Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins.Synopsis: Many proteins are chemically modified after they are synthesized in the cell. These post-translational modifications can modulate the ability of a protein to perform chemical reactions and to interact with other proteins. At the cellular level, for example, these chemical modifications are critical for allowing the cell to respond to its environment and control its division. One of the most common mechanisms by which proteins can be modified is by phosphorylation—the addition of a phosphate group to an amino acid side chain of the protein. Thousands of proteins are known to be modified by phosphorylation, but only for a small minority of these do we have any detailed understanding of how the chemical modification regulates the function of the protein. The authors describe a computational method that can make testable predictions about the structural changes that occur in a protein induced by post-translational phosphorylation. Their results show that the method can produce structural models of the phosphorylated proteins with near-atomic accuracy, and provide insight into the energetics of conformational switches driven by phosphorylation. As such, the computational method complements experiments aimed at understanding the mechanisms of protein regulation by phosphorylation.