Essential Protein Factors in pre-mRNA splicing : A Structural Study by Nuclear Magnetic Resonance Spectroscopy

This thesis describes two novel three-dimensional structures and the functional characterization of proteins that play important roles in eukaryotic RNA splicing. These results are discussed in Chapters 1 and 2, while biomolecular NMR techniques that were employed for the structure determination are outlined in Chapter 3. Materials and methods are described in Chapter 4. Chapter 1 presents the solution structure of the Tudor domain of the human Survival of Motor Neuron (SMN) protein and its molecular interaction with the spliceosomal Sm proteins. Sm proteins are common components of small nuclear ribonucleoprotein particles (snRNPs), which are assembled by a protein complex that contains SMN. The structure of the SMN Tudor domain exhibits a five stranded ?-barrel, which resembles the fold of Sm proteins. The Tudor domain of SMN binds to arginine and glycine-rich C-terminal regions of Sm proteins, where it specifically recognizes symmetrically di-methylated arginine residues. The E134K mutant Tudor domain, which corresponds to a human mutation associated with Spinal Muscular Atrophy (SMA), is structurally intact but fails to interact with Sm proteins. This provides an explanation for a molecular defect underlying SMA. In Chapter 2, the structural basis for the molecular recognition between the essential splicing factors SF1 and U2 auxiliary factor 2 (U2AF) is provided. This interaction involves the third RNA recognition motif (RRM3) of the large subunit of U2AF (U2AF65) and the N-terminal 25 residues of SF1. The structure of RRM3 exhibits the classical RNP-type fold, but contains an additional C-terminal helix. SF1 is bound by the helical surface of RRM3, opposite of the canonical RNA binding site. The molecular recognition involves insertion of a conserved tryptophan of SF1 into a hydrophobic binding pocket of RRM3. This interaction is complemented by electrostatic contacts that are mediated by acidic residues of RRM3 and basic amino acids of SF1. Surprisingly, the molecular interface is highly similar to that between the large (U2AF65) and small (U2AF35) subunits of U2AF. This RRM-mediated protein interaction provides an example of how conserved structural folds have evolved different molecular functions

In-Cell Protein Structures from 2D NMR Experiments

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 μM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures

Real-time NMR monitoring of biological activities in complex physiological environments

Biological reactions occur in a highly organized spatiotemporal context and with kinetics that are modulated by multiple environmental factors. To integrate these variables in our experimental investigations of 'native' biological activities, we require quantitative tools for time-resolved in situ analyses in physiologically relevant settings. Here, we outline the use of high-resolution NMR spectroscopy to directly observe biological reactions in complex environments and in real-time. Specifically, we discuss how real-time NMR (RT-NMR) methods have delineated insights into metabolic processes, post-translational protein modifications, activities of cellular GTPases and their regulators, as well as of protein folding events.Fil: Smith, Matthew J.. Ontario Cancer Institute; CanadáFil: Marshall, Christopher B.. Ontario Cancer Institute; CanadáFil: Theillet, Francois Xavier. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Binolfi, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Selenko, Philipp. Leibniz Institute of Molecular Pharmacology; AlemaniaFil: Ikura, Mitsuhiko. Ontario Cancer Institute; Canadá. University of Toronto; Canad

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson’s disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c’s activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits

An NMR‐based biosensor to measure stereo‐specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.Fil: Sanchez Lopez, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Labadie, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lombardo, Veronica Andrea. Universidad Nacional de Rosario. Centro de Estudios Interdisciplinarios; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Biglione, Franco Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Manta, Bruno. Harvard Medical School; Estados UnidosFil: Jacob, Reeba. Weizmann Institute Of Science.; IsraelFil: Gladyshev, Vadim. Harvard Medical School; Estados UnidosFil: Abdelilah Seyfried, Salim. Universitat Potsdam; Alemania. Leibniz Universitat Hannover; AlemaniaFil: Selenko, Philipp. Weizmann Institute Of Science.; IsraelFil: Binolfi, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Quo Vadis Biomolecular NMR Spectroscopy?

In-cell nuclear magnetic resonance (NMR) spectroscopy offers the possibility to study proteins and other biomolecules at atomic resolution directly in cells. As such, it provides compelling means to complement existing tools in cellular structural biology. Given the dominance of electron microscopy (EM)-based methods in current structure determination routines, I share my personal view about the role of biomolecular NMR spectroscopy in the aftermath of the revolution in resolution. Specifically, I focus on spin-off applications that in-cell NMR has helped to develop and how they may provide broader and more generally applicable routes for future NMR investigations. I discuss the use of ‘static’ and time-resolved solution NMR spectroscopy to detect post-translational protein modifications (PTMs) and to investigate structural consequences that occur in their response. I argue that available examples vindicate the need for collective and systematic efforts to determine post-translationally modified protein structures in the future. Furthermore, I explain my reasoning behind a Quinary Structure Assessment (QSA) initiative to interrogate cellular effects on protein dynamics and transient interactions present in physiological environments

Structural biology outside the box-inside the cell

Recent developments in cellular cryo-electron tomography, in-cell single-molecule Förster resonance energy transfer-spectroscopy, nuclear magnetic resonance-spectroscopy and electron paramagnetic resonance-spectroscopy delivered unprecedented insights into the inner workings of cells. Here, we review complementary aspects of these methods and provide an outlook toward joint applications in the future

Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins

Abstract We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni 2? -chelated DO2A. In proton-detected 1 H-15 N SOFAST-HMQC and carbon-detected (H-flip) 13 CO-15 N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmolÁL -1 of the PRE agent, we obtain a 1.7-to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach