Structural basis for ABC transporters involved in cellular stress responses
EMSL Project ID
50477
Abstract
ATP binding cassette (ABC) transporters act as vital cellular gatekeepers that both maintain the constitution of cellular barriers and control efflux of molecules across them. These molecules include critical compounds that either cause or help evade an external stress such as oxygen deprivation, nutrient deprivation, or drug treatment with severe consequences for human and environmental health through microbial pathogenesis and cancer invasion. For this proposal, we focus on two groups of unusual ABC transporters from bacteria and fungi that play roles in these stress responses. TmrAB and BmrCD are two bacterial transporters that are unusual because they lack a second functional nucleotide binding domain (NBD) found in many ABC transporters that is responsible for helping convert ATP binding to transport of substrates. With my co-mentor Yifan Cheng at UCSF, we published the TmrAB structure - first sub-nanometer structure of a transporter with the then newly developed direct electron detectors. This proposal aims to extend this work to understand both substrate coupling to TmrAB and how this translate to the entire pumping cycle, by trapping TmrAB in several intermediate states. We plan to complement this work with a conformational map of the similar transporter BmrCD, an ABC transporter from Bacillus subtilis that is well characterized multidrug resistance transporter. Our proposal also extends to higher order fungal infections, a pressing worldwide problem. We have collected significant preliminary data on two transporters, ABC protein involved in the Uptake of Sterols 1 (AUS1) and Yeast Cadmium Factors (YCF1), both transporters that provide protection from drug treatment and other stressors and make fungal infections extremely difficult to treat. In both AUS1 and YCF1, we have collected EM data to moderate resolution that reveals aspects of highly unusual ABC transporter biology in yeast. Our preliminary AUS1 class averages and 3D reconstruction suggests an unusual pairing of NBDs in a way that makes a pseudo 4-fold arrangement similar to a channel, an architecture never described to high resolution. AUS1 is also unusual in that it is an importer of sterols, a direction of transport rare for eukaryotic ABC transporters. These sterols are critical for response to hypoxia or antifungal treatment, two conditions that limit sterol biosynthesis, and our structure reveals a large central cavity that hints at a substrate binding site. Our YCF1 reconstructions hint at the location of a multi-substrate binding pocket, and single particles were amenable to high contrast imaging in vitreous ice on an FEI Talos Arctica microscope.
Altogether, our preliminary data for these transporters suggests a promising route to high-resolution reconstruction of these clinically important classes of transporters and will be greatly accelerated by access to the resources at the PNCC. Our proposed experiments to 1) map details of their highly unusual poly-specific binding sites and 2) trap them in different pumping intermediates will provide new avenues for understanding how they contribute to disease and how therapeutic intervention may target these stress responses and leave microbial infections more vulnerable to treatment.
Project Details
Start Date
2018-11-01
End Date
2019-01-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members