Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase

JOURNAL OF MOLECULAR BIOLOGY

卷:422

Article

Bastidas, Adam C.¹  Deal, Michael S.²  Steichen, Jon M.²  Keshwani, Malik M.¹  Guo, Yurong^1,2  Taylor, Susan S.^1,2,3 

[1] Univ Calif San Diego, Dept Pharmacol, San Diego, CA 92093 USA

[2] Univ Calif San Diego, Dept Chem & Biochem, San Diego, CA 92093 USA

[3] Univ Calif San Diego, Howard Hughes Med Inst, San Diego, CA 92093 USA

关键词: crystal structure;    protein kinase A;    N-myristylation (N-myristoylation);    multiple conformations;    stabilize;   

DOI  :  10.1016/j.jmb.2012.05.021

来源: Elsevier

【 摘 要 】

The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PICA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5'-(beta,gamma-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 angstrom, and K7C ternary complex, refined to 1.35 angstrom. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 angstrom, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k(cat). The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit. (C) 2012 Elsevier Ltd. All rights reserved.

【 授权许可】

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