Predicting variation of DNA shape preferences in protein-DNA interaction in cancer cells with a new biophysical model

DNA shape readout is an important mechanism of target site recognition by transcription factors, in addition to the sequence readout. Several models of transcription factor-DNA binding which consider DNA shape have been developed in recent years. We present a new biophysical model of protein-DNA interaction by considering the DNA shape features, which is based on a neighbour dinucleotide dependency model BayesPI2. The parameters of the new model are restricted to a subspace spanned by the 2-mer DNA shape features, which allowing a biophysical interpretation of the new parameters as position-dependent preferences towards certain values of the features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across cancer cell lines and cellular conditions. We find evidence of DNA shape variations at FOXA1 binding sites in MCF7 cells after treatment with steroids. The new model is useful for elucidating finer details of transcription factor-DNA interaction. It may be used to improve the prediction of cancer mutation effects in the future

Evolutionary Covariant Positions within Calmodulin EF-hand Sequences Promote Ligand Binding

Intracellular calcium signaling is an essential regulatory mechanism through calcium-mediated signal transduction pathways involved in many cell processes, such as exocytosis, motility, apoptosis, excitability, transcription, and muscle contraction. The calcium-binding, ubiquitous, and highly conserved protein calmodulin (CaM) is an important regulator of hundreds of target proteins involved in cellular calcium signaling. CaM comprises of two pairs of EF-hand calcium-binding domains and these structural regions of the protein are highly conserved. Studying the molecular mechanisms underlying the binding of calcium to the EF-hands of CaM is critical in understanding the calcium-mediated cellular processes and how improper binding of calcium can lead to various human pathologies. Previous site-specific binding measurements indicate that each of the four EF-hands of CaM have distinct affinities for calcium. In this study, we have utilized covariance patterns and site-specific mutagenesis to analyze calcium affinity in the two EF-hands of the N-lobe of CaM in order to determine the specific amino acids that are evolutionarily conserved to coordinate calcium. The specific amino acids in CaM that we studied are theorized to coevolve, which means that in their protein coding genes, when a mutation occurs, a compensatory mutation is likely to follow to conserve structure and function of CaM. Since CaM is a highly conserved protein with a known structure, covariance analyses will help in understanding which amino acid contacts are most important for the coordination of calcium in the EF-hands of CaM and to determine which amino acids are under evolutionary constraint. Covariance algorithms, multiple sequence analyses and accompanied protein structure analyses were used to identify the two high scoring amino acid pairs in the N-lobe EF-hands: positions 22 and 24 in EF-hand site 1 and positions 58 and 60 in EF-hand site 2. The amino acids in these locations were mutated and accompanied calcium binding was measured to better understand the effects of the mutations on calcium binding. We have found that both the D24N mutation in site 1 and the D58N mutation in site 2 disrupt binding likely due to the removal of a necessary aspartate in the binding site. However, the combined D58N and N60D mutations restore binding in site 2 by providing the necessary aspartate in the covariant location. The N60D mutation by itself has little impact on calcium binding in site 2. Therefore, it is evident that evolution conserves at least one aspartate in the covariant positions of the binding site and the presence of two aspartates in the covariant positions of the binding site has little affect on calcium binding. We are currently studying the covariant positions in site 1 and future work includes structurally analyzing the covariant positions in the C-lobe of CaM and studying covariance patterns of other calcium-binding proteins with EF-hand binding domains.Biochemistr

Synthetic biology—putting engineering into biology

Synthetic biology is interpreted as the engineering-driven building of increasingly complex biological entities for novel applications. Encouraged by progress in the design of artificial gene networks, de novo DNA synthesis and protein engineering, we review the case for this emerging discipline. Key aspects of an engineering approach are purpose-orientation, deep insight into the underlying scientific principles, a hierarchy of abstraction including suitable interfaces between and within the levels of the hierarchy, standardization and the separation of design and fabrication. Synthetic biology investigates possibilities to implement these requirements into the process of engineering biological systems. This is illustrated on the DNA level by the implementation of engineering-inspired artificial operations such as toggle switching, oscillating or production of spatial patterns. On the protein level, the functionally self-contained domain structure of a number of proteins suggests possibilities for essentially Lego-like recombination which can be exploited for reprogramming DNA binding domain specificities or signaling pathways. Alternatively, computational design emerges to rationally reprogram enzyme function. Finally, the increasing facility of de novo DNA synthesis—synthetic biology’s system fabrication process—supplies the possibility to implement novel designs for ever more complex systems. Some of these elements have merged to realize the first tangible synthetic biology applications in the area of manufacturing of pharmaceutical compounds.