Phosphorylation-induced Conformational Changes in a Mitogen-activated Protein Kinase Substrate

Abstract

Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems.Phosphorylation of specific amino acids near the surface of a protein is an almost universal mechanism of protein activation that has been long appreciated (1,2). Yet the mechanism whereby phosphorylation modifies the activity of the protein is not well understood (1). Analysis of phosphorylation sites from different enzymes reveals common themes. In eukaryotes, phosphorylation typically occurs at tyrosine, threonine, or serine side chains, and these phosphorylated residues often form a network of hydrogen bonds with adjacent positively charged arginine residues (1,(3)(4)(5)(6). The network of hydrogen bonds and salt bridges that form can then communicate phosphorylation to distant areas of the protein (6). In the case of yeast glycogen phosphorylase, phosphorylation occurs at a threonine residue located near the N terminus, a region that overlies the active site of the enzyme (3). The enzyme, which normally exists as a homodimer, contains two active catalytic sites, one in each monomer (3). Phosphorylation at this site causes the N-terminal region to fold into a compact structure that wedges between the dimer interface. This structural change helps to reorient the active site in a manner that facilitates enzymatic activation (3). Examples such as this suggest that large scale movements of flexible regions within a protein are an impor...

14-3-3ζ C-terminal Stretch Changes Its Conformation upon Ligand Binding and Phosphorylation at Thr232

“…Phosphorylation modulates protein function for example by affecting protein-protein interaction or by inducing conformational changes within the tertiary structure of the protein. One common mechanism of protein regulation is based on the phosphorylation of a site located within a flexible, solvent-exposed region of the protein (45)(46)(47). In such a case Ser/Thr phosphorylation can cause steric obstruction of the active site or induce major conformational change in the protein structure.…”

A proposed signaling motif for nuclear import in mRNA processing via the formation of arginine claw

“…The SR protein retains its splicing abilities even without the RS domain in vitro. Mutational studies have shown that this short motif (RS) 8 of the RS domain is phosphorylated in the cytoplasm, and the phosphorylated motif forms the nuclear localization signal (NLS) for the nuclear importation of this protein (18). The nuclear transport receptor transportin-SR2 (TRN-SR2), which is an importin-␤ family protein, binds the SR protein at the phosphorylated region of the RS domain and shuttles the SR protein into the nucleus through the nuclear pore complex (19)(20)(21).…”

“…The answers to these questions might lie in the uniqueness of the RS dipeptide repeats and the conformation of this motif after phosphorylation. We have studied the structure of the eight RS dipeptide repeats (RS) 8 of the RS domain at atomistic detail before and after phosphorylation by using molecular dynamics simulation studies and free-energy calculations. We observed very dramatic differences between the unphosphorylated (RS) 8 and the phosphorylated form, henceforth referred to as (RpS) 8 .…”

Phosphorylation-Elicited Quaternary Changes of GA Binding Protein in Transcriptional Activation

“…Besides increasing the negative charge of the protein, it can be hypothesized that phosphorylation-specific alterations of the quaternary structure of the GABP complex might prime it for further protein-protein interactions. One way of monitoring such structural changes of GABP in living cells is to measure the changes in energy transfer between the subunits upon phosphorylation, the so-called FRET signal (53). This involves the .…”

“…However, the consequences of GABP phosphorylation in transcriptional initiation have remained speculative, and the mechanism whereby phosphorylation would mediate transcriptional activation of GABP has remained unresolved. In fact, the mechanism whereby phosphorylation results in protein activation has been elucidated for only a few proteins (30,53), but it is likely that phosphorylation mediates protein activation through conformational allosteric changes within the tertiary and quaternary structures of the protein.…”