Serine/threonine protein kinases (STKs) are extremely ancient and ubiquitous signaling enzymes; despite their common name “eukaryotic protein kinases”, these protein domains are also present in archaea and bacteria suggesting their presence in the last universal common ancestor 3-4 billion years ago. It is known that tyrosine kinases (TKs) descended from this lineage much later, just prior to the emergence of the first metazoans. TKs share a great deal of structural homology with even the most distantly related STKs, however their ability to phosphorylate Tyr instead of Ser and Thr along with their unique domain organizations sets them apart from STKs in both sequence and function. This thesis explores the distinct conformational “landscapes” of these two important protein families, dealing with a ~20 residue long “activation loop” which has multiple inactive conformations but only one active conformation. By employing a statistical energy Potts Hamiltonian model of protein sequences and using molecular dynamics free-energy simulations, major sequence features of the catalytic domain were determined which control the shape of the free-energy landscape i.e., the relative depths of the “active” and “inactive” basins, a quantity termed the reorganization free-energy ΔG_reorg. A key finding from this approach is the marked divergence in the conformational landscapes of TKs from STKs that is encoded in the sequences of extant family members, which was detected by threading their Potts sequence energies over the active “DFG-in” (catalytic “Asp-Phe-Gly motif oriented “in”) basin relative to an inactive “DFG-out” basin where the activation loop is “folded up” by ~20 Å. This free-energy basin autoinhibits the kinase because the activation loop behaves as a pseudo-substrate in cis. The Potts couplings threaded over the active and inactive basins suggest that TKs evolved to have a smaller free-energy difference between the active and inactive basins compared with STKs, by 4-6 kcal/mol. The sequence and structural basis for this effect was explored in detail by decomposing the threaded Potts Hamiltonian into pairwise interactions and analyzing the statistical energy effects of natural sequence variation at evolutionary divergent positions in the sequence. These effects were then verified by performing mutations of amino acid sidechains using FEP (Free Energy Perturbation) molecular dynamics simulations in both the active and inactive conformational states and comparing the results with analogous sequence-based calculations by making mutations in the Potts model. The results are highly consistent (Pearson correlation of 0.81) suggesting that the Potts model is comparable to FEP in its ability to capture the physical free-energy balance of amino acid sidechain interactions between two different conformational basins and validates the Potts model-predicted evolutionary divergent landscapes of TKs and STKs. This divergence can in part be attributed to autoinhibitory pseudo-substrate interactions involving the activation loop; the evolved peptide-substrate specificity of TKs compared with STKs, and the functional surfaces that have been evolutionarily molded to complement Tyr vs Ser/Thr-containing peptides, appear to have energetic feedback with the propensity of the kinase’s own activation loop to “fold” against these surfaces when the DFG is flipped from “in” to “out”, and TKs have evolved to exploit this as a means of regulation. / Chemistry
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/10278 |
| Date | 05 1900 |
| Creators | McDevitt, Joan, 0000-0002-4127-2294 |
| Contributors | Levy, Ronald, Voelz, Vincent, Stanley, Robert, Dunbrack, Roland |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 152 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/10240, Theses and Dissertations |
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