Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.
Summary (2 min read)
Introduction
- The SH2 and SH3 domains bind specific phosphotyrosine and proline-rich motifs, respectively.
- The catalytic domain consists of two lobes, which form a cleft that serves as a docking site for ATP and other substrates representing the active site of the kinase.
- The crystal structures of the inactive („closed‟) forms of c-Src [6-8] and Hck [9, 10] reveal that intramolecular interactions involving the SH2 and SH3 domains are essential for suppression of kinase activity despite being far away from the active site.
- In Hck and most other SFKs the connecting linker stabilizes the SH3-SH2 domain orientation by a salt-bridge between a conserved glutamate of the linker (E147 in Hck) and a lysine (K104 in Hck) of the SH3 domain.
Preparation of starting structures
- The crystal structures of inactive Hck complexed with the nucleoside analog PP1 (Protein Data Bank ID 1QCF, [10]), was used to generate the starting structures.
- The force field parameters for PP1, which are not available in the AMBER package, were obtained as follows: ArgusLab [21] was utilized to add missing hydrogen atoms.
- Then, the structure was subjected to two consecutive geometry optimizations with Gaussian03 [22] using the ab initio methods HF/MIDI!, and HF/6-31G(d).
Molecular dynamics (MD) simulations
- The parameters for the phosphotyrosine present in the C-terminal kinase tail were assigned according to Homeyer et al. [30].
- All structures were minimized in a three-step procedure by using the SANDER module of AMBER following a previously established protocol [32].
- Subsequently, 30-ns MD simulations with standard NPT conditions were performed for data collection.
- All bonds involving hydrogen atoms were constrained using the SHAKE procedure [35].
Results and discussion
- Influence of the SH3-SH2 linker sequence on the global structure and dynamics of PP1inhibited Hck To determine the effect of the SH3-SH2 linker on the global structure of Src kinases, 30-ns molecular dynamics simulations of wildtype Hck and of Hck with a mutated SH3-SH2 linker (Hck*) were carried out.
- This rigidity is also consistent with the structural overlay showing that the geometry of the kinase domain is almost unaffected by the SH3-SH2 linker sequence (Fig. 4).
- All starting structures of the present simulations correspond to the “DFG-in” conformation, which is also retained in the simulations of PP1-bound Hck/Hck* as well as in the simulation of uninhibited wildtype Hck.
- Since their results indicate that mutations of the SH3-SH2 linker affect the active site geometry of Hck, the authors have checked the literature, whether remote effects of mutations on the active site conformation have already been reported for other SFKs in the past.
- This is reflected in the low affinity of c-Src for the drug imatinib, which exclusively binds the DFGout conformation.
Acknowledgments
- The authors thank Pia Rücker and Dr. Holger Dinkel for fruitful discussions, and A. Jens Meiselbach-Wilke for critically reading the manuscript.
- This work was supported by grants from the Deutsche Forschungsgemeinschaft.
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Citations
38 citations
Cites background from "Effect of the SH3-SH2 domain linker..."
...Molecular dynamics studies also support an important regulatory role for the Hck SH3-SH2 connector showing that connector modification influences the conformation of the kinase domain activation loop (58, 59)....
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References
2,455 citations
"Effect of the SH3-SH2 domain linker..." refers background in this paper
...The Src family of non-receptor tyrosine kinases to date comprises nine members (Src, Fyn, Lck, Hck, Yes, Fgr, Yrk, Blk, and Lyn) which play critical roles in eukaryotic signal pathways that control a diverse array of processes such as cell growth, differentiation, activation and transformation [1-3]....
[...]
1,403 citations
1,213 citations
"Effect of the SH3-SH2 domain linker..." refers methods in this paper
...The atomic charges for PP1 were then obtained following the established procedure [23, 24] by fitting the charges to the HF/6-31G(d) computed electrostatic potential using the Antechamber tool from the AMBER program suite....
[...]
1,198 citations
"Effect of the SH3-SH2 domain linker..." refers background in this paper
...The Src family of non-receptor tyrosine kinases to date comprises nine members (Src, Fyn, Lck, Hck, Yes, Fgr, Yrk, Blk, and Lyn) which play critical roles in eukaryotic signal pathways that control a diverse array of processes such as cell growth, differentiation, activation and transformation [1-3]....
[...]
1,179 citations
"Effect of the SH3-SH2 domain linker..." refers background in this paper
...Activation of Src-family kinases (SFKs) requires the disruption of the intramolecular interactions, the dephosphorylation of the C-terminal tail by phosphatases, or the binding of high-affinity ligands to the SH2 or the SH3 domain [9-12]....
[...]
...The crystal structures of the inactive („closed‟) forms of c-Src [6-8] and Hck [9, 10] reveal that intramolecular interactions involving the SH2 and SH3 domains are essential for suppression of kinase activity despite being far away from the active site....
[...]