Xavier Lee

Double Superhelix Model of High Density Lipoprotein

High density lipoprotein (HDL), the carrier of so-called “good” cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity.

The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.

Congruency between biophysical data from multiple platforms and molecular dynamics simulation of the double-super helix model of nascent high-density lipoprotein

The predicted structure and molecular trajectories from >80 ns molecular dynamics simulation of the solvated Double-Super Helix (DSH) model of nascent high-density lipoprotein (HDL) were determined and compared with experimental data on reconstituted nascent HDL obtained from multiple biophysical platforms, including small angle neutron scattering (SANS) with contrast variation, hydrogen-deuterium exchange tandem mass spectrometry (H/D-MS/MS), nuclear magnetic resonance spectroscopy (NMR), cross-linking tandem mass spectrometry (MS/MS), fluorescence resonance energy transfer (FRET), electron spin resonance spectroscopy (ESR), and electron microscopy. In general, biophysical constraints experimentally derived from the multiple platforms agree with the same quantities evaluated using the simulation trajectory. Notably, key structural features postulated for the recent DSH model of nascent HDL are retained during the simulation, including (1) the superhelical conformation of the antiparallel apolipoprotein A1 (apoA1) chains, (2) the lipid micellar-pseudolamellar organization, and (3) the solvent-exposed Solar Flare loops, proposed sites of interaction with LCAT (lecithin cholesteryl acyltransferase). Analysis of salt bridge persistence during simulation provides insights into structural features of apoA1 that forms the backbone of the lipoprotein. The combination of molecular dynamics simulation and experimental data from a broad range of biophysical platforms serves as a powerful approach to studying large macromolecular assemblies such as lipoproteins. This application to nascent HDL validates the DSH model proposed earlier and suggests new structural details of nascent HDL.

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion

Small-angle neutron scattering (SANS) with contrast variation was used to obtain the low-resolution structure of nascent HDL (nHDL) reconstituted with dimyristoyl phosphatidylcholine (DMPC) in the absence and presence of cholesterol, [apoA1:DMPC (1:80, mol:mol) and apoA1:DMPC:cholesterol (1:86:9, mol:mol:mol)]. The overall shape of both particles is discoidal with the low-resolution structure of apoA1 visualized as an open, contorted, and out of plane conformation with three arms in nascent HDL/dimyristoyl phosphatidylcholine without cholesterol (nHDLDMPC) and two arms in nascent HDL/dimyristoyl phosphatidylcholine with cholesterol (nHDLDMPC+Chol). The low-resolution shape of the lipid phase in both nHDLDMPC and nHDLDMPC+Chol were oblate ellipsoids, and fit well within their respective protein shapes. Modeling studies indicate that apoA1 is folded onto itself in nHDLDMPC, making a large hairpin, which was also confirmed independently by both cross-linking mass spectrometry and hydrogen-deuterium exchange (HDX) mass spectrometry analyses. In nHDLDMPC+Chol, the lipid was expanded and no hairpin was visible. Importantly, despite the overall discoidal shape of the whole particle in both nHDLDMPC and nHDLDMPC+Chol, an open conformation (i.e., not a closed belt) of apoA1 is observed. Collectively, these data show that full length apoA1 retains an open architecture that is dictated by its lipid cargo. The lipid is likely predominantly organized as a bilayer with a micelle domain between the open apoA1 arms. The apoA1 configuration observed suggests a mechanism for accommodating changing lipid cargo by quantized expansion of hairpin structures.

Purification and crystallization of a novel membrane-anchored protein: the Schistosoma haematobium serpin

A unique serine-protease inhibitor (serpin) of the blood fluke S. haematobium has been crystallized. It is an antitrypsin with an unusual residue (phenylalanine) at its reactive center. Unlike any known member of this gene family, it is a membrane-anchored protein on the surface of the parasite. The location of this serpin and immunological response to the protein indicate that it may play a important role in host-parasite interaction. The crystals belong to the trigonal space group P3221 or P3121 with unit-cell parameters a = b = 64.7, c = 186.7 A, alpha = 90.0, beta = 90.0, gamma = 120.0 degrees. There is one molecule per asymmetric unit and the crystals diffracted to 2.2 A.

Characterization of the secondary binding sites of Maclura pomifera agglutinin by glycan array and crystallographic analyses.

The Maclura pomifera agglutinin (MPA) recognizes the T-antigen disaccharide Galβ1,3GalNAc mainly through interaction of the α-GalNAc moiety with its primary site, but the interactions of the two flanking subsites A and B with aglycones and substituents other than Gal, respectively, are not well understood. We therefore characterized the specificity of MPA in more detail by glycan microarray analysis and determined the crystal structures of MPA without ligand and in complexes with Galβ1,3GalNAc and p-nitrophenyl α-GalNAc. In both sugar complexes, pairs of ligands created inter-tetramer hydrogen-bond bridging networks. While subsite A showed increased affinity for hydrophobic aglycones, it also accommodated several sugar substituents. Notably, a GalNAc-O-tripeptide, a Tn-antigen mimic, showed lower affinity than these compounds in surface plasmon resonance (SPR) experiments. The glycan array data that showed subsite B accepted compounds in which the O3 position of the GalNAc was substituted with various sugars other than Gal, but substitutions at O6 led to inactivity. Additions to the Gal moiety of the disaccharide also had only small effects on reactivity. These results are all compatible with the features seen in the crystal structures.

Crystal growth of human estrogenic 17β-hydroxysteroid dehydrogenase

Estrogenic 17beta-hydroxysteroid dehydrogenase from human placenta, an enzyme of low solubility, has been crystallized in the complex form with its cofactor NADP(+). These are the first crystals with X-ray diffraction quality for structure analysis from any human steroid-converting enzyme. The crystals were grown by vapor diffusion in the presence of 0.06% beta-octylglucoside, using polyethylene glycol 4000 as the precipitant (27-28%) and one of several different salts at pH 7.5 and room temperature. Crystals grown with magnesium chloride diffract up to 2.4 A. The most important steps leading to the rapid success of the crystallization of this labile enzyme were the following: preparation of a highly active and homogeneous enzyme protein using a rapid procedure; the choice of a suitable enzyme buffer system and a detergent favorable to maintaining high activity and solubility for the enzyme; and a combined screening procedure. The present study could be useful for the successful crystal growth of other hydrophobic or membrane-bound proteins.

Three-dimensional structure of a schistosome serpin revealing an unusual configuration of the helical subdomain.

Parasitic organisms are constantly challenged by the defence mechanisms of their respective hosts, which often depend on serine protease activities. Consequently, protease inhibitors such as those belonging to the serpin superfamily have emerged as protective elements that support the survival of the parasites. This report describes the crystal structure of ShSPI, a serpin from the trematode Schistosoma haematobium. The protein is exposed on the surface of invading cercaria as well as of adult worms, suggesting its involvement in the parasite-host interaction. While generally conforming to the well established serpin fold, the structure reveals several distinctive features, mostly concerning the helical subdomain of the protein. It is proposed that these peculiarities are related to the unique biological properties of a small serpin subfamily which is conserved among pathogenic schistosomes.

Primary Sequence and Refined Tertiary Structure of Pseudomonas fluorescens Holo Azurin at 2.05 A

This paper reports the primary sequence and refined crystal structure of Pseudomonas fluorescens holoazurin. The crystal structure has been determined by molecular replacement on the basis of the molecular model of azurin from Alcaligenes denitrificans, and refined by the method of molecular dynamics simulation and energy-restrained least-squares methods. P. fluorescens was crystallized in the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 31.95, b = 43.78, c = 78.81 A. The asymmetric unit is composed of only one molecule. The final R value is 16.7% for 6691 reflections to a resolution of 2.05 A. This azurin structure shows some interesting features at His35 and His83. Part of the main chain of strand 3 including His35 O are involved in the contact between two symmetrically related molecules. P. fluorescens is also compared with the other azurin structures in terms of primary sequence, crystal packing, solvent structure and Cu-site geometry. The difference in fluorescence decay behavior of two holoazurins from P. fluorescens and P. aeruginosa and the correlation between the fluorescence quenching and electron transfer are discussed.