Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A.
The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type Ialpha. The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The alpha helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit.
Sorol
M R
MR
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, Buenos Aires, Argentina.
Pastori
R L
RL
Muro
A
A
Moreno
S
S
Rossi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases