X-Ray Crystallographic Study of Na,K-ATPase in Complex with Cardiotonic Steroids

Abstract

Na,K-ATPase transports three sodium ions from the cytoplasm to the extracellular side and counter-transports two potassium ions upon hydrolysis of one ATP in each reaction cycle, thereby establishing gradients for sodium and potassium ions across the membrane. These ion gradients are used in many fundamental processes, notably, excitation of nerve cells. It is one of the most important members of the P-type ATPases. It is a hetero-trimer complex, consisting of alpha-subunit (112K), beta-subunit (55K) and FXYD regulatory protein (10K). Na,K-ATPase is also regarded as a membrane receptor for cardiotonic steroids (CTS), including digoxin prescribed for congestive heart failure and supraventricular arrhythmias for ∼200 years. Moreover, CTS have been studied as potential drugs for cancer chemotherapy, because they inhibit growth of cancer cells through the binding to Na,K-ATPase. Thus, the elucidation of the binding mechanism of CTS to Na,K-ATPase has direct medical implication. We reported a crystal structure with bound ouabain at 2.8 A resolution (Ogawa et al., PNAS 2009), but the affinity of ouabain under the conditions employed is low due to antagonism with bound potassium. As to the high affinity ouabain-bound form, a crystal structure was reported very recently by other group, but at only 3.4 A resolution with a very low completeness (Laursen et al., PNAS 2013). Thus, higher-resolution structures are still awaited. Moreover, structures bound with other CTSs having different sugar moieties and/or different lactone rings are also awaited. Here, we report our recent crystal structures with bound several CTSs, including different sugar moieties and lactone rings, at better than 3.0 A resolution. These structures provide detailed information on the interaction of Na,K-ATPase and CTS and may lead us to design better drugs for treating heart failure, arrhythmias and cancer.