Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: The SH3 domain of c-Src interacts with residues 50–79 in ChoKα, and requires prolines 61 and 62. ( A ) 1:1 mixture of purified ChoKα truncation constructs and of His-SUMO-c-Src (84–137) at 25 µM in 100 µl was washed three times with size exclusion purification buffer containing 30 mM imidazole. Both the active (WT) and kinase-dead mutants (D306A) were analyzed, demonstrating the enzyme activity has no impact on the protein binding behavior. M = protein mixture, B = bead-bound pull-down. Red boxes indicate where ChoKα would be expected. ( B ) Analysis of the proline-rich region between residues 50 and 79 uncovered several putative PxxP motifs that could be responsible for this interaction. Dual proline mutants were made in the background of ChoKαΔ49 to interrupt the predicted polyproline helix and experiments with co-precipitation experiments were repeated as described, showing a ~70% decrease in binding when prolines 61 & 62 were mutated to alanine (red box). Molecular weights for the constructs as follows: ChoKαFL = 52.2 kDa; ChoKαΔ49 = 47.7 kDa; ChoKαΔ79 = 44.4 kDa; His-SUMO-c-Src (84–137) = 19.6 kDa.

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Purification, Construct, Activity Assay, Protein Binding, Binding Assay

Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: Interaction with the c-Src SH3 domain does not affect ChoKα activity. ( A ) The Choline Kinase rate (left) and the ATPase rate (right) of the constructs are unaffected by the presence of the SH3 domain of c-Src. ( B ) Kinase dead mutants show no activity compared to similar activity rates for the Δ49 and Δ79 wild-type enzymes. ( C ) The SH3 domain of c-Src has effectively no kinase or ATPase activity measured via this assay compared to full-length wild type choline kinase.

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Activity Assay, Construct

Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: Selective interaction of ChoKα with the c-Src SH3 domain. ( A ) Alignment of the c-Src, Hck and Fgr amino acid sequences. Hck-SH3 has 51% sequence identity to c-Src-SH3; while the Fgr-SH3 has 73% identity. All three SH3 domains are known to interact with PxxP motifs and to have similar secondary structures. ( B ) SPR analysis of wild-type ChoKα interaction with the c-Src, Hck and Fgr SH3 domains. Recombinant SH3 domains were immobilized on a Biacore CM5 carboxymethyl dextran biosensor chip, and purified ChoKα was injected in triplicate over the range of concentrations shown (one trace shown at each concentration for clarity). Once equilibrium was reached, interactions were followed by a 2 min dissociation phase. A plot of the extent of interaction at each SH3 domain concentration was then fitted directly to determine the steady-state K D value for Src and Hck.

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Sequencing, Recombinant, Purification, Injection, Concentration Assay

Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: ChoKα Δ49 binding to c-Src-SH3 requires an intact polyproline sequence preceding PL repeats motif but not ChoK kinase activity. The recombinant wild-type and mutant ChoKα Δ49 proteins indicated at the top were immobilized on a Biacore CM5 carboxymethyl dextran chip, and the Src SH3 protein was injected in triplicate over the range of concentrations shown. Interaction was recorded for 2 min, followed by a 2 min dissociation phase. The resulting sensorgrams were best-fit by a two-state induced-fit model, and the resulting K D values against ChoKα Δ49-WT and Δ49–306A are 2.4 × 10 −6 M and 1.8 × 10 −6 M, respectively. Insufficient interaction was observed with the Δ49-P61/62 A mutant to allow curve fitting.

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Binding Assay, Sequencing, Activity Assay, Recombinant, Mutagenesis, Injection

Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: Crystal structure of c-Src-SH3 fused to the ChoKα (60–69) proline-rich peptide. ( A ) The crystal structure reveals a non-biological assembly unit with the peptide of domain A binding to domain B and vice-versa. ( B ) Residues 60–69 of ChoKα bind across the SH3 domain surface as a polyproline type II helix as observed in previous SH3:peptide structures (Ref ). ( C ) Key interactions between ChoKα (60–69) and c-Src-SH3 show why prolines 61 and 62 are crucial for the interaction, as described in Fig. . ( D ) Crystal contacts strengthen the interaction of the peptide and the SH3 domain. Though ChoKα (60–69) does not contain an arginine, the interaction is stabilized by Arg110 from protomer A of an adjacent unit cell.

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Molecular basis for the interaction between human choline kinase alpha and the SH3 domain of the c-Src tyrosine kinase

doi: 10.1038/s41598-019-53447-0

Figure Lengend Snippet: Comparison of c-Src SH3 in complex with optimised peptide VSL12 and ChoKα (60–69) or the linker region of c-Src. ( A ) The structure of c-Src SH3 in complex with the optimised peptide VSL12, as determined from solution NMR (PDB ID 1QWF). Note the interaction between Asp99 of the SH3 domain and Arg6’ of the peptide. ( B ) The c-Src SH3 domain in complex with ChoKα (60–69), reported here, vs. the VSL12 peptide (pink, left) or the c-Src linker (orange, right, from PDB ID 2SRC).

Article Snippet: Recombinant, purified Src-family kinase SH3 domain proteins were immobilized on a single carboxymethyl dextran CM5 biosensor chip (Biacore) with phosphate-buffered saline, pH 7.5, as running buffer at a flow rate of 30 μL/min.

Techniques: Comparison