DETERMINATION OF THE KINETIC ROLES OF THE MEMBRANE IN THE CATALYSIS OF RASGEFS BY USING AN ENGINEERED MODEL PROTEIN

Abstract

Ras guanine nucleotide exchange factors (RasGEFs), which include Son of Sevenless (SOS), Ras Guanine Nucleotide Releasing Protein (RasGRP), and Ras Guanine Nucleotide Releasing Factor (RasGRF), catalyze nucleotide exchange of Ras upon activating signals. Crystal structure analyses suggest that catalytic cavities of RasGEFs are not deep enough to uphold their substrates. Given that RasGEFs are all membrane binding proteins, a novel “membrane supplement hypothesis” is proposed that RasGEFs in cytoplasm are catalytically unformed; and RasGEFs on membrane are catalytically competent. The reason RasGEFs are catalytically unformed without the support of membrane is because the catalytic sites of RasGEFs are not sufficiently deep to hold their substrates within their catalytic sites for their catalysis. It was shown that the catalytic function of SOS, one of RasGEFs, is enhanced upon its binding to membrane, supporting this membrane supplement hypothesis. To examine the membrane supplement hypothesis, an engineered RasGRP is designed, in which a catalytically unformed RasGRP is fused with a foreign protein that mimics the plasma membrane. Comparative kinetic analyses of the catalytically unformed RasGRP with the engineered RasGRP showed that the fused-foreign protein of engineered RasGRP functioned to hold and precisely orientate the substrate Ras within the catalytic site of engineered RasGRP, thereby making the engineered RasGRP become catalytically efficient. RasGEFs are classified as amphitropic enzymes, which adapt a compact conformation in the cytosol and are fully activated when recruited to membrane. The catalytic sites of many amphitropic proteins, including RasGEFs, are brought proximally toward the membrane interface after membrane binding event, and furthermore, the catalysis of these amphitropic enzymes is enhanced upon their membrane binding. However, the role of the membrane in the kinetic action of these amphitropic enzymes has not been previously proposed. The membrane supplement hypothesis in this study provides a rationalization that these amphitropic enzymes also possess a shallow pocket at their catalytic sites, challenging them to hold the bulky substrates. Therefore, locating the catalytic site of these amphitropic enzymes close to the membrane interface strengthens their substrate binding interactions, thereby facilitating their catalytic functions.