Structural and thermodynamic studies of Bergerac-SH3 chimeras

Bergerac-type chimeras of spectrin SH3 were designed by extending a β-hairpin by eight amino acids so that the extension protruded from the domain body like a “nose” being exposed to the solvent. A calorimetric study of several Bergerac-SH3 variants was carried out over a wide range of pH values and protein concentrations and the three-dimensional structure of one of them, SHH, was determined. X-ray studies confirmed that the nose had a well defined β-structure whilst the chimera formed a stable tetramer within the crystal unit because of four tightly packed noses. In the pH range of 4–7 the heat-induced unfolding of some chimeras was complex and concentration dependent, whilst at pH values below 3.5, low protein concentrations of all the chimeras studied, including SHH, seemed to obey a monomolecular two-state unfolding model. The best set of data was obtained for the SHA variant, the unfolding heat effects of which were systematically higher than those of the WT protein (about 16.4 kJ/mol at 323 K), which may be close to the upper limit of the enthalpy gain due to 10 residue β-hairpin folding. At the same time, the chimeras with high nose stability, which, like SHH, have a hydrophobic (IVY) cluster on their surface, showed a lower apparent unfolding heat effect, much closer to that of the WT protein. The possible reasons for this difference are discussed.     

Disentangling host,pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogen''s distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.     

Crystal structures of mutant ribosomal proteins L1

Nine mutant ribosomal proteins L1 from the bacterium Thermus thermophilus and archaeon Methanococcus jannaschii were obtained and their crystal structures were determined and analyzed. The structure of the S179C TthL1 mutant, determined earlier, was also analyzed. In half of the proteins studied, point mutations of the amino acid residues exposed on the protein surface essentially changed the spatial structure of the protein. This proves that a correct study of biological processes with the help of site-directed mutagenesis requires a preliminary determination or, at least, modeling of the structures of mutant proteins. A detailed comparison of the structures of the L1 mutants and the corresponding wild-type L1 proteins demonstrated that the side chain of a mutated amino acid residue tends to adopt a location similar to that of the side chain of the corresponding residue in the wild-type protein. This observation assists in modeling the structure of mutant proteins.     

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.     

Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases

Spider venom sphingomyelinases D catalyze the hydrolysis of sphingomyelin via an Mg(2+) ion-dependent acid-base catalytic mechanism which involves two histidines. In the crystal structure of the sulfate free enzyme determined at 1.85A resolution, the metal ion is tetrahedrally coordinated instead of the trigonal-bipyramidal coordination observed in the sulfate bound form. The observed hyperpolarized state of His47 requires a revision of the previously suggested catalytic mechanism. Molecular modeling indicates that the fundamental structural features important for catalysis are fully conserved in both classes of SMases D and that the Class II SMases D contain an additional intra-chain disulphide bridge (Cys53-Cys201). Structural analysis suggests that the highly homologous enzyme from Loxosceles bonetti is unable to hydrolyze sphingomyelin due to the 95Gly-->Asn and 134Pro-->Glu mutations that modify the local charge and hydrophobicity of the interfacial face. Structural and sequence comparisons confirm the evolutionary relationship between sphingomyelinases D and the glicerophosphodiester phosphoesterases which utilize a similar catalytic mechanism.     

Structural Studies of Component of Lysoamidase Bacteriolytic Complex from <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL1

The lysoamidase bacteriolytic complex (LBC) comprising five enzymes (L1–L5) is secreted into the culture liquid by gram-negative bacterium Lysobacter sp. XL1. The medicinal agent lysoamidase has a broad-antimicrobial spectrum. Bacteriolytic protease L1 belongs to the LBC. Recombinant L1 protease of Lysobacter sp. XL1 was expressed, purified to homogeneity and crystallized. The X-ray structure of L1 at 1.35 Å resolution has been determined using the synchrotron data and the molecular replacement method. L1 protease is a thermostable whose thermal unfolding proceeds in one step without forming stable intermediates. Structural information concerning L1 will contribute to the development of new-generation antimicrobial drugs, whose application will not be accompanied by the selection of resistant microorganisms.     

Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.     

Evolution of protein 3D structures as diffusion in multidimensional conformational space

A theory of protein spatial-structure evolution in terms of random walks in multidimensional conformational space is proposed. It is shown that the spatial divergence in pairs of homologous proteins depends only on their sequence similarity and is independent of the protein size. X-ray data are reasonably well described in terms of the theory developed. Correspondence to: A.M. Gutin     

Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy.     

A highly parallel strategy for storage of digital information in living cells

Background

Encoding arbitrary digital information in DNA has attracted attention as a potential avenue for large scale and long term data storage. However, in order to enable DNA data storage technologies there needs to be improvements in data storage fidelity (tolerance to mutation), the facility of writing and reading the data (biases and systematic error arising from synthesis and sequencing), and overall scalability.

Results

To this end, we have developed and implemented an encoding scheme that is suitable for detecting and correcting errors that may arise during storage, writing, and reading, such as those arising from nucleotide substitutions, insertions, and deletions. We propose a scheme for parallelized long term storage of encoded sequences that relies on overlaps rather than the address blocks found in previously published work. Using computer simulations, we illustrate the encoding, sequencing, decoding, and recovery of encoded information, ultimately demonstrating the possibility of a successful round-trip read/write. These demonstrations show that in theory a precise control over error tolerance is possible. Even after simulated degradation of DNA, recovery of original data is possible owing to the error correction capabilities built into the encoding strategy. A secondary advantage of our method is that the statistical characteristics (such as repetitiveness and GC-composition) of encoded sequences can also be tailored without sacrificing the overall ability to store large amounts of data. Finally, the combination of the overlap-based partitioning of data with the LZMA compression that is integral to encoding means that the entire sequence must be present for successful decoding. This feature enables inordinately strong encryptions. As a potential application, an encrypted pathogen genome could be distributed and carried by cells without danger of being expressed, and could not even be read out in the absence of the entire DNA consortium.

Conclusions

We have developed a method for DNA encoding, using a significantly different fundamental approach from existing work, which often performs better than alternatives and allows for a great deal of freedom and flexibility of application.