The Rienstra Group has three Primary Interests in Structural Biology:
Alpha-Synuclein | Membrane Proteins | Blood Coagulation | Amphotericin
- Structural Dynamics of Protein Aggregation and Fibrillation
Parkinson's disease (PD) is the second most common neurodegenerative disease and one of the 15 most common causes of death in the USA. PD is pathologically characterized by the presence of Lewy Bodies (LB), aggregates found in the dopaminergic neurons during patients autopsy. In 1997, Spillantini et al. discovered that the main component of LB is fibrillar alpha-synuclein (AS). Subsequent studies demonstrated that AS in unstructured in solution and it binds to anionic phospholipids with an alpha-helical secondary structure. While most cases of PD are sporadic, three distinct mutations (A30P, E46K, A53T) in the AS gene48-50 have been identified in familial early-onset PD. Although all these findings implicate AS misfolding in PD and have motivate numerous investigations to understand the fibrils formation, up to now no high resolution structure has yet been published. MAS SSNMR spectroscopy provides high-resolution structural information of amyloidogenic fibrils, which are not accessible by traditional biophysical methods of solution NMR and X-ray diffraction.
Graphic summary of known alpha-synuclein structural information and hypothesis related to Parkinson's disease.
- Accelerating Membrane Protein Structure Determination
(DsbA/DsbB, cytochome bo3 oxidase, etc.)
Atomic-resolution structures of proteins advance our understanding of fundamental biological processes behind pathogenesis and provide a basis for rational design of therapeutics. Membrane proteins (MP) are especially important targets for structure elucidation, because they carry out many essential biological functions and thereby are targeted by ~60% of drugs. However, MPs are extraordinarily difficult to study in vitro. Interrogating the structure of MPs hinges on the ability to identify optimal conditions for expression, purification, crystallization and/or solubilization for NMR. Relatively few MP structures have been determined, and often the structures have moderate-to-low resolution (~2.5-4.0 Å) and/or missing or disordered portions, such as active site prosthetic groups. SSNMR methods can be applied in cases where sample lifetimes are too short, particle sizes too great, and/or multiple timescales of motion exist that cause broadening of the solution NMR spectra at typical data collection conditions.The membrane proteins that we are interested in are integral membrane proteins: DsbB and cytochrome bo3 (cyt bo3). DsbB (20 kDa) is responsible to reoxidize the periplasmic protein DsbA (21 kDa) to form a disulfide bond generation and transfer pathway that catalyzes protein folding in E. coli periplasm. Although DsbA/DsbB is not found in humans, its presence and role in the folding of virulence factors in several pathogenic bacteria make these proteins a potential target for therapeutics.Cyt bo3 is an integral MP that is the major terminal oxidase in the E. coli respiratory chain. The enzyme catalyzes the oxidation of ubiquinol and reduction of oxygen to water, and pumps protons across the membrane to generate a proton motive force. Cyt bo3 has a molecular weight of 144 kDa, contains four subunits and 23 transmembrane helices, and is a member of the heme/copper oxidase superfamily. DsbB and cyt bo3 share the same cofactor - ubiquinone (UQ-8), and thus the structural studies of the ubiquinone binding sites of DsbB and cytochrome bo3 provide insights into the redox states and interactions of ubiquinone with these MPs.
Pictorial representation of strucutral biology questions SSNMR is uniquely suited to address for these membrane protein complex systems.
- Membrane-Protein Interactions and Structural Rearrangementsof Blood Coagulation Proteins
(Calcium-induced Phosphatidylserine (PS) clustering, PS interactions with Tissue Factor and FVIIa)
Almost every step in blood coagulation requires assembly of multiple proteins on membrane surfaces, yet protein-membrane interactions in blood clotting remain poorly understood at the molecular level. We are using new, high-resolution technologies including magic-angle spinning solid-state NMR (SSNMR) to probe the mechanisms by which blood clotting proteins interact with phospholipid surfaces, and how changes in membrane composition regulate clotting reactions. The primary focus of our studies is the membrane-bound complex of tissue factor and factor VIIa, the two-subunit enzyme responsible for triggering blood clotting in health and disease. Disorders of the blood clotting system represent the leading cause of disability and death in the United States, but we still have a very incomplete understanding of blood clotting reactions at the molecular level. These studies will shed new light on the regulation of the blood clotting system, with a particular focus on achieving a detailed understanding of how and why blood clotting reactions occur on membrane surfaces.
Trama activates factor VIIa (FVIIa), which binds tissue factor (TF) to initiate the extrinsic blood clotting cascade. FVIIa binding is dependent on phosphatidylserine (PS) and the molecular details on these interactions are unknown.
- (Structures of Amphotericin-Sterol Complexes)
This project aims to advance understanding of the mode of action of the clinically vital but also highly toxic antifungal drug amphotericin B (AmB). Alternative to the classic ion channel model, preliminary studies in this proposal show that AmB forms large extramembranous aggregates that extract sterols from lipid bilayers and thereby kill cells. This novel "sterol sponge" model illuminates a new and more actionable roadmap to an improved therapeutic index, i.e., maximize the relative binding affinity of the AmB sterol sponge for the sterol found in fungi (ergosterol) vs. humans (cholesterol). Aim 1 is to determine the structure of the AmB sterol sponge, assembled in the presence of physiologically relevant lipid bilayers. Aim 2 is to determine the structures of the complexes of the AmB sponge with ergosterol and cholesterol. Aim 3 is to determine the structure of the sterol sponge and corresponding ergosterol complex derived from a new derivative of AmB, AmBMU, which was recently discovered and shown to bind ergosterol but not cholesterol, to be non-toxic to human cells, and to retain potent antifungal activity in vitro and in mice. Collectively, these studies wll provide a high-resolution picture of the atomistic interactions that underlie the biological activities of AmB and thus powerfully enable the rational development of less toxic derivatives of this clinically vital natural product. These studies will also further illuminate the fundamental features of how clinically validated resistance-refractory antimicrobial action can be achieved and lay the foundation for the frontier pursuit of other biologically relevant small molecule-small molecule interactions. Relevance to Human Health. Amphotericin B is the powerful but unfortunately highly toxic gold standard therapy for treatment of systemic fungal infections, and this drug has uniquely evaded the emergence of microbial resistance despite more than half a century of widespread clinical utilization. Better understanding how AmB exerts its biological activities is thus critical for guiding the rational development of derivatives with an improved therapeutic index as well as other resistance-refractory antimicrobial agents.
Check back soon as we add more about our progress in these endeavors.