Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics

Protein structural variation in computational models and crystallographic data

Normal mode analysis offers an efficient way of modeling the conformational flexibility of protein structures. Simple models defined by contact topology, known as elastic network models, have been used to model a variety of systems, but the validation is typically limited to individual modes for a single protein. We use anisotropic displacement parameters from crystallography to test the quality of prediction of both the magnitude and directionality of conformational variance. Normal modes from four simple elastic network model potentials and from the CHARMM forcefield are calculated for a data set of 83 diverse, ultrahigh resolution crystal structures. While all five potentials provide good predictions of the magnitude of flexibility, the methods that consider all atoms have a clear edge at prediction of directionality, and the CHARMM potential produces the best agreement. The low-frequency modes from different potentials are similar, but those computed from the CHARMM potential show the greatest difference from the elastic network models. This was illustrated by computing the dynamic correlation matrices from different potentials for a PDZ domain structure. Comparison of normal mode results with anisotropic temperature factors opens the possibility of using ultrahigh resolution crystallographic data as a quantitative measure of molecular flexibility. The comprehensive evaluation demonstrates the costs and benefits of using normal mode potentials of varying complexity. Comparison of the dynamic correlation matrices suggests that a combination of topological and chemical potentials may help identify residues in which chemical forces make large contributions to intramolecular coupling.Comment: 17 pages, 4 figure

Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology.

Deregulation of mini-chromosome maintenance (MCM) proteins is associated with genomic instability and cancer. MCM complexes are recruited to replication origins for genome duplication. Paradoxically, MCM proteins are in excess than the number of origins and are associated with chromatin regions away from the origins during G1 and S phases. Here, we report an unusually wide left-handed filament structure for an archaeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation. The filament has a remarkably strong electro-positive surface spiraling along the inner filament channel for DNA binding. We show that this MCM filament binding to DNA causes dramatic DNA topology change. This newly identified function of MCM to change DNA topology may imply a wider functional role for MCM in DNA metabolisms beyond helicase function. Finally, using yeast genetics, we show that the inter-subunit interactions, important for MCM filament formation, play a role for cell growth and survival

Rigidity analysis of protein biological assemblies and periodic crystal structures

Background We initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. The goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. We use experimental X-ray crystallography data from the Protein Data Bank (PDB). The analysis relies on an effcient graph-based algorithm. Computational experiments were performed using new protein rigidity analysis tools available in the new release of our KINARI-Web server http://kinari.cs.umass.edu. Results We provide two types of results: on biological assemblies and on crystals. We found that when only isolated subchains are considered, structural and functional information may be missed. Indeed, the rigidity of biological assemblies is sometimes dependent on the count and placement of hydrogen bonds and other interactions among the individual subchains of the biological unit. Similarly, the rigidity of small crystals may be affected by the interactions between atoms belonging to different unit cells. We have analyzed a dataset of approximately 300 proteins, from which we generated 982 crystals (some of which are biological assemblies). We identified two types of behaviors. (a) Some crystals and/or biological assemblies will aggregate into rigid bodies that span multiple unit cells/asymmetric units. Some of them create substantially larger rigid cluster in the crystal/biological assembly form, while in other cases, the aggregation has a smaller effect just at the interface between the units. (b) In other cases, the rigidity properties of the asymmetric units are retained, because the rigid bodies did not combine. We also identified two interesting cases where rigidity analysis may be correlated with the functional behavior of the protein. This type of information, identified here for the first time, depends critically on the ability to create crystals and biological assemblies, and would not have been observed only from the asymmetric unit. For the Ribonuclease A protein (PDB file 5RSA), which is functionally active in the crystallized form, we found that the individual protein and its crystal form retain the flexibility parameters between the two states. In contrast, a derivative of Ribonuclease A (PDB file 9RSA), has no functional activity, and the protein in both the asymmetric and crystalline forms, is very rigid. For the vaccinia virus D13 scaffolding protein (PDB file 3SAQ), which has two biological assemblies, we observed a striking asymmetry in the rigidity cluster decomposition of one of them, which seems implausible, given its symmetry. Upon careful investigation, we tracked the cause to a placement decision by the Reduce software concerning the hydrogen atoms, thus affecting the distribution of certain hydrogen bonds. The surprising result is that the presence or lack of a very few, but critical, hydrogen bonds, can drastically affect the rigid cluster decomposition of the biological assembly. Conclusion The rigidity analysis of a single asymmetric unit may not accurately reflect the protein\u27s behavior in the tightly packed crystal environment. Using our KINARI software, we demonstrated that additional functional and rigidity information can be gained by analyzing a protein\u27s biological assembly and/or crystal structure. However, performing a larger scale study would be computationally expensive (due to the size of the molecules involved). Overcoming this limitation will require novel mathematical and computational extensions to our software

A pilot study of the genotype and phenotype in Amelogenesis Imperfecta and Molar Incisor Hypomineralization

Background Enamel is an external layer of the crown, and its production can be affected by genetic, systemic or environmental causes Amelogenesis Imperfecta (AI) is an inherited defect of dental enamel, and can be autosomal dominant, recessive, x-linked or sporadic. It can present as hypoplasia, hypomineralization or both. Mutations in several genes can cause defective enamel formation and have been linked to AI, e.g: AMELX (amelogenin), ENAM (enamelin), MMP20 (enamelysin) and KLK4 (kallikrein 4), although the correlation between genotype and phenotype is poorly understood. Molar Incisal Hypomineralization (MIH) is defined as an environmentally caused enamel defect of one to four permanent first molars, frequently associated with affected incisors, although the aetiology is unknown. The presence of MIH in siblings, and lack of obvious systemic cause suggests there may be an underlying genetic defect involved. When a patient presents in the early mixed dentition, it can be difficult to distinguish between AI and MIH in the absence of a clear family or medical history. Better understanding of the relationship between phenotype and genotype is required to aid diagnoses and management of these conditions. A pilot study was set up to determine the best method to collect data from patients, and establish a database to record dental anomalies. In the second part of this study, different machines were used to determine the most appropriate method to measure the physical proprieties of AI and MIH teeth. In the third part of the study DNA was extracted from AI and MIH patients to; (i) find the most common genes related to the AI patients in UK, and (ii) to check if there is genetic association in MIH patients. This was in order to correlate phenotype and genotype in AI and MIH patients. Aims To develop a dental anomalies clinic to identify patients with AI and MIH and create a data base. Apply a method to characterize phenotype vs. genotype for AI & MIH. Method Ethical approval was obtained. A dental anomalies clinic was established to record information (using DDE index) using a database in liaison with University of Strasbourg (Phenodent database). Phenotype analysis of MIH and AI teeth was done using Scanning Electron Microscope (SEM), hardness was obtained using both a Wallace indenter and an Atomic Force Microscope (AFM). To investigate the genotype, DNA was extracted from saliva samples using TaqMan protocol, and analysed for gene markers known to occur in inherited enamel defect conditions (Enam 2 Allele C and Allele T, Enam 1 Allele A and Allele G and MMp20 for Allele A and Allele T) and was applied on MIH patients for possible genetic association. Results 57 AI patients and 58 MIH patients were identified through the anomalies clinic. 8 MIH, 4 with AI and 8 control teeth were collected and analysed using SEM. Under higher magnifications, normal enamel had well organized prism and crystal structure, while the hypomineralised enamel in (AI and MIH) had less distinct prism borders and increased interprismatic space. In the AI teeth a glass like appearance and loss of prism layer were obvious. Over all, the hypomineralised enamel appeared more porous than the adjacent normal unaffected enamel. The average hardness ranged between 2.3 to 8.0 GPa for control teeth, between 0.004 to 0.027 GPa for AI teeth and from 0.07 to 0.40 for MIH teeth. Yellow/ brown opacities had lower hardness (0.07 GPa) compared to white/cream opacity (0.40 GPa). Strong association of AI and the ENAM 1 gene in UK. Conclusion Teeth diagnosed with Amelogenesis Imperfecta and Molar Incisor Hypomineralisation has significantly lower hardness values in the hypomineralised enamel compared with normal enamel. Yellow/ brown opacities had lower hardness values than white/ cream opacities. No correlation found between the phenotypic presentation of AI and MIH and the genotype of ENAM 1 was polymorphism

Salvage enzymes in nucleotide biosynthesis

Balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, and ribonucleoside triphosphates (NTPs), the precursors of RNA, are crucial for a controlled cell proliferation. The dNTPs and NTPs are synthesized de novo via energy-consuming reactions involving low-weight molecules, and through a salvage pathway by recycling (deoxy)ribonucleosides originating from food and degraded DNA and RNA. The enzymes described in this thesis catalyze the first reaction in the salvage biosynthesis of dNTPs and NTPs. The crystal structures of three bacterial thymidine kinases (TKs) are described and the enzymes are investigated as potential targets for antibacterial therapies. TK is a deoxyribonucleoside kinase (dNK) with specificity for thymidine. In addition to the natural substrates, TK can also phosphorylate a number of nucleoside analogs used in antiviral and anticancer therapies. This thesis presents the structures of TKs from three pathogenic microorganisms: Ureaplasma urealyticum (parvum), Bacillus anthracis and Bacillus cereus, and compares them to the human thymidine kinase 1 (hTK1). The bacterial TKs and the hTK1 are structurally very similar and have a highly conserved active site architecture, which may complicate structure-based drug design. However, the different complex structures presented in this work provide information regarding the conformational changes of TK1-like enzymes during the time of reaction. The structure of human uridine-cytidine kinase 1 (UCK1) is also presented. Humans possess two uridine-cytidine kinases, UCK1 and UCK2. The expression pattern of these enzymes is tissue dependent, and despite high sequence as well as structural similarities they possess somewhat diverse substrate specificity. In addition to the natural substrates, uridine and cytidine, UCKs are able to phosphorylate a number of nucleoside analogs. The monomeric structure of UCK comprises four domains: a CORE domain, an NMP-binding domain, a LID domain and a β-hairpin domain, which upon substrate binding undergo dramatic conformational changes. In the structure described in this thesis the enzyme has been trapped in an intermediate conformation between a fully opened and fully closed form, which may represent a sequential mode of substrate binding