Structural basis for interactions of microtubule-associated proteins with microtubules by magic angle spinning NMR spectroscopy and methodological developments for biomolecular applications
Date
2017
Authors
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Publisher
University of Delaware
Abstract
Microtubules (MTs) are an important cytoskeletal component involved in various cellular activities, including intracellular transport, cell motility, maintenance of cell shape and cell organization, and separation of chromosomes during mitosis. Their associated proteins are vital to these microtubule-based functions by regulating the MT dynamics or mediating the interactions between MTs and other organelles. Despite studies with different biophysical or biochemical methods, there is a significant gap between high-resolution molecular structures and the knowledge of mechanisms of microtubule-based transport, interactions between MTs and microtubule-associated proteins (MAPs), and how the malfunctions of MAPs occur. To understand these mechanisms at a molecular level, the atomic-level structures and dynamics of MAPs and the structural basis for their interactions with MTs are needed. Due to the large molecular size, insolubility, dynamic features, and a lack of long-range order of the MT/MAPs assemblies, the most ubiquitously used structural biology techniques, such as X-ray crystallography and solution nuclear magnetic resonance (NMR) spectroscopy, are inapplicable. ☐ Magic-angle spinning (MAS) NMR is a powerful technique for gaining a structural and dynamic basis for macromolecular assemblies because it has no limitation on molecular weight, long-range order, or solubility. Therefore, it is particularly suitable for application in MTs/MAPs assemblies. In this dissertation, we utilize magic-angle spinning NMR as the key method to study structural characteristics of several microtubule-associated binding proteins in complex with microtubules, with the long-term goal of deciphering the mechanisms of various MT-related processes. ☐ This research especially focuses on the structures and dynamics of the CAP-Gly domain in the p150Glued subunit of dynactin in complex with MTs using MAS NMR spectroscopy. We determined intermolecular interfaces formed by CAP-Gly and MTs and other binding partners through comprehensive MAS NMR methods. In particular, we incorporated rotational echo double resonance (REDOR) and double-REDOR (dREDOR) filters into homo- and heteronuclear correlation experiments. These approaches are critical when one of the binding partners cannot be isotopically labeled with magnetically active nuclei, such as in the case of mammalian microtubules. Our results reveal that the binding interfaces of CAP-Gly with MTs and with EB1 partly overlap. Integration of the interface with the three-dimensional (3D) structure of CAP-Gly in MT-bound state led to the first atomic-resolution structure of a microtubule-associated protein on polymerized microtubules. The novel application of the MAS NMR approaches introduced in this work is an example of discovering the binding interactions between MAPs and MTs as well as interactions between other cytoskeleton structures and their associated proteins. ☐ The dREDOR is an alternative approach that we explore for gaining atomic-resolution information into the interfaces formed by U-13C,15N-CAP-Gly and natural abundance microtubules. In this work, we present an in-depth analysis of the dREDOR-based experiments. First we establish the dREDOR dephasing dynamics in a series of one-dimensional (1D) dREDOR-CPMAS experiments on two control samples, U-13C,15N-Histidine and U-13C,15N-dynein’s light chain 8 protein (LC8). We discuss the effects of dREDOR on quasi-uniform dipolar dephasing during the filter time in both samples. We then apply dREDOR to probe the intermolecular binding interface formed by U-13C,15N-CAP-Gly in complex with unlabeled end-binding 1 protein (EB1). Our results establish the general requirements for dREDOR-based experiments that are applicable to a wide variety of protein complexes. ☐ Fast MAS NMR has been widely applied in biomolecules because it yields large increases in resolution and sensitivity when used in combination with proton detection. However, heteronuclear detection under fast MAS (frequencies of up to 65 kHz) is sometimes advantageous, as it results in shorter experiment times. We demonstrate heteronuclear-detected dipolar-based 3D correlation spectroscopy for assignments of 1H, 13C, and 15N resonances and structural analysis in fully protonated proteins by applying this approach to an 89-residue microtubule-associated protein, LC8, an integral subunit of cytoplasmic dynein. The sensitivity and resolution of 3D HNCA, HNCO, and HNCOCX experiments are excellent, and with these three datasets, we assign the majority of the backbone and a number of side-chain 1H/13C/15N resonances in dynein’s LC8. Furthermore, we resolve many ambiguities that were persistent in our previous studies using moderate MAS frequencies and lacking the 1H dimension. In addition, we present an experiment employing a combined R-driven and radiofrequency-driven recoupling (CORD-RFDR) sequence that yields long-range cross peaks and gives rise to enhanced cross-peak intensities across the entire correlation spectrum. We demonstrate the applications of two-dimensional (2D) CORD-RFDR correlation spectroscopy in dynein’s LC8. The results reveal that the CORD-RFDR experiment is beneficial for homonuclear correlation spectroscopy in a broad range of conditions, including both moderate and high magnetic fields and MAS frequencies. ☐ In the final chapter, we discuss our preliminary studies on a p150Glued fragment, the CAP-Gly1-191 (extended CAP-Gly), which contains basic domain, serine-proline rich domain, and CAP-Gly domain. We summarize the preparation protocols of CAP-Gly1-191 protein, biochemical characterization of interactions between CAP-Gly1-191 and microtubules as well as the initial characterization of the protein by both solution and solid-state NMR spectroscopy. The results lay the foundation for future structural determination of the extended CAP-Gly construct and investigation of its binding interface with MTs.
Description
Keywords
CAP-Gly domain, Intermolecular interface, MAS NMR spectroscopy, Microtubule-associated proteins, Microtubules