Elucidation of the interaction loci of the human pyruvate dehydrogenase complex E2·E3BP core with pyruvate dehydrogenase kinase 1 and kinase 2 by H/D exchange mass spectrometry and nuclear magnetic resonance.

The human pyruvate dehydrogenase complex (PDC) comprises three principal catalytic components for its mission: E1, E2, and E3. The core of the complex is a strong subcomplex between E2 and an E3-binding protein (E3BP). The PDC is subject to regulation at E1 by serine phosphorylation by four kinases (PDK1-4), an inactivation reversed by the action of two phosphatases (PDP1 and -2). We report H/D exchange mass spectrometric (HDX-MS) and nuclear magnetic resonance (NMR) studies in the first attempt to define the interaction loci between PDK1 and PDK2 with the intact E2·E3BP core and their C-terminally truncated proteins. While the three lipoyl domains (L1 and L2 on E2 and L3 on E3BP) lend themselves to NMR studies and determination of interaction maps with PDK1 and PDK2 at the individual residue level, HDX-MS allowed studies of interaction loci on both partners in the complexes, PDKs, and other regions of the E2·E3BP core, as well, at the peptide level. HDX-MS suggested that the intact E2·E3BP core enhances the binding specificity of L2 for PDK2 over PDK1, while NMR studies detected lipoyl domain residues unique to interaction with PDK1 and PDK2. The E2·E3BP core induced more changes on PDKs than any C-terminally truncated protein, with clear evidence of greater plasticity of PDK1 than of PDK2. The effect of L1L2S paralleled HDX-MS results obtained with the intact E2·E3BP core; hence, L1L2S is an excellent candidate with which to define interaction loci with these two PDKs. Surprisingly, L3S' induced moderate interaction with both PDKs according to both methods.

Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).

Insight to the interaction of the dihydrolipoamide acetyltransferase (E2) core with the peripheral components in the Escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches.

Multifaceted structural approaches were undertaken to investigate interaction of the E2 component with E3 and E1 components from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), as a representative of the PDHc from Gram-negative bacteria. The crystal structure of E3 at 2.5 Å resolution reveals similarity to other E3 structures and was an important starting point for understanding interaction surfaces between E3 and E2. Biochemical studies revealed that R129E-E2 and R150E-E2 substitutions in the peripheral subunit-binding domain (PSBD) of E2 greatly diminished PDHc activity, affected interactions with E3 and E1 components, and affected reductive acetylation of E2. Because crystal structures are unavailable for any complete E2-containing complexes, peptide-specific hydrogen/deuterium exchange mass spectrometry was used to identify loci of interactions between 3-lipoyl E2 and E3. Two peptides from the PSBD, including Arg-129, and three peptides from E3 displayed statistically significant reductions in deuterium uptake resulting from interaction between E3 and E2. Of the peptides identified on E3, two were from the catalytic site, and the third was from the interface domain, which for all known E3 structures is believed to interact with the PSBD. NMR clearly demonstrates that there is no change in the lipoyl domain structure on complexation with E3. This is the first instance where the entire wild-type E2 component was employed to understand interactions with E3. A model for PSBD-E3 binding was independently constructed and found to be consistent with the importance of Arg-129, as well as revealing other electrostatic interactions likely stabilizing this complex.

Nuclear magnetic resonance approaches in the study of 2-oxo acid dehydrogenase multienzyme complexes--a literature review.

The 2-oxoacid dehydrogenase complexes (ODHc) consist of multiple copies of three enzyme components: E1, a 2-oxoacid decarboxylase; E2, dihydrolipoyl acyl-transferase; and E3, dihydrolipoyl dehydrogenase, that together catalyze the oxidative decarboxylation of 2-oxoacids, in the presence of thiamin diphosphate (ThDP), coenzyme A (CoA), Mg²âº and NAD⁺, to generate CO2, NADH and the corresponding acyl-CoA. The structural scaffold of the complex is provided by E2, with E1 and E3 bound around the periphery. The three principal members of the family are pyruvate dehydrogenase (PDHc), 2-oxoglutarate dehydrogenase (OGDHc) and branched-chain 2-oxo acid dehydrogenase (BCKDHc). In this review, we report application of NMR-based approaches to both mechanistic and structural issues concerning these complexes. These studies revealed the nature and reactivity of transient intermediates on the enzymatic pathway and provided site-specific information on the architecture and binding specificity of the domain interfaces using solubilized truncated domain constructs of the multi-domain E2 component in its interactions with the E1 and E3 components. Where studied, NMR has also provided information about mobile loops and the possible relationship of mobility and catalysis.

Studies of the structure of the N-terminal domain from the Y4 receptor - a G protein-coupled receptor - and its interaction with hormones from the NPY family.

Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 microM affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor, whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP, and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket.

Properties of the N-terminal domains from Y receptors probed by NMR spectroscopy.

Binding of neurohormones from the NPY family to their receptors, the so-called Y receptors, that belong to the superfamily 1b of G-protein coupled receptors might include transient binding to the N-terminal domains of the receptors. Accordingly, we have studied structural features of the N-terminal domains from the Y1, Y2, Y4, and Y5 receptor subtypes (N-Y1, N-Y2, N-Y4, N-Y5). We developed efficient strategies for their recombinant expression. N-Y4 and N-Y1 were expressed as insoluble fusions to enforce accumulation into inclusion bodies, whereas N-Y2 and N-Y5 were expressed as soluble fusion proteins. All N-terminal domains are fully flexible in aqueous buffer. In the presence of phospholipid micelles some stretches within the polypeptides adopt helical conformations, but these are too unstable to be characterized in detail. Using chemical shift mapping techniques, interactions of NPY, peptide YY (PYY), and pancreatic polypeptide (PP), the three members of the neurohormone family that are the Y receptors' natural ligands, with N-Y1, N-Y2, and N-Y5 revealed chemical shift changes in all cases, with the largest values being encountered for PP interacting with N-Y1 or N-Y5 both in the presence and in the absence of phospholipid micelles. The strength of the interactions, however, is generally weak, and the data also point to nonspecific contacts. Previously, in case of the interaction of N-Y4 with PP, the contacts were shown to be electrostatic in nature. This work indicates that association of the peptides with the N-terminal domains may generally be part of their binding trajectory.

Interactions of lipopolysaccharide and polymyxin studied by NMR spectroscopy.

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Protein minimization of the gp120 binding region of human CD4.

CD4 is an important component of the immune system and is also the cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. Constructs consisting of all four extracellular domains of human CD4 as well as the first two domains (CD4D12) have previously been expressed and characterized. All of the gp120-binding residues are located within the first N-terminal domain (D1) of CD4. To date, it has not been possible to obtain domain D1 alone in a soluble and active form. Most residues in CD4 that interact with gp120 lie within the region 21-64 of domain D1 of CD4. On the basis of these observations and analysis of the crystal structure of CD4D12, a mutational strategy was designed to express CD4D1 and region 21-64 of CD4 (CD4PEP1) in Escherichia coli. K(D) values for the binding of CD4 analogues described above to gp120 were measured using a Biacore-based solution-phase competition binding assay. Measured K(D) values were 15 nM, 40 nM, and 26 microM for CD4D12, CD4D1, and CD4PEP1, respectively. All of the proteins interact with gp120 and are able to expose the 17b-binding epitope of gp120. Structural content was determined using CD and proteolysis. Both CD4D1 and CD4PEP1 were partially structured and showed an enhanced structure in the presence of the osmolyte sarcosine. The aggregation behavior of all of the proteins was characterized. While CD4D1 and CD4PEP1 did not aggregate, CD4D12 formed amyloid fibrils at neutral pH within a week at 278 K. These CD4 derivatives should be useful tools in HIV vaccine design and entry inhibition studies.