Resonance assignments of the nucleotide-free wildtype MloK1 cyclic nucleotide-binding domain

Abstract

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of (1)H, (13)C and (15)N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

“…The final yield was roughly 7 mg∕l cell culture of [U-15 N, 13 (21). Assignments have been deposited in the BioMagResBank (accession number 16628).…”

“…All NMR experiments were performed at 298 K on Varian Unity INOVA and VNMRS instruments, equipped with a cryogenic Z-axis pulse-field-gradient (PFG) triple resonance probe at proton frequencies of 800 and 600 MHz. Backbone and side chain assignments of cAMP-free and -bound CNBD were obtained using heteronuclear standard experiments as reported previously A B 21,38). All NMR spectra were processed using NMRPipe (39) and evaluated in CARA (40).…”