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The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

Background Docking large ligands, and especially peptides, to protein receptors is still considered a challenge in computational structural biology. Besides the issue of accurately scoring the binding modes of a protein-ligand complex produced by a molecular docking tool, the conformational sampling of a large ligand is also often considered a challenge because of its underlying combinatorial complexity. In this study, we evaluate the impact of using parallelized and incremental paradigms on the accuracy and performance of conformational sampling when docking large ligands. We use five datasets of protein-ligand complexes involving ligands that could not be accurately docked by classical protein-ligand docking tools in previous similar studies. Results Our computational evaluation shows that simply increasing the amount of conformational sampling performed by a protein-ligand docking tool, such as Vina, by running it for longer is rarely beneficial. Instead, it is more efficient and advantageous to run several short instances of this docking tool in parallel and group their results together, in a straightforward parallelized docking protocol. Even greater accuracy and efficiency are achieved by our parallelized incremental meta-docking tool, DINC, showing the additional benefits of its incremental paradigm. Using DINC, we could accurately reproduce the vast majority of the protein-ligand complexes we considered. Conclusions Our study suggests that, even when trying to dock large ligands to proteins, the conformational sampling of the ligand should no longer be considered an issue, as simple docking protocols using existing tools can solve it. Therefore, scoring should currently be regarded as the biggest unmet challenge in molecular docking.

Background It has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis. Results Combining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics. Conclusions These data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Background Leucine-rich-repeat receptor-like kinases (LRR-RLKs) play central roles in sensing various signals to regulate plant development and environmental responses. The extracellular domains (ECDs) of plant LRR-RLKs contain LRR motifs, consisting of highly conserved residues and variable residues, and are responsible for ligand perception as a receptor or co-receptor. However, there are few comprehensive studies on the ECDs of LRR-RLKs due to the difficulty in effectively identifying the divergent LRR repeats. Results In the current study, an efficient LRR motif prediction program, the “Phyto-LRR prediction” program, was developed based on the position-specific scoring matrix algorithm (PSSM) with some optimizations. This program was trained by 16-residue plant-specific LRR-highly conserved segments (HCS) from LRR-RLKs of 17 represented land plant species and a database containing more than 55,000 predicted LRRs based on this program was constructed. Both the prediction tool and database are freely available at http://phytolrr.com/ for website usage and at http://github.com/phytolrr for local usage. The LRR-RLKs were classified into 18 subgroups (SGs) according to the maximum-likelihood phylogenetic analysis of kinase domains (KDs) of the sequences. Based on the database and the SGs, the characteristics of the LRR motifs in the ECDs of the LRR-RLKs were examined, such as the arrangement of the LRRs, the solvent accessibility, the variable residues, and the N-glycosylation sites, revealing a comprehensive profile of the plant LRR-RLK ectodomains. Conclusion The “Phyto-LRR prediction” program is effective in predicting the LRR segments in plant LRR-RLKs, which, together with the database, will facilitate the exploration of plant LRR-RLKs functions. Based on the database, comprehensive sequential characteristics of the plant LRR-RLK ectodomains were profiled and analyzed.

Background TgDCX is a doublecortin-domain protein associated with the conoid fibers, a set of strongly curved non-tubular tubulin-polymers in Toxoplasma . TgDCX deletion impairs conoid structure and parasite invasion. TgDCX contains two tubulin-binding domains: a partial P25α and the DCX/doublecortin domain. Orthologues are found in apicomplexans and their free-living relatives Chromera and Vitrella . Results We report that isolated TgDCX-containing conoid fibers retain their pronounced curvature, but loss of TgDCX destabilizes the fibers. We crystallized and determined the 3D-structure of the DCX-domain, which is similar to those of human doublecortin and well-conserved among TgDCX orthologues. However, the orthologues vary widely in targeting to the conoid in Toxoplasma and in modulating microtubule organization in Xenopus cells. Several orthologues bind to microtubules in Xenopus cells, but only TgDCX generates short, strongly curved microtubule arcs. EM analysis shows microtubules decorated with TgDCX bundled into rafts, often bordered on one edge by a “C”-shaped incomplete tube. A Chromera orthologue closely mimics TgDCX targeting in Toxoplasma and binds to microtubules in Xenopus cells, but does not generate arcs or “C”-shaped tubes, and fails to rescue the defects of the TgDCX-knockout parasite. Conclusions These observations suggest that species-specific features of TgDCX enable it to generate strongly curved tubulin-polymers to support efficient host-cell invasion.

Background Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail. Results In this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression ( R 2  = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles. Conclusions Our study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome.

Advances in the study of chromosome conformation capture technologies, such as Hi-C technique - capable of capturing chromosomal interactions in a genome-wide scale - have led to the development of three-dimensional chromosome and genome structure reconstruction methods from Hi-C data. The three dimensional genome structure is important because it plays a role in a variety of important biological activities such as DNA replication, gene regulation, genome interaction, and gene expression. In recent years, numerous Hi-C datasets have been generated, and likewise, a number of genome structure construction algorithms have been developed. In this work, we outline the construction of a novel Genome Structure Database (GSDB) to create a comprehensive repository that contains 3D structures for Hi-C datasets constructed by a variety of 3D structure reconstruction tools. The GSDB contains over 50,000 structures from 12 state-of-the-art Hi-C data structure prediction algorithms for 32 Hi-C datasets. GSDB functions as a centralized collection of genome structures which will enable the exploration of the dynamic architectures of chromosomes and genomes for biomedical research. GSDB is accessible at http://sysbio.rnet.missouri.edu/3dgenome/GSDB

Background Type A influenza viruses are contagious and even life-threatening if left untreated. So far, no broadly protective vaccine is available due to rapid antigenic changes and emergence of new subtypes of influenza virus. In this study, we exploited bioinformatics tools in order to design a subunit chimeric vaccine from the antigenic and highly conserved regions of HA and M2 proteins of H7N9 subtype of influenza virus. We used mucosal adjuvant candidates, including CTxB, STxB, ASP-1, and LTB to stimulate mucosal immunity and analyzed the combination of HA2, M2e, and the adjuvant. Furthermore, to improve the antigen function and to maintain their three-dimensional structure, 12 different linkers including six rigid linkers and six flexible linkers were used. The 3D structure model was generated using a combination of homology and ab initio modeling methods and the molecular dynamics of the model were analyzed, either. Results Analysis of different adjuvants showed that using CtxB as an adjuvant, results in higher overall vaccine stability and higher half-life among four adjuvant candidates. Fusion of antigens and the CTxB in the form of M2e-linker-CTxB-linker-HA2 has the most stability and half life compared to other combination forms. Furthermore, the KPKPKP rigid linker showed the best result for this candidate vaccine among 12 analyzed linkers. The changes in the vaccine 3D structure made by linker insertion found to be negligible, however, although small, the linker insertion between the antigens causes the structure to change slightly. Eventually, using predictive tools such as Ellipro, NetMHCpan I and II, CD4episcore, CTLpred, BepiPred and other epitope analyzing tools, we analyzed the conformational and linear epitopes of the vaccine. The solubility, proteasome cleavage sites, peptidase and potential chemical cutters, codon optimization, post translational modification were also carried out on the final vaccine. Conclusions It is concluded that M2e-Linker-CTxB-Linker-HA2 combination of chimeric vaccine retains its 3D structure and antigenicity when KPKPKP used as linker and CTxB used as adjuvant.

Background The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called ‘Point-of-Care tests’. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited. Results We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp . The enzyme showed optimal polymerase activity at pH 8–9 and 25–110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25–37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes. Conclusions Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

Background Transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown. Results In this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria. Conclusions This study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.