Naoki Kunishima

Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1—in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose ‘active’ and ‘resting’ conformations are modulated through the dimeric interface by a packed α-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an ‘open’ or ‘closed’ conformation. The structures imply that glutamate binding stabilizes both the ‘active’ dimer and the ‘closed’ protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+

Crystal structures of the extracellular ligand-binding region of the metabotropic glutamate receptor, complexed with an antagonist, (S)-(α)-methyl-4-carboxyphenylglycine, and with both glutamate and Gd3+ ion, have been determined by x-ray crystallographic analyses. The structure of the complex with the antagonist is similar to that of the unliganded resting dimer. The antagonist wedges the protomer to maintain an inactive open form. The glutamate/Gd3+ complex is an exact 2-fold symmetric dimer, where each bi-lobed protomer adopts the closed conformation. The surface of the C-terminal domain contains an acidic patch, whose negative charges are alleviated by the metal cation to stabilize the active dimeric structure. The structural comparison between the active and resting dimers suggests that glutamate binding tends to induce domain closing and a small shift of a helix in the dimer interface. Furthermore, an interprotomer contact including the acidic patch inhibited dimer formation by the two open protomers in the active state. These findings provide a structural basis to describe the link between ligand binding and the dimer interface.

Crystal Structure of Rat Bcl-xL IMPLICATIONS FOR THE FUNCTION OF THE Bcl-2 PROTEIN FAMILY

Bcl-xL is a member of the Bcl-2 protein family, which regulates apoptosis. Preparation of recombinant rat Bcl-xL yielded two forms, one deamidated at -Asn-Gly- sequences to produce isoaspartates and the other not deamidated. The crystal structures of the two forms show that they both adopt an essentially identical backbone structure which resembles the fold of human Bcl-xL: three layers of two α-helices each, capped at one end by two short helices. Both forms have a long disordered region, which contains the potential deamidation sites. The molecular structure exhibits a low level of interhelical interactions, the presence of three cavities, and a notable hydrophobic cleft surrounded by walls rich in basic residues. These unique structural features may be favorable for its accommodation into membranes or for possible rearrangement to modulate homo-/heterodimerization. Homology modeling of Bcl-2 and Bax, based on the Bcl-xL structure, suggests that Bax has the strongest potential for membrane insertion. Furthermore, we found a possible interface for interaction with non-Bcl-2 family member proteins, such as CED-4 homologues.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

Significance This study shows successful experimental phasing methods (single-wavelength anomalous diffraction, single isomorphous replacement, and single isomorphous replacement with anomalous scattering) for crystal structure determination of a membrane protein by serial femtosecond crystallography with X-ray free electron lasers. Our iodine-containing detergent provided strong anomalous and isomorphous difference signals, which enabled experimental phasing using lower-resolution reflections (worse than 3 Å) from fewer indexed images than phasing attempts reported previously. The findings of this study will be applicable to a wide range of target proteins in structural biology, especially membrane proteins that often diffract to low resolution. The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.

Purification, crystallization and preliminary X-ray analysis of a complex between granulocyte colony-stimulating factor and its soluble receptor

Crystals of the complex between granulocyte colony-stimulating factor and its soluble receptor were obtained by a vapour-diffusion method using ammonium sulfate as a precipitant. Addition of 1, 4-dioxane was critical in order to grow the crystals to sufficient sizes. Cryoprotection was essential in order to collect diffraction data at atomic resolution. Two kinds of crystal forms were obtained depending on the cryoprotectants. In a cryosolvent with the same salt concentration as in the crystallization conditions, the crystal belonged to the space group I4(1)22. At higher salt concentrations, the crystal was converted to a different space group P4(1)2(1)2 (P4(3)2(1)2) with the same unit-cell parameters.

Crystallization and preliminary X‐ray diffraction studies of a novel killer toxin from a halotolerant yeast Pichia farinosa

A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations. Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.46 A. The low-salt solution provided crystals that belonged to the same space group, with nearly same cell dimensions. Preliminary diffraction studies showed that the intensity distributions are significantly different between the two crystals. Both types of crystals contained either two or three molecules per asymmetric unit. They diffracted X-rays beyond 2.0 A resolution and were stable to X-ray irradiation.

Crystallization and preliminary X-ray crystallographic studies of NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific NADP-dependent dehydrogenase (HIBADH). HIBADH from Thermus thermophilus HB8 has been overexpressed in Escherichia coli and crystallized by the microbatch method using lithium chloride as a precipitant at 296 K. X-ray diffraction data have been collected to 1.80 A resolution at 100 K using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 85.878, b = 106.367, c = 168.639 A. A homotetramer of HIBADH is likely to be present in the asymmetric unit, giving a V(M) of 3.0 A(3) Da(-1) and a solvent content of 59.3%.