Crystal Structure of a Polyhistidine-Tagged Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase Complexed with the Peptide Inhibitor PKI(5-24) and Adenosine
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- 作者：Narendra Narayana ; Sarah Cox ; Shmuel Shaltiel ; Susan S. Taylor ; and Nguyen-huu Xuong
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：April 15, 1997
- 年：1997
- 卷：36
- 期：15
- 页码：4438 - 4448
- 全文大小：289K
- 年卷期：v.36,no.15(April 15, 1997)
- ISSN：1520-4995
文摘
The crystal structure of the hexahistidine-tagged mouserecombinant catalytic subunit (H6-rC)of cAMP-dependent protein kinase (cAPK), complexed with a 20-residuepeptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, wasdetermined at 2.2 Å resolution. Novelcrystallization conditions were required to grow the ternary complexcrystals. The structure was refinedto a final crystallographic R-factor of 18.2% with goodstereochemical parameters. The "active" enzymeadopts a "closed" conformation as found in rC:PKI(5-24)[Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptideproviding a major contact surface. Thisstructure clearly defines the subsites of the unique nucleotide bindingsite found in the protein kinasefamily. The adenosine occupies a mostly hydrophobic pocket at thebase of the cleft between the twolobes and is completely buried. The missing triphosphate moiety ofATP is filled with a water molecule(Wtr 415) which replaces the 
-phosphate of ATP. Theglycine-rich loop between 
1 and 2 helps toanchor the phosphates while the ribose ring is buried beneath-strand 2. Another ordered water molecule(Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49in -strand 1, Glu 127 in thelinker strand between the two lobes, Tyr 330, and a third watermolecule, Wtr 359. The conservednucleotide fold can be defined as a lid comprised of -strand 1, theglycine-rich loop, and -strand 2.The adenine ring is buried beneath -strand 1 and the linkerstrand (120-127) that joins the small andlarge lobes. The C-terminal tail containing Tyr 330, a segmentthat lies outside the conserved core,covers this fold and anchors it in a closed conformation. Themain-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a meanB-factor of 41.4 Å2. This loopisquite mobile, in striking contrast to the other conserved loops thatconverge at the active site cleft. Thecatalytic loop (residues 166-171) and the Mg2+positioning loop (residues 184-186) are a stable part ofthe large lobe and have low B-factors in all structuressolved to date. The stability of the glycine-richloop is highly dependent on the ligands that occupy the active sitecleft with maximum stability achievedin the ternary complex containing Mg·ATP and the peptideinhibitor. In this ternary complex the-phosphate is secured between both lobes by hydrogen bonds to thebackbone amide of Ser 53 in theglycine-rich loop and the amino group of Lys 168 in the catalytic loop.In the adenosine ternary complexthe water molecule replacing the -phosphate hydrogen bonds betweenLys 168 and Asp 166 and makesno contact with the small lobe. This glycine-rich loop is thus themost mobile component of the activesite cleft, with the tip of the loop being highly sensitive to whatoccupies the -subsite.
-phosphate of ATP. Theglycine-rich loop between 1 and 2 helps toanchor the phosphates while the ribose ring is buried beneath-strand 2. Another ordered water molecule(Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49in -strand 1, Glu 127 in thelinker strand between the two lobes, Tyr 330, and a third watermolecule, Wtr 359. The conservednucleotide fold can be defined as a lid comprised of -strand 1, theglycine-rich loop, and -strand 2.The adenine ring is buried beneath -strand 1 and the linkerstrand (120-127) that joins the small andlarge lobes. The C-terminal tail containing Tyr 330, a segmentthat lies outside the conserved core,covers this fold and anchors it in a closed conformation. Themain-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a meanB-factor of 41.4 Å2. This loopisquite mobile, in striking contrast to the other conserved loops thatconverge at the active site cleft. Thecatalytic loop (residues 166-171) and the Mg2+positioning loop (residues 184-186) are a stable part ofthe large lobe and have low B-factors in all structuressolved to date. The stability of the glycine-richloop is highly dependent on the ligands that occupy the active sitecleft with maximum stability achievedin the ternary complex containing Mg·ATP and the peptideinhibitor. In this ternary complex the-phosphate is secured between both lobes by hydrogen bonds to thebackbone amide of Ser 53 in theglycine-rich loop and the amino group of Lys 168 in the catalytic loop.In the adenosine ternary complexthe water molecule replacing the -phosphate hydrogen bonds betweenLys 168 and Asp 166 and makesno contact with the small lobe. This glycine-rich loop is thus themost mobile component of the activesite cleft, with the tip of the loop being highly sensitive to whatoccupies the -subsite.