Leonard J. Banaszak

Lipid-Binding Proteins: A Family of Fatty Acid and Retinoid Transport Proteins

Publisher Summary This chapter focuses on the structural analyses and comparisons between members of a multigene family of hydrophobic ligand-binding proteins. It discusses the structural motif, general characteristics of the binding cavity, ligand entry, and the portal hypothesis and provides a detailed comparison of intra- and extracellular lipid binding proteins with known crystal structures. The members of this family are referred as lipid-binding proteins (LBPs). This collection of proteins can be subdivided into two groups: the intracellular lipid-binding protein family (iLBP) and the extracellular lipid binding protein family (eLBP). The comparison primarily deals with the iLBP branch because this family is becoming structurally well characterized. However, the structural comparisons are extended to some members of the eLBP family because the basic structural motif used to bind hydrophobic ligands applies to both. The products of hydrolysis of the intestinal lipids, including fatty acids, cholesterol, monoglycerides, and lysophospholipids, have very low solubilities and are absorbed by biliary micelles in the gut. These micelles diffuse through the glycocalyx, which stabilizes an unstirred water layer at the surface of the enterocyte. The chapter concludes with a discussion of the results of site-directed mutagenesis studies, the thermodynamics of lipid binding, and considerations of protein stability and folding.

The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates.

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 Å and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the β-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains.

Polypeptide conformation of cytoplasmic malate dehydrogenase from an electron density map at 3.0 Å resolution

Abstract The polypeptide chain conformation of both subunits of eytoplasmic malate dehydrogenase has been determined from an electron density map at 3.0 A resolution. The two polypeptide chains are believed to be sequentially identical, but in the crystals studied, only one subunit will bind additional coenzyme, nicotinamide adenine dinucleotide. The present interpretation of the chain-folding places 665 alpha carbons in the dirner. A least-squares fit of these alpha carbon positions indicates that the two subunits in the dimer are closely related by 2-fold rotational symmetry. The folding of the polypeptide chains of malate dehydrogenase involves considerable amounts of secondary structure. The first 150 residues from the amino terminal end of the chain involve alternating parallel extended and helical conformations. The parallel-extended segments form a twisted sheet, one region of which includes the active site. The remaining polypeptide chain cannot be described in such a regular manner. It does, however, include helical segments and regions of anti-parallel extended polypeptide chain. The interface between the two subunits consists of three helical regions from each subunit. The polypeptide conformation of malate dehydrogenase is remarkably similar to that of dogfish lactate dehydrogenase. While the atomic co-ordinates of malate dehydrogenase will be improved by a higher resolution electron density map, it is already clear that these two different enzymes have fundamentally the same conformation. The one exception to the structural homology is the absence of the lactate dehydrogenase extended arm in the malate dehydrogenase structure. This arm in lactate dehydrogenase contains the first 22 amino acids at the N-terminal. In addition to the structural homology in the polypeptide conformation, both enzymes bind nicotinamide adenine dinucleotide in structurally homologous regions.

The structural basis of lipid interactions in lipovitellin, a soluble lipoprotein

BACKGROUND The conformation and assembly of lipoproteins, protein containing large amounts of noncovalently bound lipid, is poorly understood. Lipoproteins present an unusual challenge as they often contain varying loads of lipid and are not readily crystallized. Lipovitellin is a large crystallizable oocyte protein of approximately 1300 residues that contains about 16% w/w lipid. Lipovitellin contains two large domains that appear to be conserved in both microsomal triglyceride transfer protein and apolipoprotein B-100. To gain insight into the conformation of a lipoprotein and the potential modes of binding of both neutral and phospholipid, the crystal structure of lamprey lipovitellin has been determined. RESULTS We report here the refined crystal structure of lipovitellin at 2.8 A resolution. The structure contains 1129 amino acid residues located on five peptide chains, one 40-atom phosphatidylcholine, and one 13-atom hydrocarbon chain. The protein contains a funnel-shaped cavity formed primarily by two beta sheets and lined predominantly by hydrophobic residues. CONCLUSIONS Using the crystal structure as a template, a model for the bound lipid is proposed. The lipid-binding cavity is formed primarily by a single-thickness beta-sheet structure which is stabilized by bound lipid. This cavity appears to be flexible, allowing lipid to be loaded or unloaded.

A Common Binding Site on the Microsomal Triglyceride Transfer Protein for Apolipoprotein B and Protein Disulfide Isomerase

The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI). In the MTP complex, the amino-terminal region of MTP (residues 22–303) interacts with the amino-terminal region of apoB (residues 1–264). Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517–603 of MTP. PDI binds in close proximity to this apoB binding site on MTP. The proximity of these binding sites on MTP for PDI and amino acids 512–721 of apoB was evident from studies carried out in a yeast two-hybrid system and by co-immunoprecipitation. The expression of PDI with MTP and apoB16 (residues 1–721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73%. The interaction of residues 512–721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.

5‐Hydroxydecanoate is metabolised in mitochondria and creates a rate‐limiting bottleneck for β‐oxidation of fatty acids

5‐Hydroxydecanoate (5‐HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP‐sensitive K+ (KATP) channels. However, recent work has shown that 5‐HD is activated to 5‐hydroxydecanoyl‐CoA (5‐HD‐CoA), which is a substrate for the first step of β‐oxidation. We have now analysed the complete β‐oxidation of 5‐HD‐CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl‐CoA. At the second step of β‐oxidation, catalysed by enoyl‐CoA hydratase, enzyme kinetics were similar using either decenoyl‐CoA or 5‐hydroxydecenoyl‐CoA as substrate. The last two steps were investigated using l‐3‐hydroxyacyl‐CoA dehydrogenase (HAD) coupled to 3‐ketoacyl‐CoA thiolase. Vmax for the metabolite of 5‐HD (3,5‐dihydroxydecanoyl‐CoA) was fivefold slower than for the corresponding metabolite of decanoate (l‐3‐hydroxydecanoyl‐CoA). The slower kinetics were not due to accumulation of d‐3‐hydroxyoctanoyl‐CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5‐dihydroxydecanoyl‐CoA suggested that the 5‐hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5‐hydroxydecanoyl‐CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 μm 5‐HD‐CoA inhibited the metabolism of decanoyl‐CoA or lauryl‐carnitine. In conclusion, 5‐HD is activated, transported into mitochondria and metabolised via β‐oxidation, albeit with rate‐limiting kinetics at the penultimate step. This creates a bottleneck for β‐oxidation of fatty acids. The complex metabolic effects of 5‐HD invalidate the use of 5‐HD as a blocker of mitochondrial KATP channels in studies of preconditioning.

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.

Sequence of lamprey vitellogenin: Implications for the lipovitellin crystal structure☆

The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.

The preparation of the cytoplasmic and mitochondrial forms of malate dehydrogenase and aspartate aminotransferase from pig heart by a single procedure

Abstract A single procedure for the preparation of the mitochondrial and cytoplasmic forms of malate dehydrogenase and aspartate aminotransferase from pig heart is described. l -3-Hydroxyacyl CoA dehydrogenase may also be obtained from this procedure. The five enzymes are obtained in preparative amounts in homogeneous form with specific activities equal to or higher than those previously reported. These experiments also have established that the subforms of the mitochondrial isozyme of malate dehydrogenase are apparently the result of preparative manipulations.

Structural properties of the adipocyte lipid binding protein

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.