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Effect of Phosphorylation and Mutation to Asp and Glu on the Conformational Landscape of an Intrinsically Disordered Region Pastor, Nina


Intrinsically disordered proteins are notorious for their conformational flexibility and capacity for interaction with different targets by acquiring distinct conformations depending on the specifics of the binding site. They can also engage in specific interactions without loosing conformational freedom, forming fuzzy complexes. The particular conformations favored by these proteins can be tuned by posttranslational modifications, such as phosphorylation. A common experimental strategy to study the effect of phosphorylation is to perform mutations of the modified residues by aspartate (D) or glutamate (E), under the assumption that the main effect of phosphorylation is the inclusion of negative charge. Whether the mutation to D or E is equivalent to phosphorylation is case dependent. In this work we explore the conformational landscape of an intrinsically disordered region at the C-terminus of adenoviral protein E1B55kDa, which is regulated by phosphorylation at three residues at the C-terminus, with the aim of establishing whether mutation to D or E is equivalent to modification by phosphorylation. In the context of the complete virus, the triple mutant with Ds produces a more efficient virus compared to wild type, and mutation to alanine (A), which cannot be phosphorylated, is equivalent to not having the full protein. We chose the last 20 residues of E1B55kDa as our reference peptide, as multiple disorder predictors consider it to be disordered. This peptide has only one cationic residue (arginine 9), and the C-terminal half is enriched in negative residues. We submitted the wild type sequence to Pepfold3, and obtained 100 different structures for it. Taking these as a reference, we built versions with three phosphorylated residues (two serines and one threonine), three Ds, three Es and three As. We placed each peptide in a water box with 0.15M NaCl using Charmm-gui, and ran it in NAMD in the NPT ensemble at 298K and 1 atm with the Charmm36m forcefield for 50 ns, achieving a total simulated time of 5 µs for each peptide variant. The distribution of the radius of gyration shows the prevalence of extended structures, with a slightly expanded ensemble for the triple D and triple E variants, and a slightly compressed one for the phosphorylated version, compared to the wild type. This is reflected in almost saturated hydration for the peptide in all its residues, except for a small decrease in hydration number for arginine 9 in the phosphorylated version. A closer look at intrapeptide hydrogen bonds reveals that there are few interactions in general, but arginine 9 engages in many more interactions with the phosphorylated residues than with the other charges in the peptide; the interaction with phosphorylated threonine 19 is preferred above all. This interaction leads to the formation of a loop that prefers to adopt disordered conformations, so we propose that it engages in fuzzy complexes with other proteins. In general, phosphorylation leads to an increase in alpha helix formation in the peptide, while substitution for Ds and Es leads to a loss of this structure. We conclude that phosphorylation and the mutation to D and E are not equivalent.

Acknowledgments: This research was supported by a CONACYT scholarship for MARM and supercomputing time at the Laboratorio Nacional de Supercómputo del Sureste (LNS), LANCAD in México City, and the Laboratorio de Dinámica de Proteínas at UAEM.

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