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 prote in kinase A was cloned. The deduced amino acid sequence was highly homologo us 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, A mino acids reported to be important for interaction with cAMP, for cooperat ivity between the two cAMP binding domains, in the general folding of the d omain, 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 RI I of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit typ e I-alpha. The quality of the model is good. The two backbones could be per fectly overlapped, except for two loop regions of high divergence. The alph a helix C of domain A, proposed to have a strong interaction with the catal ytic subunit, contains a leucine replacing a basic residue (arginine or lys ine) commonly found in RI or RII, The domains A and B of the M. rouxii regu latory subunit were overexpressed as fusion proteins with GST, GST domain B protein was inactive. GST domain A was active; the kinetic parameters of a ffinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole reg ulatory subunit. (C) 2000 Academic Press.