The molecular chaperone HdeA is a stress-specific chaperone that is rapidly activated by low pH-conditions to protect proteins against widespread acid-induced protein aggregation. This stress specific activation of HdeA is essential for pathogenic bacteria such as E. coli to survive the low pH antimicrobial environment of the stomach. HdeA senses the stress conditions at the protein level and responds to it with its own very rapid unfolding, making HdeA a member of a new class of chaperones, which need to lose structure to gain activity. Similar stress-specific activation by partial unfolding has been recenty reported for other ATP-independent chaperones as well, suggesting that this mechanism may represent a new paradigm in the field of chaperones and intrinsically disordered proteins. We will now use a combination of structural, biochemical and mutational tools to elucidate i) how HdeA becomes activated, ii) the features that allow HdeA to bind to a wide variety of different client proteins under stress conditions and iii) the mechanism by which it supports refolding of its client proteins upon return to non-stress conditions. These studies will reveal the answers to two very general unanswered questions: how proteins bind multiple unrelated client proteins, and how chaperones interact with client proteins to facilitate their refolding. Thus this work will simultaneously address fundamental and as yet unresolved questions in two major fields, the field of molecular recognition and the field of chaperone action. HdeA is ideally suited for these studies. It is a small 10 kDa intrinsically disordered chaperone, which is highly amendable to structural analysis by NMR. It forms very stable interactions with a number of unrelated small client proteins with known NMR structures, and HdeA supports client refolding in the absence of co- chaperones or energy sources. Moreover, client binding and client release from HdeA is easily and precisely controlled by simple pH shifts. We will perform detailed NMR residue-level studies of dynamics and disorder in HdeA as a function of pH to assess how pH-changes are used for the controlled unfolding and activation of a chaperone. We will conduct residue-level NMR studies on HdeA and client proteins to simultaneously determine how HdeA impacts its client proteins and how client proteins affect HdeA. We will use in vivo folding biosensors to directly select for HdeA mutants with altered flexibility to assess the role that flexibility playsin chaperone function, and we will conduct refolding studies with select folding-mutants of HdeA's client protein immunity protein 7 to determine how HdeA promotes client refolding. Together, these studies will enable us to characterize the molecular mechanism that underlies HdeA's chaperone activity and test the hypothesis that flexibility is required for HdeA's molecular recognition function. With these studies, we now have an unprecedented opportunity to understand, in molecular detail, both the mechanism of chaperone action and the role of disorder in protein function and molecular recognition.
Stomach acid aids in food digestion and helps kill disease-causing bacteria, partly by causing bacterial proteins to lose structure and function. Escherichia coli protect itself against the damaging effects of stomach acid by using the small, acid-activated chaperone HdeA. We will now study how this chaperone is activated and facilitates protein folding;specifically we will study how acid activates HdeA and elucidate the mechanism by which HdeA binds proteins under low pH conditions, and releases them once neutralizing conditions are restored for efficient refolding.
|Jakob, Ursula; Kriwacki, Richard; Uversky, Vladimir N (2014) Conditionally and transiently disordered proteins: awakening cryptic disorder to regulate protein function. Chem Rev 114:6779-805|
|Groitl, Bastian; Jakob, Ursula (2014) Thiol-based redox switches. Biochim Biophys Acta 1844:1335-43|