Structure/function studies of Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis.

Many bacterial species express 'eukaryotic-like' Ser/Thr or Tyr protein kinases and phosphatases that are candidate mediators of developmental changes and host/pathogen interactions. The biological functions of these systems are largely unknown. Recent genetic, biochemical and structural studies have begun to establish a framework for understanding the systems for Ser/Thr and Tyr protein phosphorylation in Mycobacterium tuberculosis (Mtb). Ser/Thr protein kinases (STPKs) appear to regulate diverse processes including cell division and molecular transport. Proposed protein substrates of the STPKs include putative regulatory proteins, as well as six proteins containing Forkhead-associated domains. Structures of domains of receptor STPKs and all three Mtb Ser/Thr or Tyr phosphatases afford an initial description of the principal modules that mediate bacterial STPK signaling. These studies revealed that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic STPKs. Several structures also support novel mechanisms of regulation, including dimerization of STPKs, metal-ion binding to PstP and substrate mimicry in PtpB.

An alternate conformation and a third metal in PstP/Ppp, the M. tuberculosis PP2C-Family Ser/Thr protein phosphatase.

Serine/threonine protein phosphatases are central mediators of phosphorylation-dependent signals in eukaryotes and a variety of pathogenic bacteria. Here, we report the crystal structure of the intracellular catalytic domain of Mycobacterium tuberculosis PstPpp, a membrane-anchored phosphatase in the PP2C family. Despite sharing the fold and two-metal center of human PP2Calpha, the PstPpp catalytic domain binds a third Mn(2+) in a site created by a large shift in a previously unrecognized flap subdomain adjacent to the active site. Mutations in this site selectively increased the Michaelis constant for Mn(2+) in the reaction of a noncognate, small-molecule substrate, p-nitrophenyl phosphate. The PstP/Ppp structure reveals core functional motifs that advance the framework for understanding the mechanisms of substrate recognition, catalysis, and regulation of PP2C phosphatases.

Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2.

An inherited form of diabetes, maturity-onset diabetes of the young type 3 (MODY3), results from mutations in the transcriptional activator, hepatocyte nuclear factor-1alpha (HNF1alpha). Transcription by HNF1alpha is stimulated by the bifunctional coactivator DCoH (dimerization cofactor of HNF1). Strikingly, an HNF1alpha deletion in mice causes more severe phenotypes than a DCoH deletion. It has been hypothesized that a DCoH homolog, DCoH2, partially complements the DCoH deletion. To test this idea, we determined the biochemical properties and the 1.6-A-resolution crystal structure of DCoH2. Like DCoH, DCoH2 forms a tetramer, displays pterin-4alpha-carbinolamine dehydratase activity, and binds HNF1alpha in vivo and in vitro. DCoH and DCoH2 adopt identical folds with structural differences confined largely to the protein surfaces and the tetramer interface. In contrast to the hyperstable DCoH tetramer, DCoH2 readily disproportionates and forms a 2:2 complex with HNF1 in vitro. Phylogenetic analysis reveals six major subfamilies of DCoH proteins, including unique DCoH and DCoH2 branches in metazoans. These results suggest distinct roles for DCoH and DCoH2. Differences in conserved surface residues could mediate binding to different effectors. We propose that HNF1alpha binding kinetics may distinguish regulation by DCoH2, under thermodynamic control, from regulation by DCoH, under kinetic control.