Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase.
The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.
Cànaves
J M
JM
Department of Chemistry and Biochemistry, 0654, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0654, USA.
Leon
D A
DA
Taylor
S S
SS
D001665:Binding Sites; D002942:Circular Dichroism; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D014508:Urea
D021122:Protein Subunits; D014508:Urea; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases