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The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory
J C Williams1, A Weijland, S Gonfloni
1European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany.
Abstract:
The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.
Insights
The crystal structure of inactive chicken Src protein tyrosine kinase reveals how its domains interact to maintain low activity. These interactions, involving the SH3, SH2, and kinase domains, restrict access to the active conformation, offering insights into kinase regulation.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Signaling
Background:
- Src protein tyrosine kinase is crucial for cell signaling.
- Its activity must be tightly regulated for proper cellular function.
- Understanding Src regulation is key to deciphering various signaling pathways.
Purpose of the Study:
- To determine the crystal structure of chicken Src in its least active, Tyr527-phosphorylated state.
- To elucidate the domain interactions that maintain Src in an inactive conformation.
- To provide insights into the regulatory mechanisms of Src-related kinases.
Main Methods:
- X-ray crystallography to obtain the 3D structure of chicken Src.
- Structural analysis and comparison with other protein kinases (human Hck, Src, Cdk2).
- Sequence alignments and conservation analysis of key structural motifs.
Main Results:
- The crystal structure reveals interactions between the SH3, SH2, and kinase domains, stabilizing an inactive state.
- The SH3 domain coordinates the SH2-kinase linker, positioning the SH2 domain to bind the phosphorylated Tyr527 tail.
- The alphaC-helix in the N-terminal lobe is displaced, disrupting the active site, a conformation influenced by inter-lobe interactions and the conserved alphaC-beta4 loop.
Conclusions:
- The observed quaternary structure of inactive chicken Src explains its low activity through restricted domain mobility.
- Interactions involving the SH3, SH2-kinase linker, and N-terminal lobe are critical for maintaining the inactive state.
- Conserved structural features suggest similar regulatory mechanisms across Src-related kinases like Abl, Btk, and Csk.