The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

SummaryUbiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins

Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization

The Binding of Benzoarylsulfonamide Ligands to Human Carbonic Anhydrase is Insensitive to Formal Fluorination of the Ligand

Auf das Wasser kommt es an: Konservierte Bindungsgeometrie, enthalpiegetriebene Bindungsweise und nichtunterscheidbare Bindungsaffinitäten für fluorierte und nichtfluorierte Liganden von humaner Carbonsäureanhydrase (siehe Bild) stützen die Theorie, dass das Schlüssel-Schloss-Modell einen wichtigen Bestandteil des Bindungsvorgangs unbeachtet lässt: das Wasser, das die Bindungstasche des Proteins füllt und den Liganden umgibt.Chemistry and Chemical Biolog

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors

Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency ☆

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI I170V elicits behavioral abnormalities in Drosophila. An examination of hTPI I170V enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way

Structure of a highly conserved domain of rock1 required for shroom-mediated regulation of cell morphology

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology. © 2013 Mohan et al

Preliminary crystallographic studies of the regulatory domain of response regulator YycF from an essential two-component signal transduction system

The regulatory domain of response regulator YycF from an essential two-component signal transduction system has been crystallized and X-ray data have been collected at 1.95 Å resolution

Effects of horizontal distance and limb crossing on perceived hand spacing and ownership: Differential sensory processing across hand configurations

We have previously shown that, with the hands apart vertically, passively grasping an artificial finger induces a sense of ownership over the artificial finger and coming-together of the hands. The present study investigated this grasp illusion in the horizontal plane. Thirty healthy participants were tested in two conditions (grasp and no grasp) with their hands at different distances apart, either crossed or uncrossed. After 3 min, participants reported perceived spacing between index fingers, perceived index finger location, and, for the grasp condition, perceived ownership over the artificial finger. On average, there was no ownership at any of the hand configurations. With the hands uncrossed 7.5, 15 or 24 cm apart, there was no difference in perceived spacing between the grasp and no grasp conditions. With the hands crossed and 15 cm apart, perceived spacing between index fingers was 3.2 cm [0.7 to 5.7] (mean [95% CI]) smaller during the grasp condition compared to no grasp. Therefore, compared to when the hands are vertically separated, there is an almost complete lack of a grasp illusion in the horizontal plane which indicates the brain may process sensory inputs from the hands differently based on whether the hands are horizontally or vertically apart