Structural and energetic analysis of activation by a cyclic nucleotide binding domain.
MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo state and the bound state, and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand dependence in MlotiK1 and, more globally, in the class of nucleotide-dependent proteins.
Altieri
Stephen L
SL
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Clayton
Gina M
GM
Silverman
William R
WR
Olivares
Adrian O
AO
De la Cruz
Enrique M
EM
Thomas
Lise R
LR
Morais-Cabral
João H
JH
D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation; D009712:Nucleotides, Cyclic / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D054815:Cyclic Nucleotide-Gated Cation Channels; D009712:Nucleotides, Cyclic; D000242:Cyclic AMP; D006152:Cyclic GMP