Regulator of K þ conductance (RCK) domains control the activity of a variety of K þ transporters and channels, including the human large conductance Ca 2þ -activated K þ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca 2þ -gated K þ channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca 2þ . Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca 2þ -binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca 2þ -binding sites, resulting in a stoichiometry of 24 Ca 2þ ions per channel, is consistent with the steep relation between [Ca 2þ ] and MthK channel activity. Comparison of Ca 2þ -bound and unliganded RCK domains suggests a physical mechanism for Ca 2þ -dependent conformational changes that underlie gating in this class of channels.calcium | lipid bilayer | cooperativity R egulator of K þ conductance (RCK) domains are structurally conserved ligand-binding domains that control the activity of a diverse array of K þ channels and transporters (1-3). Many prokaryotic RCK domains contain a conserved sequence motif for binding of nucleotides (NAD þ or ATP) (4, 5). In some prokaryotic and most of the known eukaryotic RCK-containing K þ channels, however, the nucleotide binding motif is absent, and these channels are modulated by cytoplasmic ions such as Na þ , H þ , or Ca 2þ (6-12).MthK is a prototypical RCK-containing K þ channel that has provided insight toward the structural basis of ion channel gating by RCK domains (2,13,14). In MthK, binding of Ca 2þ to an octameric ring of RCK domains (the gating ring), which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K þ conduction (2, 15, 16). Based on X-ray structures of the Ca 2þ -bound MthK channel and the unliganded MthK gating ring (17), it has been hypothesized that the principal Ca 2þ -dependent conformational change is initiated by the movement of a Glu side chain (E212) at a single Ca 2þ -binding site within each RCK domain ( Fig. 1A; site 1, formed by D184, E210, and E212), followed by subsequent movement of a nearby Phe side chain (F232). However, the conformational changes in the immediate vicinity of site 1 are relatively subtle compared to apparent conformational changes in other regions of the RCK domains (17); thus the mechanism by which Ca 2þ binding at site 1 modulates channel gating is unclear.To gain insight toward mechanisms underlying Ca 2þ -dependent conformational changes in the MthK RCK domain, we probed MthK structure and function using electrophysiology and crystallography. Our results demonstrate that whereas site 1 contributes energetically to Ca 2þ -dependent gating, charge-neutralization of the key Ca 2þ -coordinating residues at this site do not eliminat...

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and are attractive targets for drug discovery. Haloxyfop and tepraloxydim belong to two distinct classes of commercial herbicides and kill sensitive plants by inhibiting the carboxyltransferase (CT) activity of ACC. Our earlier structural studies showed that haloxyfop is bound near the active site of the CT domain, at the interface of its dimer, and a large conformational change in the dimer interface is required for haloxyfop binding. We report here the crystal structure at 2.3 Å resolution of the CT domain of yeast ACC in complex with tepraloxydim. The compound has a different mechanism of inhibiting the CT activity compared to haloxyfop, as well as the mammalian ACC inhibitor CP-640186. Tepraloxydim probes a different region of the dimer interface and requires only small but important conformational changes in the enzyme, in contrast to haloxyfop. The binding mode of tepraloxydim explains the structure-activity relationship of these inhibitors, and provides a molecular basis for their distinct sensitivity to some of the resistance mutations, as compared to haloxyfop. Despite the chemical diversity between haloxyfop and tepraloxydim, the compounds do share two binding interactions to the enzyme, which may be important anchoring points for the development of ACC inhibitors.diabetes ͉ fatty acid metabolism ͉ herbicides ͉ structure-based drug design A cetyl-CoA carboxylases (ACCs) catalyze the production of malonyl-CoA from acetyl-CoA and CO 2 in two steps (1-3). The biotin carboxylase (BC) activity catalyzes the ATPdependent carboxylation of a biotin cofactor, and then the carboxyltransferase (CT) activity catalyzes the transfer of this activated carboxyl group to the acceptor acetyl-CoA. In bacteria, the BC and CT activities reside in separate subunits of the ACC holoenzyme. In comparison, ACCs are large, multidomain enzymes in most eukaryotes, with highly conserved BC and CT domains.ACCs are crucial enzymes for the metabolism of fatty acids. Two isoforms of ACCs are present in mammals, and mice lacking ACC2 have elevated fatty acid oxidation and reduced body fat and body weight (4). ACCs are attractive targets for the discovery of anti-diabetes and anti-obesity agents (5, 6), and currently there is significant interest in understanding their catalysis, regulation, and mechanism of inhibition.The relevance of ACCs for drug discovery is also underscored by the fact that two distinct classes of compounds, as illustrated by haloxyfop (FOPs) and tepraloxydim (DIMs) (Fig. 1A), are potent inhibitors of ACCs from sensitive plants and are in wide use as herbicides (7-12). These compounds inhibit the CT activity, as does a potent inhibitor of mammalian ACCs, CP-640186 (13), suggesting that the CT domain may be a suitable target for discovering small-molecule inhibitors against ACCs.We have reported the crystal structures of the CT domain of yeast ACC and its complex with CoA (14), haloxyfop (15), and CP-640186 (16). In comparison, the human CT domain has been ...

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