken in consideration, such as TAP1-Val348,and TAP2-Cys213, Thr217. ④Molecular docking was employed in the inter-molecule interaction studies onthe3inward-facing TAP models and nonamer (RRYQKSTEL). The results of thedocking performed on the TAP model based on TM0287/0288show that theN-terminus residues of the peptide strongly interact with the residues of the negativelycharged pocket, in close proximity to TAP1-Val348. Additionally, residues of thepositively charged groove coordinated the C-terminus of the peptide. This conformationof nonamer proposed a model for the binding of the peptide that is in agreement withthe experimental data. In the P-gp based model, the C-terminus residues are stronglybound to the positively charged pocket. Because of the larger opening of the cavity ofthis model compared to the TAP model based on TM0287/0288, the N-terminus cannotreach the negatively charged pockets, confirming that this conformation might not besuitable for the binding of the peptide. These studies suggest that1) the peptide boundto TAP as an extended and kinked conformation, with an average distance between thetwo termini of a9-mer of2.2nm, and that2) the coordination of the N-and C-terminusof the peptide with the negatively and the positively charged pocket, respectively, ispossible, even though only in the TM0287/0288based model with a close proximity.The improvment of structure determination technology for transmembrane proteinsand computer simulation will greatly promote the understanding of the transportmechanisms of TAP. At present, the research methods and results in this paper canprovide important clues and approach for the in-depth study of the peptide binding andthe dynamics of human TAP, which are of great significance in exploring themechanisms of cellular immune, immune escape of tumor, human pathogenesis and soon.