Gulnahar B. Mortuza

The intrinsically disordered protein p15^PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15^PAF–PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15^PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15^PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15^PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15^PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15^PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

ChTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis.

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

Abstract: The centrosome plays an essential role in cell cycle progression and cell polarity, organizing the microtubule network in interphase and mitosis. During cell division, the centrosome undergoes a series of structural and functional transitions and forms the two poles of the bipolar mitotic spindle. It is the microtubule cytoskeleton that is reorganized to form the two poles, ensuring accurate separation of the two daughter cells. To achieve this a large number of signalling proteins located at the centrosome, undergo precise time-dependent modulation. Protein kinases such as Aurora A, Polo and Neks, trigger and regulate events such as centrosome duplication, maturation and division. These enzymes are also involved in recruiting other proteins in cell division, thus they are likely to mediate the crosstalk between the cell and the centrosome cycle. In its function of microtubule organization, macromolecular complexes also have an important role. Tubulin polymerization confers the structural backbone to cell division, while other proteins may interact with it and/or mediate its recruitment to the centrosome. The interactions of these components regulate centrosome maturation and microtubule growth, essential mechanisms for cell division. Furthermore, dysregulation of this organelle, both at the level of signalling or as a structural element strongly correlates to aberrant proliferation, and the onset of tumours. Therefore, the centrosome represents an attractive target for anti-cancer therapy. Here we review the most important centrosomal proteins and their therapeutic potential. In addition, we summarize the current strategies of intervention and report the present stage of anticancer drug development targeting the centrosome.

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca²⁺ ion (K = 3 × 10¹⁰ M^-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca²⁺ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca²⁺ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J.230, 227−237) correspond to the Ca²⁺-loaded and -depleted forms of the enzyme.

The Fv1 protein is an endogenous factor in mice that confers resistance to infection by certain classes of murine leukemia virus, a phenomenon referred to as restriction. The mechanism of restriction is not understood, and the low endogenous level of Fv1 in cells has prevented any biochemical or biophysical analysis of the protein. We have now purified recombinant Fv1 ⁿ protein from a baculovirus system and demonstrate that Fv1 exists in a multimeric form. Furthermore, we have mapped the position of two domains within the protein using limited proteolysis. Biophysical characterization of the N-terminal domain reveals that it comprises a highly helical and extended dimeric structure. Based on these biochemical and biophysical data, we propose a model for the arrangement of domains in Fv1 and suggest that dimerization of the N-terminal domain is necessary for Fv1 function to allow the protein to interact with multiple capsid protomers in retroviral cores.

Retroviruses are the aetiological agents of a range of human diseases including AIDS and T-cell leukaemias. They follow complex life cycles, which are still only partly understood at the molecular level. Maturation of newly formed retroviral particles is an essential step in production of infectious virions, and requires proteolytic cleavage of Gag polyproteins in the immature particle to form the matrix, capsid and nucleocapsid proteins present in the mature virion. Capsid proteins associate to form a dense viral core that may be spherical, cylindrical or conical depending on the genus of the virus. Nonetheless, these assemblies all appear to be composed of a lattice formed from hexagonal rings, each containing six capsid monomers. Here, we describe the X-ray structure of an individual hexagonal assembly from N-tropic murine leukaemia virus (N-MLV). The interface between capsid monomers is generally polar, consistent with weak interactions within the hexamer. Similar architectures are probably crucial for the regulation of capsid assembly and disassembly in all retroviruses. Together, these observations provide new insights into retroviral uncoating and how cellular restriction factors may interfere with viral replication.