Nucleotide-Induced Conformational Changes in the Saccharomyces cerevisiae SR Protein Kinase, Sky1p, Revealed by X-ray Crystallography
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- 作者：Brad Nolen ; Jacky Ngo ; Sutapa Chakrabarti ; Don Vu ; Joseph A. Adams ; and Gourisankar Ghosh
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：August 19, 2003
- 年：2003
- 卷：42
- 期：32
- 页码：9575 - 9585
- 全文大小：569K
- 年卷期：v.42,no.32(August 19, 2003)
- ISSN：1520-4995
文摘
Conformational changes are thought to play a key role in the function of active protein kinases,although little is known about how these changes relate to the mechanism of phosphorylation. Here wepresent four high-resolution structures of a single crystal form of Sky1p, a constitutively active serinekinase implicated in yeast RNA processing, each in a different state of nucleotide binding. By comparingthe apoenzyme structure to the ADP- and ATP-bound Sky1p structures, we have revealed conformationalchanges caused by ATP binding or conversion from nucleotide reactant to product. Rotation of the smalllobe of the kinase closes the cleft upon binding, allowing the nucleotide to interact with residues fromboth lobes of the kinase, although some interactions thought to be important for phosphotransfer aremissing in the ATP-containing structure. In the apoenzyme, a kinase-conserved phosphate-anchoring loopis in a twisted conformation that is incompatible with ADP and ATP binding, providing a potentialmechanism for facilitating ADP release in Sky1p. The nonhydrolyzable ATP analogue AMP-PNP bindsin a unique mode that fails to induce lobe closure. This observation, along with comparisons between thetwo independent molecules in the asymmetric unit of each structure, has provided new molecular detailsabout how the nucleotide binds and induces closure. Finally, we have used mutational analysis to establishthe importance of a glycine within the linker that connects the two lobes of Sky1p.