Vishwanath Koppaka

The Structure of Human Lipoprotein A-I EVIDENCE FOR THE “BELT” MODEL

The two main competing models for the structure of discoidal lipoprotein A-I complexes both presume that the protein component is helical and situated around the perimeter of a lipid bilayer disc. However, the more popular “picket fence” model orients the protein helices perpendicular to the surface of the lipid bilayer, while the alternative “belt” model orients them parallel to the bilayer surface. To distinguish between these models, we have investigated the structure of human lipoprotein A-I using a novel form of polarized internal reflection infrared spectroscopy that can characterize the relative orientation of protein and lipid components in the lipoprotein complexes under native conditions. Our results verify lipid bilayer structure in the complexes and point unambiguously to the belt model.

BINDING OF BOVINE FACTOR VA TO PHOSPHATIDYLCHOLINE MEMBRANES

The interaction of bovine factor Va with phosphatidylcholine membranes was examined using four different fluorescence techniques: 1) changes in the fluorescence anisotropy of the fluorescent membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to monitor the interaction of factor Va with 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), 2) changes in the fluorescence anisotropy of N-(lissamine rhodamine B sulfonyl) diacyl phosphati-dylethanolamine (Rh-PE) incorporated into SUVs prepared from 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), 3) changes in the fluorescence anisotropy of fluorescein-labeled factor Va (labeled in the heavy chain) upon interaction with POPC SUVs, 4) fluorescence energy transfer from fluorescein-labeled factor Va to rhodamine-labeled POPC SUVs. In the first two sets of experiments, labeled lipid vesicles were titrated with unlabeled protein, whereas, in the latter two types of experiments, labeled factor Va was titrated with vesicles. For the weak binding observed here, it was impossible from any one binding experiment to obtain precise estimates of the three parameters involved in modeling the lipid-protein interaction, namely, the dissociation constant Kd, the stoichiometry of binding i, and the saturation value of the observable Rmax from any one experiment. However, a global analysis of the four data sets involving POPC SUVs yielded a stable estimate of the binding parameters (Kd of approximately 3.0 microM and a stoichiometry of approximately 200 lipids per bound factor Va). Binding to DMPC SUVs may be of slightly higher affinity. These observations support the contention that association of factor Va with a membrane involves a significant acidic-lipid-independent interaction along with the more commonly accepted acidic-lipid-dependent component of the total binding free energy.

Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.

Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex

Factor Va is an essential protein cofactor of the enzyme factor Xa, which activates prothrombin to thrombin during blood coagulation. Peptides with an apparent Mr of approximately 94,000 (heavy chain; HC) and approximately 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active Va. The two forms of Va-LC differ in their carboxyl-terminal C2 domain. Using Va reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and on the activity of membrane-bound Va. We found that 1) Va composed of the 72,000 LC bound only slightly more tightly to membranes composed of a mixture of neutral and acidic lipids, the Kd being reduced by a factor of approximately 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2) The two forms of Va seemed to undergo different conformational changes when bound to a membrane. 3) The activity of bovine Va varied somewhat with LC species, the difference being greatest at limiting Xa concentration. We have also addressed the role of the two Va peptides in membrane lipid rearrangements and binding: 1) Va binding increased lateral packing density in mixed neutral/acidic lipid membranes. In the solid phase, Va-HC had no effect, whereas Va-LC and whole Va had similar but small effects. In the fluid phase, Va-HC and whole Va both altered membrane packing, with Va-HC having the largest effect. 2) Va-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (Kd, approximately 0.3 microM; stoichiometry approximately 91). High ionic strength had little effect on binding. 3) The substantial effect of Va on packing within neutral phospholipid membranes was mimicked by Va-HC. 4) Based on measurements of membrane phase behavior, binding of Va or its peptide components did not induce thermodynamically discernible lateral membrane domains. These results suggest that the membrane association of factor Va is a complex process involving both chains of Va, changes in lipid packing, and changes in protein structure.

Lipoprotein A-I structure.

High density lipoproteins are produced by the liver as protein-lipid complexes with a characteristic discoidal shape. A crystal structure is available for the chief protein component of these complexes, apolipoprotein A-I, but controversy about how this protein is situated with respect to the lipid components has flourished for lack of experimental techniques that can characterize protein structure in a lipid environment. New spectroscopic techniques developed to address this problem now indicate that apolipoprotein A-I is arranged as a helical belt around a bilayer of phospholipids. This is an important step towards understanding how these lipoproteins regulate cholesterol transport.