CRYSTALLIZATION AND CRYSTALLOGRAPHIC STUDIES OF THE HUMAN PLASMA LIPOPROTEINS, LOW DENSITY LIPOPROTEIN (LDL) AND APOLIPOPROTEIN-H

R. Schwarzenbacher, K. Zeth, A.Gries, M. J. Chapman, R. Prassl and P. Laggner

¹Institute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Graz, Austria;
²Faculty of Biology, University of Konstanz, POB-55 60, 78457-Konstanz, Germany;
³Institute of Physiology, University of Graz, Harrachgasse 21, A-8010 Graz, Austria;
⁴INSERM Unite 321, Paris, France.

Knowledge of the molecular protein structure of apo-B100 (Mw=550KD) constitutes a major step towards an understanding of LDL (Mw=2.6MD) interaction with its cellular receptor and equally allows insight into mechanisms of LDL oxidation. To overcome this barrier, a systematic search was made for suitable conditions to crystallize LDL as a prerequisite for X-ray crystallographic analysis. Following this strategy, microscopic single crystals have been obtained from narrow density subspecies of the LDL spectrum. Preliminary X-ray data analysis, so far to a resolution of about 28A (14 A), indicate different crystalline forms, i.e. P1 ( a= 161 A, b= 425 A, c=180 A, a= 96.2°, b= 92.1°, g= 102°) and P4₂1₂ ( a=b=425 A, c=180 A). Further efforts are under way to construct a low resolution model in combination with data obtained from Electron Microscopy. Owing to the instability, susceptibility to oxidation and temperature sensitivity of the crystals, as well as, the heterogeneity of LDL subfractions between donors, the determination of the three-dimensional static structural domains of apo-B100 will be a sophisticated task, but the present results are strong indicators of the feasibility of the study.

Apolipoprotein-H (apo-H), also known as ß2-glycoprotein-I, present on circulating lipoproteins, preferentially binds to negatively charged phospholipids such as cardiolipin and acts as cofactor for the binding of antiphospholipid autoantibodies in patients with antiphospholipid syndrome. Apo-H is a glycoprotein with 50kDa molecular mass and shows extensive internal homology with five consecutive domains of 60 amino acid residues each. The fifth domain was suggested as lipid binding region. Nevertheless, detailed knowledge on the underlying molecular mechanisms and structural requirements for interaction are still missing. To solve the high resolution structure of apo-H we have grown single crystals of apo-H from ammonium sulfate solutions by hanging drop vapour diffusion techniques. X-ray diffraction patterns have been collected to a resolution of 3 and the primary crystallographic data have been determined as orthorhombic space group C2221, with cell dimensions of a=158.47 A, b=169.25 A, c=113.28 A (at 100 K) and one tetramer per asymmetric unit. Efforts to overcome the phase -problem with the MIRAS-approach are just under progress.