DOI: 10.1002/pro.5560030203. cAMP‐dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer

cAMP‐dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer

10.1002/pro.5560030203

Crossref journal-article

Wiley

Protein Science (311)

Abstract

AbstractThe crystal structure of ternary and binary substrate complexes of the catalytic subunit of CAMP‐dependent protein kinase has been refined at 2.2 and 2.25 Å resolution, respectively. The ternary complex contains ADP and a 20‐residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R‐factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P‐site is 2.7 Å from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine ‐OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 Å from the γ‐phosphate of ATP and supports a direct in‐line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the 6N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the γ‐phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154‐2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 Å following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine‐rich loop. The high B‐factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.

Bibliography

Madhusudan, Trafny, E. A., Xuong, N., Adams, J. A., Eyck, L. F. T., Taylor, S. S., & Sowadski, J. M. (1994). cAMPâdependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer. Protein Science, 3(2), 176â187. Portico.

Authors 7

Madhusudan (first)
Elzbieta A. Trafny (additional)
Nguyen‐Huu Xuong (additional)
Joseph A. Adams (additional)
Lynn F.Ten Eyck (additional)
Susan S. Taylor (additional)
Janusz M. Sowadski (additional)

References 39 Referenced 244

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10.1042/bj2310655 / Biochem J / An active twenty‐amino‐acid‐residue peptide derived from the inhibitor protein of the cyclic AMP‐dependent protein kinase by Cheng HC (1986)
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Dates

Type	When
Created	15 years, 1 month ago (July 12, 2010, 4:27 a.m.)
Deposited	1 year, 10 months ago (Oct. 24, 2023, 9:17 a.m.)
Indexed	2 months, 2 weeks ago (June 18, 2025, 12:08 a.m.)
Issued	31 years, 7 months ago (Feb. 1, 1994)
Published	31 years, 7 months ago (Feb. 1, 1994)
Published Online	16 years, 8 months ago (Dec. 31, 2008)
Published Print	31 years, 7 months ago (Feb. 1, 1994)

Funders 4

Lucille P. Markey Charitable Trust 10.13039/100011517
Region: Americas
pri (Trusts, charities, foundations (both public and private))
Labels1
1. Markey Trust
PHS
Awards2
1. GM 19301
2. GM 37674
NSF
Awards1
1. DIR 88-22385
PHS research fellowship
Awards1
1. GM 14528-02

BibTeX

@article{Madhusudan_1994, title={cAMP‐dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer}, volume={3}, ISSN={1469-896X}, url={http://dx.doi.org/10.1002/pro.5560030203}, DOI={10.1002/pro.5560030203}, number={2}, journal={Protein Science}, publisher={Wiley}, author={Madhusudan and Trafny, Elzbieta A. and Xuong, Nguyen‐Huu and Adams, Joseph A. and Eyck, Lynn F.Ten and Taylor, Susan S. and Sowadski, Janusz M.}, year={1994}, month=feb, pages={176–187} }

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