Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (R free = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.

Auxiliary subunits keep AMPA receptors compact during activation and desensitization

Signal transduction at vertebrate excitatory synapses involves the rapid activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors, glutamate-gated ion channels whose four subunits assemble as a dimer-of-dimers. Technical advances in cryo-electron microscopy brought a slew of full-length structures of AMPA receptors, on their own and in combination with auxiliary subunits. These structures indicate that dimers might undergo substantial lateral motions during gating, opening up the extracellular layer along the central twofold symmetry axis. We used bifunctional methanethiosulfonate cross-linkers to calibrate the conformations found in functional AMPA receptors in the presence and absence of the auxiliary subunit Stargazin. Our data indicate that extracellular layer of AMPA receptors can get trapped in stable, opened-up conformations, especially upon long exposures to glutamate. In contrast, Stargazin limits this conformational flexibility. Thus, under synaptic conditions, where brief glutamate exposures and the presence of auxiliary proteins dominate, extracellular domains of AMPA receptors likely stay compact during gating.

Mechanism of single- and double-sided inhibition of dual topology fluoride channels by synthetic monobodies

The Fluc family of proteins comprises small, electrodiffusive fluoride channels, which prevent accumulation of toxic F− ions in microorganisms. Recent crystal structures have confirmed their unusual architecture, in which a pair of antiparallel subunits convenes to form a dimer with a twofold symmetry axis parallel to the plane of the membrane. These structures have also revealed the interactions between Fluc channels and several different fibronectin domain monobodies that inhibit Fluc-mediated F− currents; in all structures, each channel binds to two monobodies symmetrically, one on either side of the membrane. However, these structures do not reveal the mechanism of monobody inhibition. Moreover, the results appear to diverge from a recent electrophysiological study indicating that monobody binding is negatively cooperative; that is, a bound monobody on one side of a Fluc channel decreases the affinity of an oppositely bound monobody by ∼10-fold. In this study, we reconcile these observations by probing the mechanism of monobody binding and its negative cooperativity using electrophysiological experiments in planar lipid bilayers. Our results indicate that monobody inhibition occurs via a pore-blocking mechanism and that negative cooperativity arises from electrostatic repulsion between the oppositely bound monobodies. A single glutamate residue, on a loop of the monobody that extends into the channel interior, is responsible for negatively cooperative binding. This glutamate side chain also confers voltage dependence and sensitivity to the concentration of trans-F− ion to monobody binding. Neutralization by mutation to glutamine abolishes these electrostatic effects. Monobodies that are amenable to cocrystallization with Fluc channels lack an analogous negatively charged side chain and bind independently to opposite sides of the channel. Thus, this work reveals the source of voltage dependence and negative cooperativity of monobody binding to Fluc channels along with the pore-blocking mechanism.

MASSHA– a graphics system for rigid-body modelling of macromolecular complexes against solution scattering data

A program,MASSHA, for three-dimensional rendering and rigid-body refinement is presented. The program allows display and manipulation of high-resolution atomic structures and low-resolution models represented as smooth envelopes or ensembles of beads.MASSHAis coupled with a computational module to align structural models of different nature and resolution automatically. The main feature is the possibility for rigid-body refinement of the quaternary structure of macromolecular complexes. If high- or low-resolution models of individual subunits are available, the complex can be constructed on the computer display and refined to fit the experimental solution scattering data.MASSHAprovides both interactive and automated refinement modes to position subunits in a heterodimeric complex (general case) and in a homodimeric complex with a twofold symmetry axis. Examples of application of the program (running on IBM PC compatible machines under Windows 9x/NT/2000) are given.

Electronic excitations of the free-base porphine–Ar van der Waals complex

In this paper we report on the excited-state energetics of the two lowest-lying intravalence electronic excitations S0 → S1x and S0 → S1yof a large van der Waals complex, consisting of Ar bound to free-base porphine (H2P). H2P•Arn complexes were synthesized and interrogated by laser-induced fluorescence in seeded supersonic expansions of Ar and He/Ar. Diagnostic methods, which were based on the dependence of the intensity of the spectral features on the stagnation pressure and on spectroscopy in He(99%)/Ar(1%) mixtures, were utilized for the identification of the vibrationless electronic excitations of H2P•Ar1. The spectroscopic data were supplemented by model calculations of the potential surface, which demonstrate that the single equilibrium configuration of H2P•R1 (R = Ne, Ar, Kr, and Xe) corresponds to the R atom being located at the twofold symmetry axis perpendicular to the porphyrin ring. While the intense S0 → S1y transition of H2P•Ar, exhibits a red, dispersive, microscopic solvent shift (δv = −24 cm−1), the S0 → S1x transition is characterized by a blue microscopic spectral shift (δv = +8 cm−1), which provides a unique example for excited-state destabilization, originating from intramolecular configurational changes induced by van der Waals binding in a large complex.

Der Zeeman-Effekt im Rotationsspektrum von Orthodifluorbenzol

The linear and quadratic rotational Zeeman effect in orthodifluorobenzene has been observed in magnetic fields up to 25 kG. The magnetic susceptibility anisotropies are χaa- (χbb -χcc)/2 = (25.4 ± 0.8)* 10-6 erg/(G2 * mole) and χbb - (χcc - χaa)/2 = (29.0±0.5)* 10-6 erg/ (G2 * mole) ; (the c-axis is perpendicular to the molecular plane and the a-axis coincides with the twofold symmetry axis of the molecule); the molecular ^-values are gaa= -0.0412 ± 0.00'I2, gbb=0.0371± 0.0008 and gcc= +0.0163 ±0.0007. A comparison of ⊿χ = χcc- (χaa + χbb)/2 with the corresponding values of related compounds, part of which were determined by the Cotton-Mouton technique, indicates that the effect of the magnetic field on the electric polarizability of benzene - like molecules is in the order of - 1 * 10-39 cm3/G2 and should be accounted for in the evaluation of Cotton- Mouton data.

The Croonian Lecture, 1968 - The haemoglobin molecule

Haemoglobin is the respiratory protein of the red blood cells which carries oxygen from the lungs to the tissues and facilitates, both directly and indirectly, the return transport of carbon dioxide. Mammalian haemoglobin has a molecular weight of 64500 and contains two pairs of polypeptide chains: the α -chains with 141 amino acid residues each and the β -chains with 146. Each chain is combined with one haem. Myoglobin, the oxygen carrier of muscle, is closely related to haemoglobin, but has a simpler constitution: it consists of only one polypeptide chain of 153 residues and a single haem. The amino acid sequences of the myoglobins and haemoglobins of man and of several animals have been determined (Dayhoff & Eck 1968). By means of the method of isomorphous replacement with heavy atoms, X-ray analysis of sperm whale myoglobin at 2·0 Å resolution provided the first solution of the structure of a protein (Kendrew et al . 1960; Watson 1969). All but 21 of its 153 residues form part of helices; over most of their length these helices have conformations closely resembling the right-handed α -helix of Pauling & Corey (1951). The chain is divided into 8 helical segments, separated by corners or non-helical regions. Together these form a kind of basket into which the haem group fits neatly, so that only its propionic acid side-chains protrude into the surrounding liquid (figures 1, 2). X-ray analysis at 5·5 Å resolution showed each chain of horse haemoglobin to be folded in much the same way as the single chain of sperm whale myoglobin. The 4 chains are arranged tetrahedrally, each carrying one haem in a pocket near the protein surface. The chemically identical halves of the molecule are related by a twofold symmetry axis (figure 3, plate 18; Cullis et al . 1962).