J. G. Lindsay

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050.

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.

A new level of architectural complexity in the human pyruvate dehydrogenase complex

Mammalian pyruvate dehydrogenase multienzyme complex (PDC) is a key metabolic assembly comprising a 60-meric pentagonal dodecahedral E2 (dihydrolipoamide acetyltransferase) core attached to which are 30 pyruvate decarboxylase E1 heterotetramers and 6 dihydrolipoamide dehydrogenase E3 homodimers at maximal occupancy. Stable E3 integration is mediated by an accessory E3-binding protein (E3BP) located on each of the 12 E2 icosahedral faces. Here, we present evidence for a novel subunit organization in which E3 and E3BP form subcomplexes with a 1:2 stoichiometry implying the existence of a network of E3 “cross-bridges” linking pairs of E3BPs across the surface of the E2 core assembly. We have also determined a low resolution structure for a truncated E3BP/E3 subcomplex using small angle x-ray scattering showing one of the E3BP lipoyl domains docked into the E3 active site. This new level of architectural complexity in mammalian PDC contrasts with the recently published crystal structure of human E3 complexed with its cognate subunit binding domain and provides important new insights into subunit organization, its catalytic mechanism and regulation by the intrinsic PDC kinase.

Solution structure and characterisation of the human pyruvate dehydrogenase complex core assembly.

Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2 + E3BP) core organisation: the ‘addition’ model (60 + 12) and the ‘substitution’ model (48 + 12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the ‘substitution’ model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.

Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis.

We show that the blue native gel polyacrylamide electrophoresis system (BN‐PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN‐PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN‐PAGE, immunoblots of second‐dimensional sodium dodecyl sulfate‐PAGE (SDS‐PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, ρ0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second‐dimensional SDS‐PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one‐dimensional SDS‐PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.

Crystallization of the B800–850-complex from Rhodopseudomonas acidophila Strain 7750

Recently,methods have become available for producing good quality three-dimensional crystals of integral membrane proteins, in the presence of detergents [1, 2]. We were especially encouraged in this respect by the successful determination of the structure of the reaction centre from Rhodopseudomonas viridis [3], which has been so well described at this meeting.

The Pyruvate Dehydrogenase Complex and Related Assemblies in Health and Disease

The family of 2-oxoacid dehydrogenase complexes (2-OADC), typified by the pyruvate dehydrogenase multi-enzyme complex (PDC) as its most prominent member, are massive molecular machines (Mr, 4-10 million) controlling key steps in glucose homeostasis (PDC), citric acid cycle flux (OGDC, 2-oxoglutarate dehydrogenase) and the metabolism of the branched-chain amino acids, leucine, isoleucine and valine (BCOADC, branched-chain 2-OADC). These highly organised mitochondrial arrays, composed of multiple copies of three separate enzymes, have been widely studied as paradigms for the analysis of enzyme cooperativity, substrate channelling, protein-protein interactions and the regulation of activity by phosphorylation . This chapter will highlight recent advances in our understanding of the structure-function relationships, the overall organisation and the transport and assembly of PDC in particular, focussing on both native and recombinant forms of the complex and their individual components or constituent domains. Biophysical approaches, including X-ray crystallography (MX), nuclear magnetic resonance spectroscopy (NMR), cryo-EM imaging, analytical ultracentrifugation (AUC) and small angle X-ray and neutron scattering (SAXS and SANS), have all contributed significant new information on PDC subunit organisation, stoichiometry, regulatory mechanisms and mode of assembly. Moreover, the recognition of specific genetic defects linked to PDC deficiency, in combination with the ability to analyse recombinant PDCs housing both novel naturally-occurring and engineered mutations, have all stimulated renewed interest in these classical metabolic assemblies. In addition, the role played by PDC, and its constituent proteins, in certain disease states will be briefly reviewed, focussing on the development of new and exciting areas of medical and immunological research.

The Peroxiredoxin Family: An Unfolding Story

Peroxiredoxins (Prxs) are a large and conserved family of peroxidases that are considered to be the primary cellular guardians against oxidative stress in all living organisms. Prxs share a thioredoxin fold and contain a highly-reactive peroxidatic cysteine in a specialised active-site environment that is able to reduce their peroxide substrates. The minimal functional unit for Prxs are either monomers or dimers, but many dimers assemble into decameric rings. Ring structures can further form a variety of high molecular weight complexes. Many eukaryotic Prxs contain a conserved GGLG and C-terminal YF motif that confer sensitivity to elevated levels of peroxide, leading to hyperoxidation and inactivation. Inactive forms of Prxs can be re-reduced by the enzyme sulfiredoxin, in an ATP-dependent reaction. Cycles of hyperoxidation and reactivation are considered to play an integral role in a variety of H2O2-mediated cell signalling pathways in both stress and non-stress conditions. Prxs are also considered to exhibit chaperone-like properties when cells are under oxidative or thermal stress. The roles of various types of covalent modifications, e.g. acetylation and phosphorylation are also discussed. The ability of Prxs to assemble into ordered arrays such as nanotubes is currently being exploited in nanotechnology.