Are acidic lipid domains induced by extrinsic protein binding to membranes?

Abstract

A widely accepted model for the association of extrinsically bound proteins with acidic lipid-containing membranes has been that approach of the protein to the membrane induces a domain of acidic lipids that serves as the protein binding site. This model has been applied to a variety of membrane proteins including those that participate in the proteolytic complex that converts prothrombin to thrombin during the final stages of the blood coagulation cascade. The ‘prothrombinase complex’ consists of a serine protease (factor X_a), its protein co-factor (factor V_a) and the substrate itself (prothrombin), all bound to phosphatidyl-serine (PS)-containing membranes derived from stimulated platelets. We have used three approaches to test the domain model as it applies to the proteins of this complex. First, phase diagrams describing the mixing of acidic and neutral lipids have failed to provide evidence for extensive acidic lipid domains (on the order of 50 or more lipid molecules) induced by protein biding. Second, pyrene-containing neutral and acidic phospholipids have been used to test for the occurrence of domains of as few as 20–3.

Iipids associated with binding of the membrane-binding fragment 1 region of prothrombin. Again, no evidence for domains was obtained. Finally, we have shown that binding of these proteins can be described in terms of a generalized model that presumes an acidic-lipid-independent surface adsorption combined with specific binding of acidic lipids to ‘m’ sites on a protein. Our results suggest that the concept of a protein-induced domain should not be applied indistriminately to explain binding of extrinsic membrane proteins such as the protein kinase C. The generalized model we have put forward probably has application to the binding of other extrinsic membrane proteins and can be used to test the consistency of the domain model with raw binding data if expereiments are performed at several acidic lipid surface concentrations.

Key Words:

• Blood coagulation proteins
• membrane structure
• binding