Molecular level regulation of BIP, a central molecular chaperone in the ER

The molecular chaperone BIP (Binding Immunoglobulin Protein, or Grp78, or Hsp5A) is the only Hsp70 chaperone in the endoplasmic reticulum (ER), a cellular organelle acting as a manufacturing and packaging site for one-third of cellular proteins (including the majority of secreted and membrane proteins). BIP is a central chaperone in the ER, which assists in protein synthesis, folding, maturation and degradation in the ER. Growing evidence suggests that the regulation of BIP activity can result in therapeutic benefits for diseases associated with problems in protein folding (e.g., Alzheimer's and Parkinson's diseases, diabetes, and cardiovascular diseases). Moreover, many cancer cells are addicted to BIP and can be treated by withdrawing BIP activity, suggesting that the regulation of BIP activity is a rational and highly attractive way to treat and/or prevent several devastating pathological processes.

How to regulate the chaperone activity of BIP is a crucial unresolved question that is essential for our fundamental understanding of this chaperone system and the future development of pharmacological tools. BIP is an ATP dependent machine that continuously binds and realizes unfolded (or misfolded) proteins to rescue them from aggregation and promote correct folding. 70 kDa BIP consists of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD), which communicate with each other to mutually regulate substrate binding and ATP hydrolysis. How this communication occurs has yet to be exposed.

To obtain a detailed mechanistic understanding of this ATP-dependent chaperone machine and its interdomain communication, we will use cutting-edge advances in biomolecular nuclear magnetic resonance (NMR) spectroscopy, thereby allowing site-specific characterization of changes in chaperone structure and dynamics. We will also utilize isothermal titration calorimetry (ITC), which provides thermodynamic features for these changes and computational molecular dynamics (MD), which facilitate the analysis and interpretation of experimental data. A synergy of these state-of-the-art techniques will result in detailed characterization of unique chaperone structural and dynamic features responsible for the regulation of ATP hydrolysis and the affinity of substrate binding.

We will next characterize the mechanistic basis on how physiological factors control and fine-tune BIP ATPase activity and substrate binding and release to achive the most effective protein folding in the constantly changing ER environment. We will utilize NMR, ITC and MD to elucidate how changes in the Ca2+ concentration and post-translational modifications affect BIP structure and dynamics and finally, how these structural and dynamic perturbations are coupled with changes in BIP ATPase activity and substrate binding.

We will further exploit electrospray ionisation-mass spectrometry coupled with ion mobility spectrometry (ESI-IMS-MS), DMSO-quenched NMR H/D exchange and real-time methyl NMR to examine the molecular mechanisms of BIP oligomerization-a unique physiological process for the reversible regulation of BIP activity upon fluctuations in concentrations of unfolded proteins and/or ATP. We will characterize size, shape and structural organisation of oligomeric BIP species and elucidate why and how substrate and ATP binding stabilize the active monomeric form of BIP.

To perform its functions in the living cells, BIP always collaborates with two types of co-chaperones, J-domain proteins and a nucleotide exchanges factors, which significantly enhance BIP activity. To elucidate the role of BIP co-chaperones, in the last part of the project, we will utilize methyl NMR to monitor how BIP, governed by its two co-chaperones (ERj3 and Grp170), binds and releases its authentic protein, an intrinsically disordered CH1 domain of antibodies. We will thus 'watch' the BIP chaperone machinery in action in real time with the atomic resolution.

Molecular chaperones are ubiquitous gatekeepers of protein homeostasis, capable to maintain protein folding in a constantly changing environment. Being in the highest demand, the characterization of these complex molecular machines is extremely challenging due to their large size and the co-existence of several functionally and structurally distinct conformations. We propose to synergize recent advances in solution NMR, mass spectrometry and molecular dynamic simulations to elucidate the mechanisms of regulation in the BIP molecular chaperone. To this end, we will utilize:

(a) Methyl 2D and multidimensional NMR, which allows for detailed characterization of large systems (up to 500 kDa); relaxation dispersion NMR, which provides information about us-ms conformational transitions; and time-resolved 2D NMR with non-uniform sampling, which enables the characterization of functional processes on the minutes-to-hours time-scale;

(b) DMSO-quenched H/D exchange NMR experiments, which will be used to obtain residue-specific deuterium incorporation patterns for large systems (e.g., in oligomers), and, thus, characterize oligomerization interfaces;

(c) Electrospray ionisation (ESI) non-covalent mass spectrometry (MS) coupled with ion mobility spectrometry (IMS), which will be exploited to characterize conformational properties of individual oligomeric conformations (i.e., size, shape, and population);

(b) Full-atom and coarse-grained molecular dynamics (MD), which will be used to explore conformational space and characterize conformational transitions, thereby facilitating the interpretation of experimental data.

The proposed approach can be easily applied for the characterization of other multidomain proteins and even more complex systems (e.g., large protein-protein complexes). During the last stage of this project, as proof of principles, we will test this approach for the characterization of communication between BIP and its co-chaperones.

Scientific beneficiaries:
The mechanistic understanding of function and specific regulation of BIP chaperone activity is of great biophysical and biomedical interest. Despite its biomedical importance, little is known about molecular mechanisms of the complex and multilevel regulation of BIP. Our project will address the key fundamental questions regarding how BIP activity can be regulated in the ER environment, thereby generating a wealth of new, significant information concerning the specific molecular mechanisms of action and regulation for this important molecular chaperone. In addition, we will develop and make widely available for future research several experimental approaches for the characterization of conformational changes in large multidomain and multicomponent protein systems. The results will be of interest to a broad spectrum of life-scientists, pharmacists and medical researchers in both academia and industry. To maximize the impact of the research, we have requested funding to organize a one-day international workshop in Leeds, which will focus on new biophysical methodologies developed for structural characterization of multidomain proteins.

Industrial beneficiaries:
We expect to develop long lasting industrial impact through the fundamental scientific insights obtained. Indeed, the understanding of specific mechanisms of BIP activation is a project of high importance. Our experimental developments will open up a new area of designing of allosteric drugs that specifically target the BIP system, which currently cannot be exploited. Thus, the pharmaceutical industry would benefit significantly; in the longer term, this would lead to new and improved medicines. For the last decade ACSMB has built strong collaborations with pharmaceutical and biomedical companies, culminating in the establishment of the pharmaceutical and biopharmaceutical innovation hub within the University with an industrial advisory board that includes 18 industrial partners. We will use the hub to promote our research and establish suitable industrial collaborators if/when appropriate.

Delivering highly skilled people:
The proposed project will train a PDRA in a wide range of cutting-edge technologies, including cutting-edge biomolecular nuclear magnetic resonance, mass spectrometry, molecular dynamic simulations, the handling and analysis of large multidomain protein systems, and a range of other biophysical, biochemical and molecular biology techniques. In addition, this multidisciplinary approach will significantly raise the PDRA's mathematical and computational skills and will facilitate implementations of these skills to tackle challenging biomedical problems. Altogether, this will prepare the PDRA for a successful future career, whether in academia or industry. At Leeds, we actively promote research-led teaching for both undergraduate and postgraduate students. Hence, progress made in the molecular chaperone field would enhance many aspects of the teaching delivered as well as contribute to the students' learning experience. In terms of our own research, once the experimental tools have been developed, we intend to exploit them to study other large protein systems of biological and medical interest. Thus, we anticipate opportunities to apply for further grant funding from research councils and charities.

Wider impacts for the general public:
BiP has been identified as a target for pharmaceutical intervention in prevalent diseases including neurodegenerative diseases, diabetes and cancers. Improved knowledge of molecular chaperone BIP will lead to improved drug developments, which will improve the quality of life.