C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.

Background: The need to restructure the world’s energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. Results: Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. Conclusions: Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast.