Atomic Structure of the E2 Inner Core of Human Pyruvate Dehydrogenase Complex

Pyruvate dehydrogenase complex (PDC) is a large multienzyme complex that catalyzes the irreversible conversion of pyruvate to acetyl-coenzyme A with reduction of NAD⁺. Distinctive from PDCs in lower forms of life, in mammalian PDC, dihydrolipoyl acetyltransferase (E2; E2p in PDC) and dihydrolipoamide dehydrogenase binding protein (E3BP) combine to form a complex that plays a central role in the organization, regulation, and integration of catalytic reactions of PDC. However, the atomic structure and organization of the mammalian E2p/E3BP heterocomplex are unknown. Here, we report the structure of the recombinant dodecahedral core formed by the C-terminal inner-core/catalytic (IC) domain of human E2p determined at 3.1 Å resolution by cryo electron microscopy (cryoEM). The structure of the N-terminal fragment and four other surface areas of the human E2p IC domain exhibit significant differences from those of the other E2 crystal structures, which may have implications for the integration of E3BP in mammals. This structure also allowed us to obtain a homology model for the highly homologous IC domain of E3BP. Analysis of the interactions of human E2p or E3BP with their adjacent IC domains in the dodecahedron provides new insights into the organization of the E2p/E3BP heterocomplex and suggests a potential contribution by E3BP to catalysis in mammalian PDC

Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains

<div><p>Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO₂ transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.</p> </div

Purification and EM of bovine AE1.

<p>(<b>a</b>) SDS-PAGE of bovine AE1 after ion-exchange chromatography. Lane 1: Coomassie stained gel; Lanes 2,3: immunoblotting, 2: immune serum; 3: preimmune serum. (<b>b,c</b>) Size-exclusion chromatography examination of bovine AE1 sample after the ion-exchange chromatography step (top) and the dimer fraction after the size-exclusion chromatography step (<b>c</b>) showing a peak corresponding to dimeric AE1. (<b>d</b>) A representative area of transmission EM micrograph of bovine dimeric AE1 stained with 1% uranyl formate. (<b>e</b>) Representative class averages (CA) and the corresponding raw particle (RP) images of AE1. Each class average is obtained by averaging about 100 particle images. The side length of each box is 26 nm.</p

Single-particle reconstruction of bovine AE1.

<p>(<b>a</b>) Schematic illustration of the OTR data collection method. For each target sample area, two micrographs were recorded with the grid tilted at −45° and +45°, respectively. (<b>b</b>) 3D map generated by averaging 25 OTR maps. Two orthogonal views, defined as front view (left panel) and side view (right panel), are shown. (<b>c</b>) Final map obtained by merging 174,197 particle images with single particle reconstruction method. The map in (<b>c</b>) is shown in the same orientations as in (<b>b</b>). (<b>d</b>) Fourier shell correlation (FSC) coefficient between two reconstructions obtained from even- and odd-numbered particle images. The effective resolution is estimated to be 2.4 nm using the 0.5 FSC cut-off. (<b>e</b>) Comparisons of the computed projection, class average, and raw particle. Four representative views (top, tilt, front, and side) are show from left to right, respectively. (<b>f</b>) Euler angle distribution of classified particles. The brightness of each point indicates the number of particles used in the class average in that orientation.</p

Fitting of the atomic structure of cytoplasmic domain

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055408#pone.0055408-Zhang1" target="_blank">[<b>14</b>] </a><b>and the 7.5 Å resolution 2D crystal structure of membrane domain </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055408#pone.0055408-Yamaguchi1" target="_blank">[<b>23</b>] </a><b>into our 3D map of full length AE1 dimer.</b> (<b>a</b>) Shaded surface views of the atomic structure of cytoplasmic domain (PDB ID: 1HYN) filtered to 2.4 nm resolution (green) compared to the corresponding views of cytoplasmic domain resolved in the EM single-particle reconstruction (gold) of full-length AE1 dimer. In the EM map, the membrane domain of AE1 dimer is removed for clarity. The two structures are similar in size and in having a double-humped shape on their cytoplasmic side. (<b>b</b>) Shaded surface views of AE1 membrane domain resolved from 2D crystals embedded in trehalose (EMDB ID: 1645) filtered to 2.4 nm resolution (blue), as compared to the corresponding views of membrane domain resolved in the EM single-particle reconstruction (gold). The extracellular and intracellular sides identified in the published 2D crystal structure were used to define the orientation for comparison. (<b>c</b>) Superposition of the two structures of membrane domains described in (<b>b</b>) viewed from the cytoplasmic side (top view). The EM single-particle reconstruction is rendered at higher density threshold to show the deep canyon, which is consistent with the membrane domain structure from 2D crystals. (<b>d</b>) Fitting the EM single-particle reconstruction of full-length AE1 dimer with the crystal structure of cytoplasmic domain (red and cyan) and 2D crystal structure of membrane domain (blue). The single-particle reconstruction is rendered in two density threshold values: at low threshold (gray mesh) and a high threshold (yellow). The approximate positions of N-terminus and C-terminus of the cytoplasmic domain are labeled with diamond and triangle, respectively.</p

Mutation of gp42 I159 does not affect binding affinity or kinetics with gHgL or HLA-DQ2.

<p>Kinetic binding experiments were conducted similarly to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-g002" target="_blank">Figure 2</a> on the OctetRED96. For panels A and B site-specifically biotinylated HLA-DQ2 (CLIP1) was immobilized on the streptavidin (SA) biosensor tip surfaces. Binding kinetics of HLA-DQ2 (ligand) with the single mutant gp42 C114S (A) or the gp42 I159C double mutant (B) are depicted. Global curve fitting with a 2∶1 heterogeneous ligand model closely matched the experimental data. The calculated binding curves are shown overlaid on the data from the experiment. For panels C and D, biotinylated EBV gHgL was immobilized on the streptavidin (SA) biosensor surfaces. Binding kinetics of EBV gHgL with the single mutant gp42 C114S (C) or I159C double mutant (D) are depicted. Overlay curves show the global fitting results using a 1∶1 Langmuir binding model. Kinetic and thermodynamic binding constants are similar to binding gp42 wildtype (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-g002" target="_blank">Figure 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-t002" target="_blank">Table 2</a>). (E) Representative class averages of gHgL/gp42-PEG2K/HLA-DQ2 complexes. The gp42 I159C mutant was labeled with PEG-2K maleimide and used to form complexes with gHgL and HLA-DQ2, which were by purified gel filtration chromatography. Representative angles between the two arms of the complexes formed by gHgL and gp42/HLA are indicated as well as the number of particles included in each class.</p

Modifications of gp42 residue I159 disrupt membrane fusion with B cells.

<p>In panels (A) and (B), CHO cells were transfected with gB, gHgL, and a T7-Luciferase reporter and either 0.1 µg or 1.0 µg soluble purified gp42 or gp42 mutant was added 24 hours post transfection along with T7 expressing Daudi B cells. The negative control (F12 media) was similarly transfected and mixed with Daudi cells but no soluble gp42 protein was added. (A) The wildtype gp42 residue C114 is not important for membrane fusion activity. Treatment of wt gp42 with reducing (TCEP) and alkylating (IAA) agents, to irreversibly block the C114 –SH group, does not affect gp42 membrane fusion activity. Mutation of C114 to serine also does not affect membrane fusion activity, it also has no unpaired cysteines due to the mutation and hence is not treated with reducing agent or alkylating agent. (B) Mutation and chemical modifications of gp42 residue 159 block membrane fusion activity. Fusion assay results of wt gp42, I159C (reduced with TCEP), I159C (reduced and alkylated with IAA) and I159C site-specifically pegylated with maleimidePEG 2000 and 10000 MW_avg (denoted as mPEG2K and mPEG10K). (C) Gel filtration traces of gp42 I159C along with gp42 I159C modified with mPEG2K or mPEG10K. The shift in elution volume (V_e) with increasing molecular weights of PEGylation is evident, allowing purification of the appropriately modified gp42 protein for fusion assays and complex formation with gHgL and HLA for EM imaging. The peaks for the purified fraction of the gp42 mutant protein are denoted as I159C-Untreated, I159C-mPEG2K and I159C-mPEG10K from right to left respectively. Abbreviations: TCEP is Tris(2-carboxyethyl)phosphine, hydrochloride; IAA is Iodoacetamide and, mPEG stands for maleimide-polyethylene glycol and mPEG2K or mPEG10K denotes this chemical with a MW_avg 2000 or 10,000 Da.</p

Biochemical assembly of the EBV B cell triggering complex.

<p>(A) <i>In-vitro</i> assembly of the EBV gHgL/gp42/HLA-DQ2 triggering complex (red, indicated by arrow) using size exclusion chromatography (S200). The triggering complex elutes at 10.7 ml (Ve, elution volume) with an estimated apparent MW of 255 kDa (also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-t001" target="_blank">Table 1</a>). This complex is formed from EBV gHgL/gp42 complex (brown) mixed with excess HLA-DQ2 (green). Excess HLA-DQ2 can be seen as a second individual peak in the red trace. EBV gHgL/gp42 complex (brown) is formed quantitatively from 1∶1 molar mixture of gHgL (blue) and gp42 (purple). (B) Thermodynamic cycle linking the two pathways to the formation of the triggering complex. The horizontal (top and bottom) reactions represent the binding of HLA to either free gp42 or to gHgL/gp42 complexes, respectively. Similarly, the vertical (left and right) reactions represent the binding of gHgL to either gp42 or gp42/HLA complexes.</p

Mutations in gH at the interface with the gp42 HP affect membrane fusion.

<p>Cell surface expression and fusion assay results with B cells with gH mutants in the R488, S507 and D511 regions. Cell surface expression was measured using the anti-gHgL monoclonal antibody E1D1. (A) The gH D-II and D-III sequence observed proximal to the gp42 HP from our EM model has been highlighted to show the sequence and secondary structure indicating the mutual positions of the mutations in gH that would validate the EM model. The residue mutated to Asn (N) that gets potentially glycosylated is shown in bold text, the NX[S/T] motif is underlined. Cystine bridge between C278–C335 is highlighted; EBV gH D-II and D-III mutants to validate the position of residues in gH close to HP as revealed in our EM model are studied in the following panels (B) and (C); (B) gH G276N in the background of the disulfide bond mutant C278A/C335A does not have the glycosylation motif NX[S/T] and exhibits near wildtype fusion levels with B cells. By contrast, the G276N/C278S that introduces the glycosylation motif at 276 reduces fusion levels to lower than 40%. (C) The R488A mutation does not have a significant effect on gHgL expression or B cell fusion, while the R488N/K90T mutation reduces expression and membrane fusion. The D511N/F513S mutant and its control (F513S) show drastically reduced surface expression and B cell fusion. Both gH507N/A509S and the control gH A509S are expressed near wildtype levels and the gH S507N glycosylation mutant shows a reduction in membrane fusion activity. The mutants where the surface expression is as good as the gp42 wildtype but the fusion levels are down (below ∼40% or less) are highlighted in the rounded rectangular box. Mutants that result in potential glycosylation due to the introduced residue change are shown with a check mark.</p

The pseudoatomic model of the triggering complex places the gp42 HP in contact with gH.

<p>(A) Pseudo-atomic model of the gHgL/gp42/HLA-DQ2 complex obtained from the EM envelope fitting. The individual domains of each protein are marked and the hydrophobic pocket (HP) in gp42 is highlighted with an arrow. The C-termini of gH and HLA chains are similarly marked and lie on one side of the complex at ∼70 Å of each other. (B) The gp42 HP interaction site with gHgL. Two sides of the gp42 HP interact with gH between D-II and D-III, including a loop between helices 2α-6 and 2α-7 from D-II and helix 3α-9 from D-III. Residues that have been previously mutated in the gp42 HP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat.1004309-Silva1" target="_blank">[30]</a> are indicated, H206 and T193 that had linker insertions as Cα spheres (dark blue) and F210 as sticks (dark blue) within the hydrophobic pocket (light blue Cα spheres) as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat.1004309-Mullen1" target="_blank">[27]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat.1004309-Kirschner3" target="_blank">[28]</a>, which disrupt membrane fusion activity. I159 is also shown as sticks (light blue). The gp42 HP faces away from the observer in this orientation. The gp42 interaction with the HLA-class II β-chain (orange) brings gH and HLA into close proximity. Residue C114 which is the only unpaired cysteine in wt gp42 is shown as sticks (light blue) (C) Close-up view of gp42 residue I159 (blue), located in the gp42 158 loop <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat.1004309-Kirschner3" target="_blank">[28]</a>, at the junction of the HLA and gH contact sites. The locations of gH mutant residues that were tested are indicated, including G276 (cyan) in the 2α-6/2α-7 loop (D-II) and D511 and S507 in the 3α- helix 9 (D-III). The view is rotated 180° along vertical axis and then rotated 45° counter-clockwise with the horizontal axis compared to the orientation shown in (A) and (B). Images were rendered using MacPyMol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat.1004309-Schrodinger1" target="_blank">[55]</a>.</p