Caracterização de sítios conformacionais de fosforilação em proteínas

Protein phosphorylation is the most frequent type of post-translational modiﬁcation in signaling pathway, developing a key role in a wide range of cell events. The full understanding of the circumstances that coordinate the phosphorylation event remains a challenge for science, despite the growing number of approaches and studies on the subject. A broadly described and accepted mechanism as essential for the coordination of protein phosphorylation is the existence of amino acids sequences that contribute to phosphorylation occurrence, known as phosphorylation consensus. In this model, each protein kinase is able to recognize phosphorylation sites inserted in a speciﬁc sequence on the primary structure. However, as the data about phosphorylation sites increases, it is possible to notice that there are sites that are validated experimentally as phosphorylated by a particular protein kinase, which do not have the consensus phosphorylation. In this work, it was tested and proved that phosphorylation sites without the sequence consensus presents anchors residues, that are close to the phosphorylation site on the tertiary structure, creating a structural conformation that mimics the stereochemical features of a substrate peptide containing the phosphorylation consensus. For this evaluation, using substrates of PKA, it was found that more than 90% of phosphorylation sites that have no consensus on the primary structure, presented this kind of disposition on the tertiary structure. Distant residues in the primary structure are spatially close on the three-dimensional structure, in a conformation similar to a phosphorylation site containing the consensus. Thus we proposed the existence of conformational phosphorylation sites. To conﬁrm that these conformational sites could be crucial in substrate recognition, it was built kinase-substrate models, aiming to demonstrate the feasibility of residues forming the conformational consensus on the substrate to interact with the kinase analogously to a substrate with consensus phosphorylation. For experimental veriﬁcation of this phenomenon, we used the phosphorylation model of α -Tubulin, in which we observed a phosphorylation at residue T253 that depends of residues K163 and K164 to interact with the protein kinase, conﬁrming the consistency of proposed model. Faced with the novelty of the proposal, the computational data and the experimental validation, it becomes clear the importance of studying the three dimensional structure of phosphorylation sites, not only as a way of achieving deeper knowledge on phosphorylation ﬁeld, but also as a potential prospect to be explored on the development of new technologies. do após o da do Identiﬁcação substrato para auxílio na comparação. Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr 253 in a -tubulin as a site that is phosphorylated by protein kinase C b I (PKC b I). Thr 253 is not part of a linear PKC consensus motif. Instead, Thr 253 occurs within a region on the surface of a -tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKC b I. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this “ structurally formed ” consensus motif and its importance for the protein kinase – substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs. Data materials availability: Three substrates with linear motifs, three substrates with formed consensus motifs, the a -tubulin – PKC complex models are available as