Nobutaka Numoto

Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase

V‐type ATPases (V‐ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V‐ATPases are distinct from F‐ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V‐ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V1‐ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 Å resolution reveals inter‐subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V1‐ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V1‐ATPases and F1‐ATPases.

Origin of Asymmetry at the Intersubunit Interfaces of V1-ATPase from Thermusthermophilus

V-type ATPase (V-ATPase) is one of the rotary ATPase complexes that mediate energy conversion between the chemical energy of ATP and the ion gradient across the membrane through a rotary catalytic mechanism. Because V-ATPase has structural features similar to those of well-studied F-type ATPase, the structure is expected to highlight the common essence of the torque generation of rotary ATPases. Here, we report a complete model of the extra-membrane domain of the V-ATPase (V1-ATPase) of a thermophilic bacterium, Thermus thermophilus, consisting of three A subunits, three B subunits, one D subunit, and one F subunit. The X-ray structure at 3.9Å resolution provides detailed information about the interactions between A3B3 and DF subcomplexes as well as interactions among the respective subunits, which are defined by the properties of side chains. Asymmetry at the intersubunit interfaces was detected from the structural differences among the three AB pairs in the different reaction states, while the large interdomain motion in the catalytic A subunits was not observed unlike F1 from various species and V1 from Enterococcus hirae. Asymmetry is mainly realized by rigid-body rearrangements of the relative position between A and B subunits. This is consistent with the previous observations by the high-resolution electron microscopy for the whole V-ATPase complexes. Therefore, our result plausibly implies that the essential motion for the torque generation is not the large interdomain movement of the catalytic subunits but the rigid-body rearrangement of subunits.

Structure of the C subunit of V-type ATPase from Thermus thermophilus at 1.85 A resolution.

The V-type H(+)-ATPases are similar to the F-type ATP synthases in their structure and functional mechanism. They hydrolyze ATP coupled with proton translocation across a membrane, but in some archaea and eubacteria they also synthesize ATP in the reverse reaction. The C subunit is one of the components of the membrane-bound V(0) moiety of V-type ATPases. The C subunit of V-type H(+)-ATPase from Thermus thermophilus was crystallized in a monoclinic form and its crystal structure was determined at 1.85 A resolution by the MAD method using selenomethionyl protein. The structure has a cone (tapered cylinder) shape consisting of only two types of helix (long and short) as secondary-structure elements. The molecule is divided into three similar domains, each of which has essentially the same topology. On the basis of the structural features and molecular-surface charge distribution, it is suggested that the bottom side of the C subunit is a possible binding site for the V(0) proteolipid L-subunit ring and that the C subunit might function as a spacer unit between the proteolipid L-subunit ring and the rotating V(1) central shaft.

Structural Basis for the Heterotropic and Homotropic Interactions of Invertebrate Giant Hemoglobin

The oxygen binding properties of extracellular giant hemoglobins (Hbs) in some annelids exhibit features significantly different from those of vertebrate tetrameric Hbs. Annelid giant Hbs show cooperative oxygen binding properties in the presence of inorganic cations, while the cooperativities of vertebrate Hbs are enhanced by small organic anions or chloride ions. To elucidate the structural basis for the cation-mediated cooperative mechanisms of these giant Hbs, we determined the crystal structures of Ca2+- and Mg2+-bound Hbs from Oligobrachia mashikoi at 1.6 and 1.7 A resolution, respectively. Both of the metal-bound structures were determined in the oxygenated state. Four Ca2+-binding sites and one Mg2+-binding site were identified in each tetramer subassembly. These cations are considered to stabilize the oxygenated form and increase affinity and cooperativity for oxygen binding, as almost all of the Ca2+ and Mg2+ cations were bound at the interface regions, forming either direct or hydrogen bond-mediated interactions with the neighboring subunits. A comparison of the structures of the oxygenated form and the partially unliganded form provides structural insight into proton-coupled cooperativity (Bohr effect) and ligand-induced transitions. Two histidine residues are assumed to be primarily associated with the Bohr effect. With regard to the ligand-induced cooperativity, a novel quaternary rotation mechanism is proposed to exist at the interface region of the dimer subassembly. Interactions among conserved residues Arg E10, His F3, Gln F7, and Val E11, together with the bending motion of the heme molecules, appear to be essential for quaternary rearrangement.

Crystal structure of the co-chaperonin Cpn10 from Thermus thermophilus HB8

Introduction. Chaperonins are ubiquitous proteins that control the folding of other proteins denatured by various stresses, such as heat shock. They consist of two types of proteins, cpn60 (60 kDa) and cpn10 (10 kDa). The GroEL (cpn60)-GroES (cpn10) complex from Escherichia coli is the most thoroughly studied chaperonin system. GroEL forms a tetradecameric oligomer with a cylinder shape, and GroES forms a dome-shaped heptamer. The end of the large central cavity of the GroEL tetradecamer binds to the unfolding substrate polypeptide. Afterwards ATP binds to GroEL, and then this large cavity is closed upon capping by GroES. Then, ATP is hydrolyzed to ADP, and the refolded polypeptide is released into solution. The structures of cpn60 of several species, including the E. coli GroEL-GroES complex (PDB code: 1AON), have been reported, and the crystal structures of cpn10 from E. coli, Mycobacterium tuberculosis (PDB code: 1HX5, 1P3H), Mycobacterium leprae (PDB code: 1LEP), and bacteriophage T4 Gp31 (PDB code: 1G31) have been reported. All cpn10 structures are homo-heptameric and dome-shaped, and each monomer form similar folds as small -barrel with a highly flexible long loop, the so-called “mobile loop.” In the crystal structure of the E. coli GroEL-GroES complex, this mobile loop makes contact with the GroEL tetradecamer. It has also been reported that some cpn10s have additional functions; for example, the M. tuberculosis and M. leprae cpn10s are strong immunogens and M. tuberculosis cpn10 forms a tetradecamer with divalent cations. M. tuberculosis cpn10 plays an important role during infection. Chaperonin from a thermophilic bacterium, Thermus thermophilus, is stable up to 80°C. Here, we report the crystal structure of cpn10 from Thermus thermophilus HB8. In the present structure, the mobile loops were completely disordered in five of the seven subunits, but in the remaining two subunits, the electron density of the mobile loop was partially observed.

Structure of the partially unliganded met state of 400 kDa hemoglobin: insights into ligand-induced structural changes of giant hemoglobins.

Recent crystallographic studies have revealed the structures of some invertebrate extracellular giant hemoglobins of 3,600 kDa or 400 kDa and their common quaternary structure of dodecameric subassembly composed of four kinds of globin subunits (A1, A2, B1, and B2). These results have provided insight into the mechanisms of their unique functional properties of oxygen binding and sulfide binding. All of these structures were solved with oxygenated or CO‐liganded forms at low or moderate resolutions. We have determined the crystal structure of 400 kDa Hb from a polychaete Oligobrachia mashikoi at 1.95 Å resolution. The electron densities at higher resolution confirm the existence of an isoform of the B1 subunit because of the inconsistency with the model that was built from the formerly known amino acid sequence. The brownish color of the crystals used in this study and the absorption spectrum from the dissolved crystals strongly indicated that the obtained structure was a ferric met state, whereas completele absence of electron density around the distal heme pockets were observed at the A2, B1, and B2 subunits. We concluded that the obtained structure was in unliganded met forms at three of four globin subunits in the 24mer assembly and in oxygenated forms at the remaining A1 subunits. The partially unliganded structure showed remarkable structural changes at the AB loop regions causing quaternary rearrangements of the EF‐dimer structure. In contrast, few changes occurred at the interface regions composed of the E and F helices. These results suggest that the ligand‐induced structural changes of Oligobrachia Hb are quite different from those of the well‐studied mollusk Hb having the same EF‐dimer structure. The structural rearrangements make the dodecameric subassembliy form a tighter conformation than those of fully oxygenated or CO‐liganded dodecamer structure. Proteins 2008.

A P39R mutation at the N-terminal domain of human αB-crystallin regulates its oligomeric state and chaperone-like activity

Recent structure analyses of αB-crystallin have proposed some models of the N-terminal domain and the manner of oligomerization, whereas the effects of the significantly high content of Pro residues at the N-terminal domain remain unclear. We report the properties of a novel P39R mutant of αB-crystallin. The content of α-helix was increased, and the molecular size of the P39R mutant was larger than that of wild-type αB-crystallin. A slight loss of chaperone-like activity was observed using alcohol dehydrogenase (ADH), while a significant increase was detected by insulin assay. The Pro residue at the N-terminal domain of αB-crystallin is important for oligomerization and function.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for “difficult” proteins, such as membrane proteins and supermolecular complexes.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Crystal structures of dengue virus protein revealed ED3 sero-specificity

Manjiri Ravindra Kulkarni1, Monirul Islam2, Nobutaka Numoto3, Nobutoshi Ito3, Yutaka Kuroda4 1Department Of Comparative Biosciences, University Of Illinois, Urbana-Champaigne, Urbana, United States, 2Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh, 3Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, 4Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology, Tokyo, Japan E-mail: jaimalhar.mrk@gmail.com