Structural studies of signal transduction in bacterial chemotaxis and a metabolism-related cancer target

Thesis

Transmission electron microscopy (TEM) has had a growing impact on the life sciences as both technological and methodological developments have allowed for cells and macromolecules to be resolved at increasingly higher resolutions. Variations of TEM can be used to visualize cellular ultrastructure as well as to determine macromolecular structure in a near-native state.

In this work, a number of TEM-based methods were utilized to structurally characterize proteins from different biological systems. The areas of study were the chemotaxis systems of Escherichia coli and Rhodobacter sphaeroides as well as human cancer metabolism. In each system, a key protein of interest was selected for investigation.

Bacteria use their chemotaxis systems to dynamically sense and respond to their environment by biasing their movements towards favorable directions. The E. coli serine chemotaxis receptor (Tsr) was studied using cryo-electron tomography of arrays formed by overexpressed Tsr in a thin E. coli cell strain and in membrane isolates. Subsequent sub-volume averaging yielded structures of the full-length receptor which showed that serine binding is accompanied by expansion of the signaling domain.

The protein selected for study in the R. sphaeroides chemotaxis systems, PpfA, is an ATPase responsible for segregating the cluster of cytoplasmic chemosensory proteins. Immuno-gold labeling of cryo-sections showed that both wild-type and mutant PpfA demonstrated some degree of clustering into foci.

The enzyme pyruvate kinase M2 (PKM2) plays an important role in various cancers by regulating metabolism to promote cell survival and propagation. Single particle cryo-electron microscopy was used to perform a structural comparison of wild-type PKM2 with a patient-derived mutant form of the protein. At the resolution of the structures solved, the molecular architecture of the patient and mutant forms of the protein were the same, except for the mutated residue. The structures revealed the potential presence of a stabilizing stacking interaction in the mutant that may explain differences in its biochemical behavior.

These studies illustrate the range of biological questions and systems to which TEM- based techniques can be applied with the goal of gaining structure-based mechanistic insights. For macromolecular targets of clinical relevance such as PKM2, such information can be used for rational drug design.

Authors: Venkatesh, M

• DOI: 10.5287/ora-xvgz0m25m
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford