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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 seriesin vitroof 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.

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca

The sarcoplasmic/endoplasmic reticulum Ca(2+)‐ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca(2+) and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA‐stabilized E2 [Formula: see text] form (3.3 Å) and the Ca(2+)‐occluded [Ca(2)]E1‐AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform‐specific residues are acceptor sites for post‐translational modifications. In addition, molecular dynamics simulations predict that isoform‐specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform‐specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.