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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.

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ∼80 μm) and a high pH optimum (pH ∼9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.