Backbone dynamics of the intrinsically disordered HIV-1 Tat protein
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Date
2017
Authors
To, Vu
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Abstract
The type 1 Human Immunodeficiency Virus (HIV-1) transactivator of transcription (Tat) is a 101-residue protein that significantly increases the viral transcription. The full-length Tat protein (Tat101) is encoded by two exons yielding the first 72 and the last 29 residues of the protein, respectively. The function and intrinsic disorder of the first 72 residues have been studied in great detail but relatively little is known about the structure and function of the second exon product despite its conserved expression in all lentiviruses.
My thesis aims to study the impact of Tat’s second exon product on the full-length protein in terms of disorder, dynamics and structural propensity. NMR spectroscopy has been used to study Tat101 protein in a fully reduced state. Multiple isotope labeling strategies were applied and the labeled protein was expressed and purified in high yield. Backbone resonance assignment, chemical shift analysis, structure propensity analysis, fast (ps-ns) and slow (ms) timescale dynamics and hydrogen exchange studies confirm the intrinsically disordered nature of the second exon product and full-length Tat. The NMR results revealed Tat’s propensities to adopt different local conformations. Reduced spectral density mapping and model-free analysis show that the fast internal motion on the ps-ns timescale dominates the relaxation, and that Tat101 has no slow motion / conformational exchange on the ms timescale. The hydrogen exchange measurements yield protection factors below 1, which can be explained by the higher local pH compared with the bulk solvent. Classical molecular dynamics simulations were used as a complementary technique to verify the NMR results and to sample the protein conformers that are invisible to NMR due to ensemble averaging. The two 100 ns trajectories from the simulations of Tat’s first exon product are dominated by non-structural elements such as coils, turns, and bends. The order parameters derived from the simulations are below 0.8 and in agreement with the NMR results, confirming the flexibility of the protein. The combination of NMR dynamics and simulation results indicate that some regions of the protein likely bind partners through a conformational-selection mechanism while other parts of protein bind their targets through an induced-fit mechanism.
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Keywords
HIV, Intrinsically disordered protein, Transactivator of transcription, Protein dynamics, Molecular dynamics simulation, NMR