The kinase homology domain of receptor guanylyl cyclase C: ATP binding and identification of an adenine nucleotide sensitive site.
The role of the kinase homology domain (KHD) in receptor guanylyl cyclases is to regulate the activity of the catalytic guanylyl cyclase domain. The KHD lacks many of the amino acids required for phosphotransfer activity and, therefore, is not expected to possess kinase activity. Guanylyl cyclase activity of the receptor guanylyl cyclase C (GC-C) is modulated by ATP, and computational modeling showed that the KHD can adopt a structure similar to protein kinases, suggesting that the KHD is the site for ATP interaction. A monoclonal antibody, GCC:4D7, raised to the KHD of GC-C, fails to react with GC-C in the presence of ATP and ATP analogues that regulate GC-C catalytic activity, indicating that a conformational change occurs in the KHD on ATP binding. Mapping of the epitope of the antibody through the use of recombinant protein constructs and phage display showed that the epitope for GC-C:4D7 lies immediately C-terminal to a critical lysine residue (Lys516 in GC-C), required for ATP interaction in protein kinases. By employing a novel approach utilizing ATP-agarose affinity chromatography, we demonstrate that the intracellular domain of GC-C and the KHD bind ATP. Mutation of Lys516 to Ala abolishes ATP binding. Thus, this report is the first to show direct ATP binding to the pseudokinase domain of receptor guanylyl cyclase C, as well as to identify dramatic conformational changes that occur in this domain on ATP binding, akin to those seen in catalytically active protein kinases.
Jaleel
Mahaboobi
M
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Saha
Sayanti
S
Shenoy
Avinash R
AR
Visweswariah
Sandhya S
SS
D000227:Adenine Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000227:Adenine Nucleotides; D000911:Antibodies, Monoclonal; D000939:Epitopes; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D006162:Guanylate Cyclase; D008239:Lysine