Sequence Divergence in the Arginase Domain of Ornithine Decarboxylase/Arginase in Fusobacteriacea Leads to Loss of Function in Oral Associated Species.

A number of species within the Fusobacteriaceae family of Gram-negative bacteria uniquely encode for an ornithine decarboxylase/arginase (ODA) that ostensibly channels l-ornithine generated by hydrolysis of l-arginine to putrescine formation. However, two aspartate residues required for coordination to a catalytically obligatory manganese cluster of arginases are substituted for a serine and an asparagine. Curiously, these natural substitutions occur only in a clade of Fusobacterium species that inhabit the oral cavity. Herein, we expressed and isolated full-length ODA from the opportunistic oral pathogen Fusobacterium nucleatum along with the individual arginase and ornithine decarboxylase components. The crystal structure of the arginase domain reveals that it adopts the classical α/ß arginase-fold, but metal ions are absent in the active site. As expected, the ureohydrolase activity with l-arginine was not detected for wild-type ODA or the isolated arginase domain. However, engineering of the complete metal coordination environment through site-directed mutagenesis restored Mn2+ binding capacity and arginase activity, although the catalytic efficiency for l-arginine was low (60-100 M-1 s-1). Full-length ODA and the isolated ODC component were able to decarboxylate both l-ornithine and l-arginine to form putrescine and agmatine, respectively, but kcat/KM of l-ornithine was ∼20-fold higher compared to l-arginine. We discuss environmental conditions that may have led to the natural selection of an inactive arginase in the oral associated species of Fusobacterium.

Biophysical characterization of the ETV6 PNT domain polymerization interfaces.

ETV6 is an E26 transformation specific family transcriptional repressor that self-associates by its PNT domain to facilitate cooperative DNA binding. Chromosomal translocations frequently generate constitutively active oncoproteins with the ETV6 PNT domain fused to the kinase domain of one of many protein tyrosine kinases. Although an attractive target for therapeutic intervention, the propensity of the ETV6 PNT domain to polymerize via the tight head-to-tail association of two relatively flat interfaces makes it challenging to identify suitable small molecule inhibitors of this protein-protein interaction. Herein, we provide a comprehensive biophysical characterization of the ETV6 PNT domain interaction interfaces to aid future drug discovery efforts and help define the mechanisms by which its self-association mediates transcriptional repression. Using NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations, along with amide hydrogen exchange measurements, we demonstrate that monomeric PNT domain variants adopt very stable helical bundle folds that do not change in conformation upon self-association into heterodimer models of the ETV6 polymer. Surface plasmon resonance-monitored alanine scanning mutagenesis studies identified hot spot regions within the self-association interfaces. These regions include both central hydrophobic residues and flanking salt-bridging residues. Collectively, these studies indicate that small molecules targeted to these hydrophobic or charged regions within the relatively rigid interfaces could potentially serve as orthosteric inhibitors of ETV6 PNT domain polymerization.

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin.

Flavodoxins are small flavin mononucleotide (FMN)-containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate-binding loop (T/S-X-T-G-X-T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The Eox/sq (oxidized/semiquinone; -43 mV) and Esq/hq (semiquinone/hydroquinone; -256 mV) are the highest recorded reduction potentials of known long-chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70-fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive Eox/sq . The more electropositive Esq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.

Staphylococcus aureus heme and siderophore-iron acquisition pathways.

Staphylococcus aureus is a versatile opportunistic human pathogen. Infection by this bacterium requires uptake of iron from the human host, but iron is highly restricted in this environment. Staphylococcus aureus iron sufficiency is achieved primarily through uptake of heme and high-affinity iron chelators, known as siderophores. Two siderophores (staphyloferrins) are produced and secreted by S. aureus into the extracellular environment to capture iron. Staphylococcus aureus expresses specific uptake systems for staphyloferrins and more general uptake systems for siderophores produced by other microorganisms. The S. aureus heme uptake system uses highly-specific cell surface receptors to extract heme from hemoglobin and hemoglobin-haptoglobin complexes for transport into the cytoplasm where it is degraded to liberate iron. Initially thought to be independent systems, recent findings indicate that these iron uptake pathways intersect. IruO is a reductase that releases iron from heme and some ferric-siderophores. Moreover, multifunctional SbnI produces a precursor for staphyloferrin B biosynthesis, and also binds heme to regulate expression of the staphyloferrin B biosynthesis pathway. Intersection of the S. aureus iron uptake pathways is hypothesized to be important for rapid adaptation to available iron sources. Components of the heme and siderophore uptake systems are currently being targeted in the development of therapeutics against S. aureus.

The Concurrent Control of Motion and Contact Force in the Presence of Predictable Disturbances.

The simultaneous control of force and motion is important in everyday activities when humans interact with objects. While many studies have analyzed the control of movement within a perturbing force field, few have investigated its dual aspects of controlling a contact force in nonisometric conditions. The mechanism by which the central nervous system controls forces during movements is still unclear, and it can be elucidated by estimating the mechanical properties of the arm during tasks with concurrent motion and contact force goals. We investigate how arm mechanics change when a force control task is accomplished during low-frequency positional perturbations of the arm. Contrary to many force regulation algorithms implemented in robotics, where contact impedance is decreased to reduce force fluctuations in response to position disturbances, we observed a steady increase of arm endpoint stiffness as the task progressed. Based on this evidence, we propose a theoretical framework suggesting that an internal model of the perturbing trajectory is formed. We observed that force regulation in the presence of predictable positional disturbances is implemented using a position control strategy together with the modulation of the endpoint stiffness magnitude, where the direction of the endpoint stiffness ellipse's major axis is oriented toward the desired force.

On force regulation strategies in predictable environments.

This paper is focused on investigating force regulation strategies employed by human central nervous system (CNS). The mechanism responsible for force control is extremely important in people's lives, but not yet well understood. We formulate the general model of force regulation and identify several possible control strategies. An experimental approach is used to determine which of the force control strategies could actually be used by the CNS. Obtained results suggest that the force regulation process involves not only the pure force controller, but also a coupled motion controller, relying on the internal model of the environment.