Yohei Miyanoiri

Myosin VI Undergoes Cargo-Mediated Dimerization

Myosin VI is the only known molecular motor that moves toward the minus ends of actin filaments; thus, it plays unique roles in diverse cellular processes. The processive walking of myosin VI on actin filaments requires dimerization of the motor, but the protein can also function as a nonprocessive monomer. The molecular mechanism governing the monomer-dimer conversion is not clear. We report the high-resolution NMR structure of the cargo-free myosin VI cargo-binding domain (CBD) and show that it is a stable monomer in solution. The myosin VI CBD binds to a fragment of the clathrin-coated vesicle adaptor Dab2 with a high affinity, and the X-ray structure of the myosin VI CBD in complex with Dab2 reveals that the motor undergoes a cargo-binding-mediated dimerization. The cargo-binding-induced dimerization may represent a general paradigm for the regulation of processivity for myosin VI as well as other myosins, including myosin VII and myosin X.

Expression and purification of a GRAS domain of SLR1, the rice della protein

GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in Escherichia coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.

Use of H/D isotope effects to gather information about hydrogen bonding and hydrogen exchange rates

Polar side-chains in proteins play important roles in forming and maintaining three-dimensional structures, and thus participate in various biological functions. Until recently, most protein NMR studies have focused on the non-exchangeable protons of amino acid residues. The exchangeable protons attached to polar groups, such as hydroxyl (OH), sulfhydryl (SH), and amino (NH2) groups, have mostly been ignored, because in many cases these hydrogen atoms exchange too quickly with water protons, making NMR observations impractical. However, in certain environments, such as deep within the hydrophobic interior of a protein, or in a strong hydrogen bond to other polar groups or interacting ligands, the protons attached to polar groups may exhibit slow hydrogen exchange rates and thus become NMR accessible. To explore the structural and biological implications of the interactions involving polar side-chains, we have developed versatile NMR methods to detect such cases by observing the line shapes of (13)C NMR signals near the polar groups, which are affected by deuterium-proton isotope shifts in a mixture of H2O and D2O. These methods allow the detection of polar side-chains with slow hydrogen-deuterium exchange rates, and therefore provide opportunities to retrieve information about the polar side-chains, which might otherwise be overlooked by conventional NMR experiments. Future prospects of applications using deuterium-proton isotope shifts to retrieve missing structural and dynamic information of proteins are discussed.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13C-methyl labeled leucines and valines, instead of the commonly used 13C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Molecular bases of multimodal regulation of a fungal transient receptor potential (TRP) channel

Background: Multimodality of TRP channels underlies their diverse physiological functions. Results: We identified a fungal multimodal TRP channel whose cytosolic domain (CTD) mediates various channel regulation. Conclusion: CTD has an oligomerization module critical for osmoreception, yet its flexible structure allows dynamic regulations with other functional modalities. Significance: This work proposes structural and biophysical principles for multimodality of a TRP channel family member. Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca(2+) elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Stereo-Array Isotope Labeling Method for Studying Protein Structure and Dynamics

The stereo-array isotope labeling (SAIL) method utilizes proteins with isotope labeling patterns optimized with regard to an intended NMR study. The SAIL proteins are prepared by incorporating chemically synthesized amino acids into target proteins, using a cell-free protein synthesis system or a cellular expression system. Over the past decade, the SAIL method has been facilitating a wide variety of new investigations, including high-resolution structure determinations of large proteins and investigations of protein dynamics. In this chapter, the applications of SAIL-related approaches are introduced.

13C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)

Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14–Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D 13C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically 13C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14–Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (mc14 and mc38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for 13C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the mc38 isomerization, the 1H-13C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.

Structural and Functional Analysis of the C-Terminal Region of FliG, an Essential Motor Component of Vibrio Na+-Driven Flagella

The flagellar motor protein complex consists of rotor and stator proteins. Their interaction generates torque of flagellum, which rotates bidirectionally, clockwise (CW) and counterclockwise. FliG, one of the rotor proteins, consists of three domains: N-terminal (FliGN), middle (FliGM), and C-terminal (FliGC). We have identified point mutations in FliGC from Vibrio alginolyticus, which affect the flagellar motility. To understand the molecular mechanisms, we explored the structural and dynamic properties of FliGC from both wild-type and motility-defective mutants. From nuclear magnetic resonance analysis, changes in signal intensities and chemical shifts between wild-type and the CW-biased mutant FliGC are observed in the Cα1-6 domain. Molecular dynamics simulations indicated the conformational dynamics of FliGC at sub-microsecond timescale, but not in the CW-biased mutant. Accordingly, we infer that the dynamic properties of atomic interactions around helix α1 in the Cα1-6 domain of FliGC contribute to ensure the precise regulation of the motor switching.

Isotope-Aided Methods for Biological NMR Spectroscopy: Past, Present, and Future

This chapter starts by providing a historical background of our research endeavors over the past half-century to develop various isotope-aided methods in biological NMR spectroscopy, since innovations bloom only on the rich ground cultivated by previous investigators. We then focused on the stereo-array isotope-labeling (SAIL) method, one of our recent accomplishments, which culminates the isotope-aided NMR technologies for structural studies of proteins from various aspects: accurate structural determinations of large proteins, elaboration for automated structural determination, highly efficient and versatile residue-selective methyl labeling with newly developed auxotrophic E. coli strains, large-amplitude slow-breathing motion (LASBM) as revealed by the aromatic ring flipping of the residues in ligand-binding interfaces, and applications of the deuterium-induced 13C-NMR isotope shift to investigate the hydrogen exchange phenomena of side-chain polar groups. Meanwhile, the expected role of NMR spectroscopy has been rapidly shifting from structure determinations to dynamics studies of biologically interesting targets, such as membrane proteins and larger protein complexes. The dynamic aspects of protein–protein and protein–ligand interactions are closely related to their biological functions and can be efficiently studied by using proteins residue selectively labeled with amino acids bearing optimized labeling patterns, prepared by cellular expression. We are absolutely confident that biological NMR spectroscopy will continually develop with further innovations of isotope-labeling technologies in the coming era, featuring ultrahigh field spectrometers beyond 1 GHz.

Biochemical characterization of the flagellar stator-associated inner membrane protein FliL from Vibrio alginolyticus

The flagellar motor is embedded in the cell envelope and rotates upon interaction between the stator and the rotor. The rotation is powered by ion flow through the stator. A single transmembrane protein named FliL is associated with torque generation in the flagellar motor. We established an Escherichia coli over-expression system for FliL of Vibrio alginolyticus, a marine bacterium that has a sodium-driven polar flagellum. We successfully expressed, purified, and crystallized the ca. 17 kDa full-length FliL protein and generated a construct that expresses only the ca. 14 kDa periplasmic region of FliL (ΔTM FliL). Biochemical characterization and NMR analysis revealed that ΔTM FliL weakly interacted with itself to form an oligomer. We speculate that the observed dynamic interaction may be involved in the role of FliL in flagellar motor function.