Evaluation of the interaction between phosphohistidine analogues and phosphotyrosine binding domains - PubMed (original) (raw)

Evaluation of the interaction between phosphohistidine analogues and phosphotyrosine binding domains

Tom E McAllister et al. Chembiochem. 2014.

Abstract

We have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding SH2 domain from the eukaryotic cell signalling protein Grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. These investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo.

Keywords: cell signaling; phosphohistidine; phosphotyrosine; protein modifications; synthetic analogues.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Scheme 1

Scheme 1

Structures of τ-phosphohistidine (1) and the corresponding triazole-phosphonate mimic, 2, which can be incorporated into peptides by using precursors 3 a and 3 b.

Scheme 2

Scheme 2

Structures of phosphotyrosine 4 and analogues 5 and 6, which have previously been incorporated into recombinant proteins.

Scheme 3

Scheme 3

Proposed route to generate a protein containing the putative phosphotyrosine analogue 10.

Scheme 4

Scheme 4

Synthesis of Fmoc-/benzyl-protected homotriazole 14 from Fmoc-azidohomoalanine 12 and alkyne 13, as well as peptides 11, 15 and 16, which were used in binding experiments. For full experimental procedures see the Supporting information.

Figure 1

Figure 1

Isothermal titration calorimetry of peptides A) 11, B) 15 and C) 16 into the Grb2 SH2 domain reveals equilibrium binding constants of (385±41) n

m

for 11 and (719±28) μ

m

for 16. D) A competitive fluorescence polarisation assay indicates an IC50 of (442±80) n

m

for pTyr peptide 11 (▪) and (363±15) μ

m

for pTz peptide 16 (□). See the Supporting Information for control dilution experiments and full fitting of polarisation data.

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