Jeffry B. Stock

The Histidine Protein Kinase Superfamily

Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.

Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins

The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 A resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C‐terminal leucine residue. Here we show that methylation plays an essential role in promoting PP2A holoenzyme assembly and that demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers.

Response regulator output in bacterial chemotaxis

Chemotaxis responses in Escherichia coli are mediated by the phosphorylated response‐regulator protein P‐CheY. Biochemical and genetic studies have established the mechanisms by which the various components of the chemotaxis system, the membrane receptors and Che proteins function to modulate levels of CheY phosphorylation. Detailed models have been formulated to explain chemotaxis sensing in quantitative terms; however, the models cannot be adequately tested without knowledge of the quantitative relationship between P‐CheY and bacterial swimming behavior. A computerized image analysis system was developed to collect extensive statistics on freeswimming and individual tethered cells. P‐CheY levels were systematically varied by controlled expression of CheY in an E.coli strain lacking the CheY phosphatase, CheZ, and the receptor demethylating enzyme CheB. Tumbling frequency was found to vary with P‐CheY concentration in a weakly sigmoidal fashion (apparent Hill coefficient ∼2.5). This indicates that the high sensitivity of the chemotaxis system is not derived from highly cooperative interactions between P‐CheY and the flagellar motor, but rather depends on nonlinear effects within the chemotaxis signal transduction network. The complex relationship between single flagella rotation and free‐swimming behavior was examined; our results indicate that there is an additional level of information processing associated with interactions between the individual flagella. An allosteric model of the motor switching process is proposed which gives a good fit to the observed switching induced by P‐CheY. Thus the level of intracellular P‐CheY can be estimated from behavior determinations: ∼30% of the intracellular pool of CheY appears to be phosphorylated in fully adapted wild‐type cells.

Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C‐terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild‐type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B′ regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

beta-lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins.

The β‐lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of β‐lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no β‐lactamase‐producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the β‐lactams, the penicillin‐binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non‐PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non‐PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non‐PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during β‐lactam treatment counteract the effects caused by the inhibition of penicillin‐binding proteins.

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N‐terminal extracytoplasmic sensing domains connected by transmembrane helices to C‐terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with ∼14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus‐induced changes in lateral packing interactions within an array of receptor‐sensing domains at the cell surface perturb an equilibrium between active and inactive receptor–kinase complexes within the cytoplasm.

Influence of topology on bacterial social interaction

The environmental topology of complex structures is used by Escherichia coli to create traveling waves of high cell density, a prelude to quorum sensing. When cells are grown to a moderate density within a confining microenvironment, these traveling waves of cell density allow the cells to find and collapse into confining topologies, which are unstable to population fluctuations above a critical threshold. This was first observed in mazes designed to mimic complex environments, then more clearly in a simpler geometry consisting of a large open area surrounding a square (250 × 250 μm) with a narrow opening of 10–30 μm. Our results thus show that under nutrient-deprived conditions bacteria search out each other in a collective manner and that the bacteria can dynamically confine themselves to highly enclosed spaces.

Structural Mechanism of Demethylation and Inactivation of Protein Phosphatase 2A

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that plays a role in many biological processes. Reversible carboxyl methylation of the PP2A catalytic subunit is an essential regulatory mechanism for its function. Demethylation and negative regulation of PP2A is mediated by a PP2A-specific methylesterase PME-1, which is conserved from yeast to humans. However, the underlying mechanism of PME-1 function remains enigmatic. Here we report the crystal structures of PME-1 by itself and in complex with a PP2A heterodimeric core enzyme. The structures reveal that PME-1 directly binds to the active site of PP2A and that this interaction results in the activation of PME-1 by rearranging the catalytic triad into an active conformation. Strikingly, these interactions also lead to inactivation of PP2A by evicting the manganese ions that are required for the phosphatase activity of PP2A. These observations identify a dual role of PME-1 that regulates PP2A activation, methylation, and holoenzyme assembly in cells.

Dimerization Is Required for the Activity of the Protein Histidine Kinase CheA That Mediates Signal Transduction in Bacterial Chemotaxis

The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a γ-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA2 ↔ 2CheA; KD = 0.2-0.4 μM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (KD values ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (kcat ≈ 10 min−).