# Protein-Lipid Interactions

Peripheral membrane proteins bind to lipid headgroups in the biomembrane or penetrate slightly into one side of the biomembrane (Figure 1), as opposed to integral membrane proteins, such as ion channels, that fully penetrate and span the membrane. Biochemical reactions that are catalyzed by peripheral membrane proteins are involved in many vital life processes. We have collaborated with Wonhwa Cho (UIC Chemistry) and Ka Yee Lee (U Chicago Chemistry) to investigate the interactions of peripheral membrane proteins in an aqueous solution with a monolayer of lipids at the water surface. Combined monolayer and cell studies have demonstrated the relevance of this model system.

The membrane binding of peripheral proteins involves interactions that depend upon the physico-chemical properties of both membrane and protein. Extensive biophysical and structure-function studies have shown that the membrane binding of peripheral protein is driven by a combination of electrostatic interactions, hydrophobic interactions, and complex interactions involving aromatic side chains.

# Protein Binding Domains

The binding domain is the structural unit of the protein that binds to the lipids. The study of a single type of binding domain is relevant for many proteins because structurally similar binding domains are common to many proteins. For example, we have studied the C2 binding domain of the protein cytosolic phospholipase A2 (cPLA2$$\alpha$$-C2) and the protein kinase C (PKC$$\alpha$$-C2) illustrated in Figure 1. C2 domains are present in a diverse set of proteins that are involved in many different cell functions. The proposed binding mechanism for many C2 containing proteins, including PKC$$\alpha$$-C2, is a Ca2+ induced electrostatic attraction for negatively charged acidic phospholipids. This binding can be considered to be ion pairing between a multivalent cationic protein and negatively charged lipids. In contrast to this, cPLA2$$\alpha$$-C2 represents a different group of C2 domains that bind to neutral zwitterionic lipids.

Part of our contribution to this field has been to develop a new methodology to analyze x-ray reflectivity measurements of protein binding domains bound to Langmuir monolayers of lipids. This methodology incorporates structural information provided by protein crystallography or NMR studies into the analysis, and leads to an understanding of the orientation of the bound protein, as well as its distance of penetration into the lipid layer.

As an example of the application of this methodology, our studies of the cPLA2$$\alpha$$-C2 domain, bound to a layer of SOPC lipids, determined that the Ca2+ is located within 1Å ($$\pm$$3 Å along the layer normal) of the negatively charged phosphate in the lipid. The amino acid residues of the protein that penetrate deepest into the lipid layer are hydrophobic and are located just within the hydrophobic tailgroup region. In addition, our measurements revealed that at least five water molecules hydrated to the lipid headgroup are released upon protein binding. This structural information suggests that the binding mechanism involves electrostatic, hydrophobic, and entropic contributions. Although not surprising in hindsight, the role of entropy, due to the release of water molecules upon binding, was not anticipated.

# References

[1] X-ray Reflectivity Studies of cPLA2$$\alpha$$-C2 Domains Adsorbed onto Langmuir Monolayers of SOPC, Sarka Malkova, Fei Long, Robert V. Stahelin, Sai V. Pingali, Diana Murray, Wonhwa Cho, and Mark L. Schlossman, Biophysical Journal 89, 1861-1873 (2005)

[2] X-ray Reflectivity Studies of p40phox-PX Domains Adsorbed onto Langmuir Monolayers of SOPC/SOPS/PI3P, Sarka Malkova, Robert V. Stahelin, Sai V. Pingali, Wonhwa Cho, and Mark L. Schlossman, Biochemistry 45, 13566-13575 (2006)

[3] Configuration of PKC$$\alpha$$-C2 Domain Bound to Mixed SOPC/SOPS Lipid Monolayers, Chiu-Hao Chen, Sarka Malkova, Sai Venkatesh Pingali, Fei Long, Shekhar Garde, Wonhwa Cho, Mark Schlossman, Biophysical Journal 97, 2794-2802 (2009)

[4] Configuration of Membrane-Bound Proteins by X-ray Reflectivity, Chiu-Hao Chen, Sarka Malkova, Wonhwa Cho, and Mark L. Schlossman, Journal of Applied Physics 110, 102215 (2011)

[5] Molecular Mechanism for Differential Recognition of Membrane Phosphatidylserine by the Immune Regulatory Receptor Tim4, Gregory T. Tietjen, Zhiliang Gong, Chiu-Hao Chen, Ernesto Vargas, James E. Crooks, Kathleen D. Cao, J. Michael Henderson, Charles T. R. Heffern, Mati Meron, Binhua Lin, Benoît Roux, Mark L. Schlossman, Theodore L. Steck, Ka Yee C. Lee, and Erin J. Adams, Proceedings of the National Academy of Sciences (USA) 111, E1463-E1472 (2014)