Molecular Mechanism For The Specific Interaction Of K-RasB With Calmodulin

Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood. Here, by applying chemical cross-linking, fluorescence spectroscopy and isothermal titration calorimetry (ITC), we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca2+/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca2+/CaM formed a1:1complex with an equilibrium association constant around105M-1, whereas no binding reaction of K-RasB-(DESGPC) with Ca2+/CaM is detected. In addition, an H-Ras mutant in this region, H-Ras-(D175A, E176A), binds to Ca2+/CaM with moderate affinity. Furthermore, the interaction of K-RasB with Ca2+/CaM is found to be enhanced by the farnesylation of K-RasB. We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca2+/CaM, but also defines its isoform specific interaction with Ca2+/CaM. The famesylation of K-RasB is also important for its specific interaction with Ca2+/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.We have studied the interaction of CaM with C terminal hexahis tagged K-RasB and K-RasB-(1-166) by using chemical cross-linking, ITC, fluorescence titration and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Cross-linking data indicate a1:1complex of K-RasB-His and Ca2+/CaM formed, which is confirmed by MADLI-TOF MS using K-RasB-(1-166)-His and Ca2+/CaM. ITC and fluorescence titration experiments have further revealed moderate, micromolar binding affinity of Ca2+/CaM with K-RasB-His and K-RasB-(1-166)-His. These results provide a new application for studying noncovalent protein complex by MADLI-TOF MS.Intracellular environment is highly crowded due to the presence of high concentrations of soluble and insoluble macromolecules including proteins, nucleic acids, ribosomes, and carbohydrates. β-Arrestin-1binds to G protein coupled receptors (GPCRs) and sequests downstream signal and facilitate the desensitization and interlization of GPCRs. We have studied the interaction between β-arrestin-1and Arf6in dilute solution and crowded environment by using fluorescence titration. We have found that β-arrestin-1binds to both Arf6-GTP and Arf6-GDP. In addition, we also study the effect of macromolecular crowding agent PEG20000, dextran70, Ficoll400and Ficoll70, as well as osmotic agent glucose, sucrose, glycerol and sarbitol on the interaction. We have found that these agents remarkably promote the interaction between β-arrestin-1and Arf6-GDP, but significantly inhibit the interaction between β-arrestin-1and Arf6-GTP. These results indicate that intracellular crowded environment could regulate the interaction between β-arrestin-1and Arf6, and this kind of regulation could be universal to intracellular protein-protein interactions.