Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Evidence for a mechanism of amyloid formation involving molecular reorganisation within native-like precursor aggregates.

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.

Amyloid fibril formation can proceed from different conformations of a partially unfolded protein.

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high beta-sheet content from partially unfolded states with very different structural features. Moreover, the presence of alpha-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.

Investigation of the effects of copper ions on protein aggregation using a model system.

Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.

Designing conditions for in vitro formation of amyloid protofilaments and fibrils.

We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.