Computational studies of protein regulation by post-translational phosphorylation
Introduction
Protein function is regulated at several levels in order to control cellular growth, division, and response to external stimuli. One important mechanism of protein regulation is by post-translational chemical modification. Since the addition and removal of a modifying group are enzyme catalyzed, the cell can finely tune the magnitude and duration of the regulatory effect and can construct complex molecular switches with rapid temporal responses.
Phosphorylation is a ubiquitous mechanism of post-translational modification. It is estimated that the fraction of proteins that are phosphorylated in vivo may be up to 30% [1]. In eukaryotes, the phosphate group is added to Ser, Thr, and Tyr side chains by protein kinases and removed by phosphatases. The phosphate predominantly carries a −2 charge at physiologically relevant pH, and the resulting large electrostatic perturbation modulates the energy landscapes governing protein folding, protein–protein and protein–ligand interactions, catalytic activity, and conformational dynamics. In many cases, phosphorylation results in switch-like changes in protein function. Numerous pathologies result from aberrant regulation of these electrostatic switches, including several cancers (e.g. [2, 3]).
Mass-spectrometric studies have identified many thousands of sites of post-translational phosphorylation [1]. However, obtaining an atomic-level understanding of the ways in which phosphorylation alters protein structure and function is difficult experimentally. X-ray crystallography and NMR provide the highest resolution structural information but are also the most difficult and time-consuming to obtain. A particular challenge can be obtaining sufficient quantities of pure protein with a particular post-translational modification. Electron paramagnetic resonance (EPR), circular dichroism, Förster resonance energy transfer (FRET), and small-angle X-ray scattering all can provide low-resolution information about structural changes due to phosphorylation but do not provide atomic detail.
Given the wide gap between the number of known post-translational modifications and the number that have been structurally or functionally characterized, we believe that computational methods can play a valuable role in elucidating principles and consequences of post-translational phosphorylation. The major focus of this review is atomically detailed simulations of phosphorylated proteins, and how computation can make sense of, complement, and drive experiment. In conclusion, we highlight challenges and problems to be tackled in the future of this young field.
Section snippets
Brief overview of computational methods to study phosphorylation
We first briefly discuss the main computational approaches referred to in this review to give a sense for the strengths, weaknesses, and computational costs of each particular class of methods. Because our focus is on atomically detailed simulations, we do not discuss coarse-grained methods.
As with other aspects of protein function, molecular dynamics (MD) has been widely used to study effects of phosphorylation. MD involves the integration of Newton's equation of motion for a system of atoms.
Structural control of kinases by phosphorylation
Many protein kinases, responsible for the phosphorylation of specific protein substrates, themselves are regulated by phosphorylation as part of signal transduction and regulatory cascades. Over the past decade, X-ray crystallographic studies have contributed tremendously to our understanding of the functioning of protein kinases; protein kinase A (PKA), the cyclin-dependent kinase (CDK) family and particularly CDK2, and the Src-family kinases are especially well studied.
Phosphorylation sites
Structural switching of peptides
Several recent computational studies have examined structural changes in peptides upon phosphorylation. Peptides are very amenable to computational studies as much greater sampling can be performed owing to the relatively small size of the system. In many cases, including the ones discussed here, the peptides are portions of a larger protein. Clearly, studying phosphorylation, either computationally or experimentally, of one small portion of a larger protein has limitations; it is not always
Phosphoregulation of membrane proteins
Many membrane proteins are regulated through phosphorylation. In some cases, phosphorylation regulates membrane protein trafficking, while in others, phosphorylation directly affects membrane protein function. As with other classes of proteins, the function and mechanism of many phosphorylation sites in membrane proteins are unknown. High-resolution structural analysis of membrane proteins is notoriously difficult experimentally, and computation has proven useful to study membrane protein
Challenges and future directions
As we have shown here, computational simulations, primarily using molecular dynamics, have already had a significant impact on elucidating mechanisms of regulation by post-translational phosphorylation. Two of the major challenges facing this area of research are those that underlie all computational structural biology: the accuracy of the energy model used and obtaining sufficient sampling. Some issues regarding the energetics of phosphorylated amino acids have been explored in model systems [
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Sam Pfaff for critical reading of the manuscript. This work was supported partly by NSF grant MCB-0346399. MPJ is a consultant to Schrodinger LLC.
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