Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src

Abstract

Site‐directed mutagenesis is a traditional approach for structure–function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96‐well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a protein tyrosine phosphatase (PTP)‐expressing host and a non‐PTP‐expressing host. Because high activity Src mutants are toxic to the host, the PTP system allowed the identification of Src mutants with high activity, while the non‐PTP system identified Src mutants with low activity. This approach was applied to Src mutant libraries randomized in the highly conserved HRD motif in the catalytic loop, and revealed that structurally diverse residues can replace the His and Arg residues, while the Asp residue is irreplaceable for catalytic activity.

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

“…In line with previous reports (Albanese et al, 2018;Cui & Sun, 2018;Seeliger et al, 2005), phosphatase co-expression improved Src KD yield from 0.4 mg/L to 6.2 mg/L after affinity purification (Table 1A). By contrast, expression of Lyn KD alone performed two-fold better (6.8 mg/L) than its co-expression with PTP1B (3.7 mg/L).…”

“…PTP1B had earlier been identified as the primary human phosphatase targeting and activating c-Src (Bjorge, Pang, & Fujita, 2000) and other PTKs (Fan, Lin, Lucito, & Tonks, 2013) and was used to improve yield and homogeneity of recombinant c-Src, c-Abl and c-Met KDs (W. . Phosphatase co-expression was then reported in subsequent studies using either PTP1B (Cui & Sun, 2018;Tu, Wang, Cai, Zhou, & Zhang, 2014;Y.-H. Wang, Ayrapetov, Lin, & Sun, 2006) or YopH (Albanese et al, 2018;Wilson et al, 2015;Filippakopoulos et al, 2008). With one dissenting opinion (W. , there seems to be a consensus that the active Src KD is cytotoxic to E. coli and that its recombinant expression benefits from phosphatase co-expression.…”

Engineering synthetic phosphorylation signaling networks in human cells

“…To test whether we could tune synSub phosphorylation levels by altering component biophysical properties, we constructed sets of synKin variants: binding affinity to synSub was tuned by introducing LZ sequence variants 45 , catalytic activity was adjusted by making active site mutations of the kinase domains 47 , and synKin expression level was tuned by inserting Kozak sequence variants into the expression construct, leading to differential rates of protein translation 48 . When we tested each set of synKin parts, we observed modulation of synSub phosphorylation across 10-20 fold range ( Fig.…”

“…We constructed several sets of synKin variants: LZ sequence variants were introduced to tune binding affinity to synSub ( 45 ), catalytic turnover rate was adjusted via previously reported pY kinase domain active site mutations ( fig. S4 ) ( 47 ), and expression level was tuned by introducing Kozak sequence variants to synKin expression constructs ( fig. S1 ), resulting in differential rates of protein translation ( 48 ).…”

Crystal Structures Reveal Hidden Domain Mechanics in Protein Kinase A (PKA)

“…The loop contains a GxGxxG consensus motif where the second and third glycine positions flank a regulatory phosphorylation site in some kinases (S35 in PKA) [28,29]. The active site contains a HRD motif (residues Y164, R165, D166 in PKA) that harbors the catalytic D166 required for phosphotransfer [30,31]. While the HRD motif is named based on the sequence similarity of the broader kinase family, the AGC kinases, including PKA, in fact have a Y-substitution at the first position.…”