No abstract

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane. Here, we describe a 4.0 Å resolution crystal structure of the pig kidney Na+,K+-ATPase stabilized by beryllium fluoride (denoted E2-BeFx). The structure shows high resemblance to the E2P phosphoenzyme obtained by phosphorylation from inorganic phosphate (Pi) and stabilised by cardiotonic steroids, and reveals a Mg2+ bound near the ion binding site II. Anomalous Fourier analysis of the crystals soaked in Rb+ (K+ congener) followed by a low resolution rigid-body refinement (6.9-7.5 Å) revealed pre-occlusion transitions leading to activation of the desphosphorylation reaction. Mg2+ location indicates a site of an initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Despite the overall structural resemblance to the Pi-induced E2P phosphoform, BeFx inhibited enzyme is able to bind both ADP/ATP and ions, the features that relate E2-BeFx complex to an intermediate of the functional cycle of the Na+,K+-ATPase prior to E2P.

No abstract

The p90 ribosomal S6 kinases (RSKs) contain two distinct catalytic kinase domains, the N-terminal and C-terminal kinase domains (NTKD and CTKD, respectively). The activation of CTKD is regulated by phosphorylation by extracellular signal–regulated kinase (ERK1/2) and an autoinhibitory αL helix. Through a mutational series in vitro of the RSK CTKDs, we found a complex mechanism lifting autoinhibition that led us to design constitutively active RSK CTKDs. These are based on a phosphomimetic mutation and a C-terminal truncation (e.g., RSK2 T577E D694*) where a high activity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50values of ATP-competitive inhibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.

The p90 ribosomal S6 kinases (RSKs) contain two distinct catalytic kinase domains, the N-terminal and C-terminal kinase domains (NTKD and CTKD, respectively). The activation of CTKD is regulated by phosphorylation by extracellular signal-regulated kinase (ERK1/2) and an autoinhibitory αL helix. Through a mutational series in vitro of the RSK CTKDs, we found a complex mechanism lifting autoinhibition that led us to design constitutively active RSK CTKDs. These are based on a phosphomimetic mutation and a C-terminal truncation (e.g. RSK2 T577E D694*) where a high activity in absence of ERK phosphorylation is obtained. Using these constructs, we characterize IC50 values of ATP-competitive inhibitors and provide a setup for determining specificity constants (kinact/Ki) of covalent CTKD inhibitors.