Naoya Shibayama

The structural basis for an essential subunit interaction in influenza virus RNA polymerase

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 310 helix.

Oxygen equilibrium study and light absorption spectra of Ni(II)Fe(II) hybrid hemoglobins

Ni(II)-Fe(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunit are substituted with Ni(II) protoporphyrin IX, have been prepared and characterized. Since Ni(II) protoporphyrin IX binds neither oxygen nor carbon monoxide, the oxygen equilibrium properties of the Fe subunit in these hybrid hemoglobins were specifically determined. K1 values, namely the equilibrium constants for the first oxygen molecule to bind to hemoglobin, agreed well for these hybrid hemoglobins with the K1 value of native hemoglobin A in various conditions. Therefore, Ni(II) protoporphyrin IX in these hybrid hemoglobins behaves like a permanently deoxygenated heme. Both Ne-Fe hybrid hemoglobins bound oxygen non-co-operatively at low pH values. When the pH was raised, alpha 2 (Fe) beta 2 (Ni) showed co-operativity, but the complementary hybrid, alpha 2 (Ni) beta 2 (Fe), did not show co-operativity even at pH 8.5. The light absorption spectra of Ni(II)-Fe(II) hybrid hemoglobins indicated that the coordination states of Ni(II) protoporphyrin IX in the alpha subunits responded to the structure of the hybrid, whereas those in the beta subunits were hardly changed. In a deoxy-like structure (the structure that looks like that observed in deoxyhemoglobin), four-co-ordinated Ni(II) protoporphyrin IX was dominant in the alpha (Ni) subunits, while under the conditions that stabilized an oxy-like structure (the structure that looks like that observed in oxyhemoglobin), five-co-ordinated Ni(II) protoporphyrin IX increased. The small change observed in the absorption spectrum of the beta (Ni) subunits is not related to the change of the co-ordination number of Ni(II) protoporphyrin IX. Non-co-operative binding of oxygen to the beta subunits in alpha 2 (Ni) beta 2 (Fe) accompanied the change of absorption spectrum in the alpha (Ni) subunits. We propose a possible interpretation of this unique feature.

Properties of chemically modified (Ni(II)―Fe(II) hybrid hemoglobins: Ni(II) protoporphyrin IX as a model for a permanent deoxy-heme

Chemical modifications, NES-Cys(beta 93), des-Arg(alpha 141), and both modifications on the same molecule, were made to Ni-Fe hybrid hemoglobins, and their effect on individual subunits was investigated by measuring oxygen equilibrium curves, the Fe(II)-N epsilon (His F8) stretching Raman lines, and light-absorption spectra. The oxygen equilibrium properties indicated that modified Ni-Fe hybrid hemoglobins remain good models for the corresponding deoxy ferrous hemoglobins, although K1, the dissociation equilibrium constant for the first oxygen to bind to hemoglobin, was decreased by the chemical modifications. Resonance Raman spectra of deoxy alpha 2 (Fe) beta 2 (Ni) and light-absorption spectra of deoxy alpha 2 (Ni) beta 2 (Fe), revealed that the state of alpha hemes in both hybrid hemoglobins underwent a transition from a deoxy-like state to an oxy-like state caused by these chemical modifications when K1 was about 3 mm Hg (1 mm Hg approximately 133.3 Pa). On the other hand, the state of beta hemes in hybrid hemoglobins was little affected, when K1 was larger than 1 mm Hg. Modified alpha 2 (Fe) beta 2 (Ni) gave a Hill coefficient greater than unity with a maximum of 1.4 when K1 was about 4 mm Hg. The two-state model predicts that the K1 value at the maximum Hill coefficient should be much larger than this value. For oxygen binding to unmodified alpha 2 (Ni) beta 2 (Fe), oxygen equilibrium data suggested no structural change, while the spectral data showed a structural change around Ni(II) protoporphyrin IX in the alpha subunits. A similar situation was encountered with modified alpha 2 (Ni) beta 2 (Fe), although K1 was decreased as a result of the structural changes induced by the modifications.

Rate constants for O2 and CO binding to the alpha and beta subunits within the R and T states of human hemoglobin.

Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual α and β subunits in human hemoglobin. To address this question systematically, we prepared a series of hybrid hemoglobins in which heme was replaced by chromium(III), manganese(III), nickel(II), and magnesium(II) protoporphyrin IXs in either the α or β subunits to produce α2(M)β2(Fe)1and α2(Fe)β2(M) tetramers. None of the abnormal metal complexes react with dioxygen or carbon monoxide. The O2 affinities of the resultant hemoglobins vary from 3 μm −1 (Cr(III)/Fe(II) hybrids) to 0.003 μm −1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A0. The α and β subunits in hemoglobin have similar O2 affinities in both quaternary states, despite the fact that the R to T transition causes significantly different structural changes in the α and β heme pockets. This functional equivalence almost certainly evolved to maintain high n values for efficient O2 transport.

Structure of deoxy-quaternary haemoglobin with liganded beta subunits.

We have determined the structure of a T-state haemoglobin in which the haem groups of the beta subunits have carbon monoxide bound, and the alpha subunits have nickel replacing the haem iron and are ligand-free. The structural adjustments on binding ligand in the T state are in the same direction as those associated with the quaternary transition, and a translational shift of the haem is severely restricted. We explain how these observations may account for the low ligand affinity of the beta haem of T-state haemoglobin.

Direct observation of photolysis-induced tertiary structural changes in hemoglobin

Human Hb, an α2β2 tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25–35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the α subunit than in the β subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state β heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.

Direct observation of two distinct affinity conformations in the T state human deoxyhemoglobin

The main features of cooperative oxygenation of human hemoglobin have been described by assuming the equilibrium between two affinity conformations of the entire molecule, T and R. However, the molecular basis for explaining the wide variation in the O2 affinities of the deoxy T state has remained obscure. We address this long‐standing issue by trapping the conformational states of deoxyhemoglobin molecules within wet porous transparent silicate sol‐gels. The equilibrium O2 binding measurements of the encapsulated deoxyhemoglobin samples showed that deoxyhemoglobin free of anions coexists in two conformations that differ in O2 affinity by 40 times or more, and addition of inositol hexaphosphate to this anion‐free deoxyhemoglobin brings about a very slow redistribution of these affinity conformations. These results are the first, direct demonstration of the existence of equilibrium between two (at least two) functionally distinguishable conformational states in the T state deoxyhemoglobin.

Structure of haemoglobin in the deoxy quaternary state with ligand bound at the α haems

Abstract We report the X-ray crystal structure of two analogues of human haemoglobin in the deoxy quaternary (T) state with ligand bound exclusively at the α haems. These models were prepared from symmetric, mixed-metal hybrid haemoglobin molecules. The structures of α Fe(II) β Co(II) , its carbonmonoxy derivative α Fe(II) CO β CoII , and α FeII O 2 β Ni(II) are compared with native deoxy haemoglobin by difference Fourier syntheses at 2·8, 2·9 and 3·5 A resolution, respectively, and the refined α Fe(II) CO β Co(II) structure is analysed. In both the native deoxy and liganded T molecules, the mean plane of the α-subunit haem is parallel with the axis of the F helix, but this plane is tilted with respect to the helix axis in the oxy-quaternary R state. The side-chains of LeuFG3 and ValFG5 sterically restrict haem tilting in the T state. We propose that strain energy develops at the contact between the haem and these residues in the liganded T-state haemoglobin, and that the strain is, in part, responsible for the low affinity of the T-state α haem.

Direct Determination of Protonation States of Histidine Residues in a 2 Å Neutron Structure of Deoxy-Human Normal Adult Hemoglobin and Implications for the Bohr Effect

We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.

Capturing the hemoglobin allosteric transition in a single crystal form

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.