De Novo Design of a Molecular Switch: Phosphorylation-dependent Association of Designed Peptides

Signarvic, Rachel S.; DeGrado, William F.

doi:10.1016/j.jmb.2003.09.041

Cited by 57 publications

(70 citation statements)

References 27 publications

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“…In addition, an energy-minimized model revealed that Ser 185 phosphorylation of human PGI may not affect the topography of the active-site residues, suggesting that phosphorylation may interfere with isomerization, not with binding of the sugar substrates. This notion is strengthened by the facts that phosphorylation alters its homodimerization ability, similar to the serine phosphorylation of the tau protein (43), and that phosphorylation increases the stability of the individual protein helices that should relieve an unfavorable electrostatic interaction in the tetramer (45).…”

Section: Phosphorylation Of Pgi/amf Regulates Its Enzymatic Activitymentioning

confidence: 99%

Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation

Yanagawa

Funasaka

Tsutsumi

et al. 2005

Journal of Biological Chemistry

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Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser 185 by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by sitedirected mutagenesis, we substituted Ser 185 with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.

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Section: Phosphorylation Of Pgi/amf Regulates Its Enzymatic Activitymentioning

confidence: 99%

Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation

Yanagawa

Funasaka

Tsutsumi

et al. 2005

Journal of Biological Chemistry

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“…In another native protein model, it has also been shown that phosphorylation of serine and threonine can have a destabilizing affect within α-helix system, particularly with phosphothreonine 7,8. The DeGrado laboratory has used phosphorylation as a molecular switch to promote the self assembly of de novo designed helical bundles 9. Work from the Zondlo laboratory has elucidated that the proline rich regions of the naturally disordered Tau peptide when phosphorylated adopts a polyproline II helix structure 10.…”

Section: Introductionmentioning

confidence: 99%

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

Riemen

Waters

2009

J. Am. Chem. Soc.

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Phosphorylated amino acids were incorporated into a designed β-hairpin peptide to study the effect on β-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue on the neighboring strand. The three commonly phosphorylated residues in biological systems, serine, threonine, and tyrosine, were studied in same β-hairpin system. It was found that phosporylation destabilizes the hairpin structure by approximately 1.0 kcal/mol regardless of the type of phosphorylated residue. In contrast, destabilization due to glutamic acid was about 0.3 kcal/mol. Double mutant cycles and pH studies are consistent with a repulsive interaction as the source of destabilization. These findings demonstrate a novel mechanism by which phosphorylation may influence protein structure and function.

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“…In a model peptide and in a designed four-helix bundle, De Grado found that an Arg-X-pSer sequence was highly stabilizing at the N-terminus of the α-helix, with phosphorylation at all four sites of the tetramer resulting in a significant stabilization of the helical bundle relative to the nonphosphorylated bundle. 15 In contrast, Vinson found substantial destabilization of a coiled coil upon phosphorylation at an interior helical position, with greater destabilization due to phosphothreonine (pThr) than phosphoserine. 12 Interestingly, when the internal serine was placed within the structural context of multiple arginine residues, serine phosphorylation was found to be stabilizing to the coiled coil, although notably phosphothreonine here was still destabilizing.…”

mentioning

confidence: 99%

OGlcNAcylation and Phosphorylation Have Similar Structural Effects in α-Helices: Post-Translational Modifications as Inducible Start and Stop Signals in α-Helices, with Greater Structural Effects on Threonine Modification

2014

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OGlcNAcylation and phosphorylation are the major competing intracellular post-translational modifications of serine and threonine residues. The structural effects of both post-translational modifications on serine and threonine were examined within Baldwin model α-helical peptides (Ac-AKAAAAKAAAAKAAGY-NH2 or Ac-YGAKAAAAKAAAAKAA-NH2). At the N-terminus of an α-helix, both phosphorylation and OGlcNAcylation stabilized the α-helix relative to the free hydroxyls, with a larger induced structure for phosphorylation than for OGlcNAcylation, for the dianionic phosphate than for the monoanionic phosphate, and for modifications on threonine than for modifications on serine. Both phosphoserine and phosphothreonine resulted in peptides more α-helical than alanine at the N-terminus, with dianionic phosphothreonine the most α-helix-stabilizing residue here. In contrast, in the interior of the α-helix, both post-translational modifications were destabilizing with respect to the α-helix, with the greatest destabilization seen for threonine OGlcNAcylation at residue 5 and threonine phosphorylation at residue 10, with peptides containing either post-translational modification existing as random coils. At the C-terminus, both OGlcNAcylation and phosphorylation were destabilizing with respect to the α-helix, though the induced structural changes were less than in the interior of the α-helix. In general, the structural effects of modifications on threonine were greater than the effects on serine, because of both the lower α-helical propensity of Thr and the more defined induced structures upon modification of threonine than serine, suggesting threonine residues are particularly important loci for structural effects of post-translational modifications. The effects of serine and threonine post-translational modifications are analogous to the effects of proline on α-helices, with the effects of phosphothreonine being greater than those of proline throughout the α-helix. These results provide a basis for understanding the context-dependent structural effects of these competing protein post-translational modifications.

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De Novo Design of a Molecular Switch: Phosphorylation-dependent Association of Designed Peptides

Cited by 57 publications

References 27 publications

Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation

Differential Regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Activities by Protein Kinase CK2 Phosphorylation

Controlling Peptide Folding with Repulsive Interactions between Phosphorylated Amino Acids and Tryptophan

OGlcNAcylation and Phosphorylation Have Similar Structural Effects in α-Helices: Post-Translational Modifications as Inducible Start and Stop Signals in α-Helices, with Greater Structural Effects on Threonine Modification

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