Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

Incorporation of divalent metal ions into an active site is a fundamental catalytic tool used by diverse enzymes. Divalent cations are used by protein kinases to both stabilize ATP binding and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent kinase 2 (CDK2) requires simultaneous binding of two Mg(2+) ions for catalysis of phosphoryl transfer.

Briefly bound to activate: transient binding of a second catalytic magnesium activates the structure and dynamics of CDK2 kinase for catalysis.

We have determined high-resolution crystal structures of a CDK2/Cyclin A transition state complex bound to ADP, substrate peptide, and MgF(3)(-). Compared to previous structures of active CDK2, the catalytic subunit of the kinase adopts a more closed conformation around the active site and now allows observation of a second Mg(2+) ion in the active site. Coupled with a strong [Mg(2+)] effect on in vitro kinase activity, the structures suggest that the transient binding of the second Mg(2+) ion is necessary to achieve maximum rate enhancement of the chemical reaction, and Mg(2+) concentration could represent an important regulator of CDK2 activity in vivo.

Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution.

Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well.

Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway.

Secreted prokaryotic effector proteins have evolved to modulate the cellular functions of specific eukaryotic hosts. Generally, these proteins are considered virulence factors that facilitate parasitism. However, in certain plant and insect eukaryotic/prokaryotic relationships, effector proteins are involved in the establishment of commensal or symbiotic interactions.