Jeffrey G. Pelton

Conformational Coupling across the Plasma Membrane in Activation of the EGF Receptor

How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation, we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. Although the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane, and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Crystal structure of an activated response regulator bound to its target

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3−-activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the β4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8–15 form two turns of helix and pack against the H4-β5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.

Understanding the impact of ionic liquid pretreatment on eucalyptus

Background: The development of cost-competitive biofuels necessitates the realization of advanced biomass pretreatment technologies. Ionic liquids provide a basis for one of the most promising pretreatment technologies and are known to allow effective processing of cellulose and some biomass species. Results & discussion: Here, we demonstrate that the ionic liquid 1-ethyl-3-methyl imidazolium acetate, [C2mim][OAc], induces structural changes at the molecular level in the cell wall of Eucalyptus globulus. Deacetylation of xylan, acetylation of the lignin units, selective removal of guaiacyl units (increasing the syringyl:guaiacyl ratio) and decreased β-ether content were the most prominent changes observed. Scanning electron microscopy images of the plant cell wall sections reveal extensive swelling during [C2mim][OAc] pretreatment. X-ray diffraction measurements indicate a change in cellulose crystal structure from cellulose I to cellulose II after [C2mim][OAc] pretreatment. Enzymatic saccharification of the pretreated material produced increased sugar yields and improved hydrolysis kinetics after [C2mim][OAc] pretreatment. Conclusion: These results provide new insight into the mechanism of ionic liquid pretreatment and reaffirm that this approach may be promising for the production of cellulosic biofuels from woody biomass.

Phosphorylation-induced Conformational Changes in the Retinoblastoma Protein Inhibit E2F Transactivation Domain Binding

Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2FTD) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2FTD binding ∼250-fold and that phosphorylation at Ser608/Ser612 and Thr356/Thr373 is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2FTD binding. We find that phosphorylation at Ser608/Ser612 promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2FTD binding site. We also find that phosphorylation of Thr356/Thr373 inhibits E2FTD binding in a manner that requires the Rb N-terminal domain. Taken together, our results suggest two distinct mechanisms for how phosphorylation of Rb modulates association between E2FTD and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.

Lignin fate and characterization during ionic liquid biomass pretreatment for renewable chemicals and fuels production

The fate of lignin from wheat straw, Miscanthus, and Loblolly pine after pretreatment by a non-toxic and recyclable ionic liquid (IL), [C2mim][OAc], followed by enzymatic hydrolysis was investigated. The lignin partitioned into six process streams, each of which was quantified and analyzed by a combination of a novel solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) method, and size exclusion chromatography (SEC). Pretreatment of biomass samples by [C2mim][OAc] at 120 and 160 °C enhances hydrolysis rates and enzymatic glucan digestions compared to those of untreated biomass samples. Lignin partitioning into the different streams can be controlled by altering the ionic liquid pre-treatment conditions, with higher temperatures favoring higher lignin partitioning to the IL stream. 2D NMR bond abundance data and SEC results reveal that lignin is depolymerized during ionic liquid pretreatment, and lignin of different molecular masses can be isolated in the different process streams. SEC suggested that higher molecular mass lignin was precipitated from the ionic liquid, leaving smaller molecular mass lignin in solution for further extraction. Lignin obtained as a residue of enzymatic hydrolysis contained the highest molecular mass molecules, similar in structure to the control lignin. The results suggest that isolated lignins via IL pretreatment from all three feedstocks were both depolymerized and did not contain new condensed structures. This finding leads to the possibility that lignin obtained from this IL pretreatment process may be more amenable to upgrading, thereby enhancing biorefinery economics.

The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems

The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of (18)O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.

Phosphoaspartates in bacterial signal transduction

Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.

The novel catabolic pathway of 3,6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4s active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.

Identification of a DNA primase template tracking site redefines the geometry of primer synthesis

Primases are essential RNA polymerases required for the initiation of DNA replication, lagging strand synthesis and replication restart. Many aspects of primase function remain unclear, including how the enzyme associates with a moving nucleic acid strand emanating from a helicase and orients primers for handoff to replisomal components. Using a new screening method to trap transient macromolecular interactions, we determined the structure of the Escherichia coli DnaG primase catalytic domain bound to single-stranded DNA. The structure reveals an unanticipated binding site that engages nucleic acid in two distinct configurations, indicating that it serves as a nonspecific capture and tracking locus for template DNA. Bioinformatic and biochemical analyses show that this evolutionarily constrained region enforces template polarity near the active site and is required for primase function. Together, our findings reverse previous proposals for primer–template orientation and reconcile disparate studies to re-evaluate replication fork organization.