Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification

Rad18 is a ubiquitin E3 ligase that monoubiquitinates PCNA on stalled replications forks. This allows recruitment of damage-tolerant polymerases for damage bypass and DNA repair. In this activity, the Rad18 protein has to interact with Rad6, the E2 ubiquitin-conjugating enzyme, ubiquitin, PCNA and DNA. Here we analyze the biochemical interactions of specific domains of the Rad18 protein. We found that the Rad6/Rad18 complex forms stable dimers in vitro. Consistent with previous findings, both the Ring domain and a C-terminal region contribute to the Rad6 interaction, while the C-terminus is not required for the interaction with PCNA. Surprisingly we find that the C2HC zinc finger is important for interaction with ubiquitin, apparently analogous to the interactions of classical zinc fingers with ubiquitin such as found in the UBZ and UBM domains in Y-family polymerases. Finally we find that the SAP domain, but not the zinc finger domain, is capable of DNA binding in vitro

Crystallographic studies of carbohydrate modifying proteins, mechanisms of action in polysaccharide binding and catalysis

grantor: University of TorontoEnzymatic degradation of cellulose and hemicellulose abundant in plant biomass is achieved by the activity of diverse hydrolytic enzyme systems secreted by microbial organisms. Such enzymes can be found incorporated within a cellulosome or expressed in soluble form. Subtle differences in specificities among the enzymes complement each other to degrade the diverse polymeric substrates efficiently. These enzymes frequently embody a modular structure, consisting of one or more substrate binding domains and a catalytic domain of the glycosyl hydrolase enzyme class. This thesis explores the three-dimensional structures and mechanisms of action in examples of each of these two types of modules, a cellulose binding domain expressed by the thermostable 'Thermotoga maritima' and a ß-1,4-glycanase from 'Cellulomonas fimi' with xylanase and cellulase activity. The first part of this manuscript describes the X-ray crystal structure of the 17 kDa family 9 binding module from 'T. maritima' (CBM9-2) to 1.9 Å resolution, as determined by multiple-wavelength anomalous dispersion using selenium-substituted methionine residues as the source for anomalous scattering. The structure reveals a ß-sandwich fold stabilized by three calcium ions, and suggests a novel mode of substrate binding through a sandwich-like binding pocket formed by two tryptophan side chains. The crystal structures of CBM9-2 in complex with several ligands allows for interpretation of the mechanism of binding in this module, which was determined to interact specifically with reducing ends. The remainder of the thesis addresses mechanistic aspects of the ' C. fimi' family 10 catalytic domain, Cex. Cex hydrolyzes xylan, and to a lesser extent cellulose, with retention of ß-configuration at the anomeric centre of the cleaved O-glycosidic bond. This implies a 2-step SN1-like catalytic mechanism involving a covalent enzyme-substrate intermediate flanked by energetically unfavourable transition states with significant oxocarbenium cationic character. Through extensive analysis by X-ray crystallography as well as kinetic studies with mutants, a detailed mechanistic picture of Cex has emerged describing unique enzyme characteristics such as substrate specificity and transition state stabilization. Furthermore, insights gained throughout the project have led to the design of strong mechanism-based inhibitors, whose crystal structures bound to the enzyme in turn have been characterized and described.Ph.D

Colloidal Liquid Crystals Confined to Synthetic Tactoids

When a liquid crystal forming particles are confined to a spatial volume with dimensions comparable to that of their own size, they face a complex trade-off between their global tendency to align and the local constraints imposed by the boundary conditions. This interplay may lead to a non-trivial orientational patterns that strongly depend on the geometry of the confining volume. This novel regime of liquid crystalline behavior can be probed with colloidal particles that are macro-aggregates of biomolecules. Here we study director fields of filamentous fd-viruses in quasi-2D lens-shaped chambers that mimic the shape of tactoids, the nematic droplets that form during isotropic-nematic phase separation. By varying the size and aspect ratio of the chambers we force these particles into confinements that vary from circular to extremely spindle-like shapes and observe the director field using fluorescence microscopy. In the resulting phase diagram, next to configurations predicted earlier for 3D tactoids, we find a number of novel configurations. Using Monte Carlo Simulations, we show that these novel states are metastable, yet long-lived. Their multiplicity can be explained by the co-existence of multiple dynamic relaxation pathways leading to the final stable states.status: publishe

Colloidal Liquid Crystals Confined to Synthetic Tactoids

When a liquid crystal forming particles are confined to a spatial volume with dimensions comparable to that of their own size, they face a complex trade-off between their global tendency to align and the local constraints imposed by the boundary conditions. This interplay may lead to a non-trivial orientational patterns that strongly depend on the geometry of the confining volume. This novel regime of liquid crystalline behavior can be probed with colloidal particles that are macro-aggregates of biomolecules. Here we study director fields of filamentous fd-viruses in quasi-2D lens-shaped chambers that mimic the shape of tactoids, the nematic droplets that form during isotropic-nematic phase separation. By varying the size and aspect ratio of the chambers we force these particles into confinements that vary from circular to extremely spindle-like shapes and observe the director field using fluorescence microscopy. In the resulting phase diagram, next to configurations predicted earlier for 3D tactoids, we find a number of novel configurations. Using Monte Carlo Simulations, we show that these novel states are metastable, yet long-lived. Their multiplicity can be explained by the co-existence of multiple dynamic relaxation pathways leading to the final stable states.BN/Gijsje Koenderink La

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: Experimental procedures, database tracking and case studies

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells