Enoch P. Baldwin

Ligand binding and activation of the Ah receptor

The Ah receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and naturally-occurring chemicals. Although a significant amount of information is available with respect to the planar aromatic hydrocarbon AhR ligands, the actual spectrum of chemicals that can bind to and activate the AhR is only now being elucidated. In addition, the lack of information regarding the actual three-dimensional structure of the AhR ligand binding domain (LBD) has hindered detailed analysis of the molecular mechanisms by which these ligands bind to and active AhR signal transduction. In this review we describe the current state of knowledge with respect to naturally occurring AhR ligands and present and discuss the first theoretical model of the AhR LBD based on crystal structures of homologous PAS family members.

Quantitative analysis of TALE–DNA interactions suggests polarity effects

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ∼ NN ≫ NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches ‘standard’ RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5′-T. Another surprising observation was that base mismatches at the 5′ end of the target site had more disruptive effects on affinity than those at the 3′ end, particularly in designed TALEs. These results provide evidence that TALE–DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

Large-scale filament formation inhibits the activity of CTP synthetase

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpSs product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001

Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent‐binding sites. Others were intended to displace solvent from preexisting sites. In one case Val‐149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild‐type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr‐152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7Å, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent‐binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen‐bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen‐bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

Mechanism of DNA Compaction by Yeast Mitochondrial Protein Abf2p

We used high-resolution atomic force microscopy to image the compaction of linear and circular DNA by the yeast mitochondrial protein Abf2p, which plays a major role in packaging mitochondrial DNA. Atomic force microscopy images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At a high concentration of Abf2p DNA collapses into a tight nucleoprotein complex. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of Abf2p. We also derived a polymer statistical mechanics model that provides a quantitative description of compaction observed in our experiments. This model shows that sharp bends in the DNA backbone are often sufficient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for Abf2p. These studies indicate that Abf2p compacts DNA through a simple mechanism that involves bending of the DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance and organization.

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation.

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1R398A-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Δ ura8Δ mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser462 and Thr455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser462 and Thr455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367–5377). These data indicated that protein kinase C phosphorylation at Ser462 stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr455 inhibits activity.

Human CTP synthase filament structure reveals the active enzyme conformation

The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.

Two Surfaces of a Conserved Interdomain Linker Differentially Affect Output from the RST Sensing Module of the Bacillus subtilis Stressosome

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the σ(B) stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue α-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a σ(B)-dependent reporter fusion. Substitutions at four conserved residues increased output 4- to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.

Substitutions in the Presumed Sensing Domain of the Bacillus subtilis Stressosome Affect Its Basal Output But Not Response to Environmental Signals

The stressosome is a multiprotein, 1.8-MDa icosahedral complex that transmits diverse environmental signals to activate the general stress response of Bacillus subtilis. The way in which it senses these cues and the pathway of signal propagation within the stressosome itself are poorly understood. The stressosome core consists of four members of the RsbR coantagonist family together with the RsbS antagonist; its cryo-electron microscopy (cryoEM) image suggests that the N-terminal domains of the RsbR proteins form homodimers positioned to act as sensors on the stressosome surface. Here we probe the role of the N-terminal domain of the prototype coantagonist RsbRA by making structure-based amino acid substitutions in potential interaction surfaces. To unmask the phenotypes caused by single-copy rsbRA mutations, we constructed strains lacking the other three members of the RsbR coantagonist family and assayed system output using a reporter fusion. Effects of five individual alanine substitutions in the prominent dimer groove did not match predictions from an earlier in vitro assay, indicating that the in vivo assay was necessary to assess their influence on signaling. Additional substitutions expected to negatively affect domain dimerization had substantial impact, whereas those that sampled other prominent surface features had no consequence. Notably, even mutations resulting in significantly altered phenotypes raised the basal level of system output only in unstressed cells and had little effect on the magnitude of subsequent stress signaling. Our results provide evidence that the N-terminal domain of the RsbRA coantagonist affects stressosome function but offer no direct support for the hypothesis that it is a signal sensor.