Here, we present a protocol for the in vitro phosphorylation of Src kinase domain (SrcKD), preparation of phospho-SrcKD in complex with the D1 domain of rPTP epsilon (rPTPεD1), and binding assays using biolayer interferometry (BLI). We describe steps for the in vitro phosphorylation of SrcKD and preparation of the phospho-SrcKD: rPTPεD1 complex for small-angle X-ray scattering (SAXS) experiments. We then detail instructions for the BLI binding assay to determine the binding affinity between phospho-SrcKD and rPTPεD1. For complete details on the use and execution of this protocol, please refer to EswarKumar et al..
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| http://dx.doi.org/10.1016/j.xpro.2024.103046 | DOI Listing |
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Protocol for phospho-SrcKD: rPTPεD1 complex preparation and BLI binding assays to demonstrate their exosite interface.
STAR Protoc
September 2024
Institute of Biological Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan. Electronic address:
Here, we present a protocol for the in vitro phosphorylation of Src kinase domain (SrcKD), preparation of phospho-SrcKD in complex with the D1 domain of rPTP epsilon (rPTPεD1), and binding assays using biolayer interferometry (BLI). We describe steps for the in vitro phosphorylation of SrcKD and preparation of the phospho-SrcKD: rPTPεD1 complex for small-angle X-ray scattering (SAXS) experiments. We then detail instructions for the BLI binding assay to determine the binding affinity between phospho-SrcKD and rPTPεD1.
View Article and Find Full Text PDFAn integrative approach unveils a distal encounter site for rPTPε and phospho-Src complex formation.
Structure
December 2023
Institute of Biological Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan; Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan. Electronic address:
The structure determination of protein tyrosine phosphatase (PTP): phospho-protein complexes, which is essential to understand how specificity is achieved at the amino acid level, remains a significant challenge for protein crystallography and cryoEM due to the transient nature of binding interactions. Using rPTPεD1 and phospho-SrcKD as a model system, we have established an integrative workflow to address this problem, by means of which we generate a protein:phospho-protein complex model using predetermined protein structures, SAXS and pTyr-tailored MD simulations. Our model reveals transient protein-protein interactions between rPTPεD1 and phospho-SrcKD and is supported by three independent experimental validations.
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