Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly

The outer shell of the papillomavirus particle is comprised of 72 pentamers of the major capsid L1 protein arranged on a T = 7 icosahedral lattice. The recombinant L1 can form T = 7 virus-like particles in vitro. The crystal structure of a T = 7 papilloma virion has not yet been determined; however, the crystal structure of a T = 1 particle containing 12 pentamers is known. The T = 1 structure reveals that helix-helix interactions, through three helices–h2, h3, and h4–near the C-terminus of L1, mediate the inter-pentameric bonding that is responsible for T = 1 assembly. Based on the T = 1 crystal structure, we have generated a set of internal deletions to test the role of the three C-terminal helices in T = 7 assembly. We have demonstrated that the h2, h3, and h4 near the C-terminal end of L1 are important for the L1 structure and particle assembly. In particular, we found that h2 and h3 are essential for L1 folding and pentamer formation, whereas h4 is indispensable for the assembly of not only T1, but also of the T7 virus-like particle

Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

<p>Abstract</p> <p>Background</p> <p>The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon <it>Sulfolobus solfataricus </it>has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal <it>Methanothermobacter thermautotrophicus </it>(mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.</p> <p>Results</p> <p>In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.</p> <p>Conclusions</p> <p>These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.</p

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

Cell-Cycle-Regulated Interaction between Mcm10 and Double Hexameric Mcm2-7 Is Required for Helicase Splitting and Activation during S Phase

Mcm2-7 helicase is loaded onto double-stranded origin DNA as an inactive double hexamer (DH) in G1 phase. The mechanisms of Mcm2-7 remodeling that trigger helicase activation in S phase remain unknown. Here, we develop an approach to detect and purify the endogenous DHs directly. Through cellular fractionation, we provide in vivo evidence that DHs are assembled on chromatin in G1 phase and separated during S phase. Interestingly, Mcm10, a robust MCM interactor, co-purifies exclusively with the DHs in the context of chromatin. Deletion of the main interaction domain, Mcm10 C terminus, causes growth and S phase defects, which can be suppressed through Mcm10-MCM fusions. By monitoring the dynamics of MCM DHs, we show a significant delay in DH dissolution during S phase in the Mcm10-MCM interaction-deficient mutants. Therefore, we propose an essential role for Mcm10 in Mcm2-7 remodeling through formation of a cell-cycle-regulated supercomplex with DHs

Simple isatin derivatives as free radical scavengers: Synthesis, biological evaluation and structure-activity relationship

To develop more potent small molecules with enhanced free radical scavenger properties, a series of N-substituted isatin derivatives was synthesized, and the cytoprotective effect on the apoptosis of PC12 cells induced by H2O2 was screened. All these compounds were found to be active, and N-ethyl isatin was found with the most potent activity of 69.7% protective effect on PC12 cells. Structure-activity relationship analyses showed the bioactivity of N-alkyl isatins decline as the increasing of the chain of the alkyl group, furthermore odd-even effect was found in the activity, which is interesting for further investigation

Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC50 values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. © 2009 Elsevier Ltd. All rights reserved

Progressive Cognitive Deficit, Motor Impairment and Striatal Pathology in a Transgenic Huntington Disease Monkey Model from Infancy to Adulthood

One of the roadblocks to developing effective therapeutics for Huntington disease (HD) is the lack of animal models that develop progressive clinical traits comparable to those seen in patients. Here we report a longitudinal study that encompasses cognitive and motor assessment, and neuroimaging of a group of transgenic HD and control monkeys from infancy to adulthood. Along with progressive cognitive and motor impairment, neuroimaging revealed a progressive reduction in striatal volume. Magnetic resonance spectroscopy at 48 months of age revealed a decrease of N-acetylaspartate (NAA), further suggesting neuronal damage/loss in the striatum. Postmortem neuropathological analyses revealed significant neuronal loss in the striatum. Our results indicate that HD monkeys share similar disease patterns with HD patients, making them potentially suitable as a preclinical HD animal model