Studies Of SNARE Protein Ykt6 Conformation Regulation

In eukaryotic cells, the dynamic trafficking of macromolecule such as proteins and lipids between organelles is closely linked to vesicle trafficking. Membrane-bound vesicles can pinch off from one membrane and fuse with another. The whole process of vesicle trafficking contains budding of transport vesicles, directional movement of vesicles along the cytoskeleton, vesicles anchoring and membrane fusion. To different classes of transport vesicles, the specific targeting and fusion to their distinct membrane destinations, relies on the precise pairing of cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) anchored separately to vesicle membrane and target membrane, which can ensure the integrity and functions of organelles.Ykt6 is the most conserved and versatile SNARE, which is an essential protein involved in multiple membrane fusion reactions. It is consisted of an N-terminal regulatory longin domain, a conserved central 60-70 amino acid "SNARE core" and a C-terminal "CCAIM" motif. In the structure, the SNARE core mediates the specific targeting and fusion of different classes of transport vesieles to their distinct membraned destinations, and the longin domain is able to regulate the activity of SNARE core. Ykt6 has large conformational changes during the process of its functions. It was found that addition of a long acyl chain fatty acid:DPC into Ykt6, the homogenous conformation is attributed to a closed state, which is similar to farnesylated Ykt6 under physiological conditions. In this structure, the SNARE core folds around the longin domain and forms a hydrophobic groove at their interface to accommodate the entire hydrophobic tail of DPC. But the lipidation of CCAIM sequence can be induced to be open conformation, regulate proteins interconverting conformational states between closed and open conformation. Because the crystal structure of Ykt6 is in DPC-binding state, the conformation regulatory mechanism of Ykt6 still has many unsolved problems. Firstly, whether the crystal structure of Ykt6 in DPC-binding state is the structure of farnesylated Ykt6 under physiological conditions in a closed state is not clear. Secondly, it is not clear the mechanism of farneysl side chain and DPC stabilizing Ykt6 protein structure. Thirdly, the process of SNARE core dissociating from longin domain when Ykt6 completes the transition from closed state to open state is not clear.We characterized the unlipidated state, the DPC-binding state and the famesylated state of Ykt6 by molecular dynamics simulations and studied the effect of farneysl side chain and DPC on Ykt6 conformation in detail. In our work, we used steered molecular dynamics simulation in addition to conventional molecular dynamics simulation, with additional force to accelerate the conformation changes of protein conformation. We found the unlipidated state is alternating among different conformational states as experiments revealed and Ykt6 shows a collapsed hydrophobic groove because of lacking in hydrophobic chain. DPC interacts with Ykt6 by both its hydrophobic and hydrophilic end and concentrates the conformation, thus localizing the protein in one of the unlipidated conformational state. Famesylated state is similar to DPC-binding state by stabilizing the closed state and also makes the protein in a local area in the conformational space. We propose this process fits the "conformational selection" mechanism. That is to say, the population of conformational states is changed by lipidation and one of the states is selected to take the dominance. The steered molecular dynamics simulation of Ykt6 in the unlipidated state indicates the process of SNARE core dissociating from longin domain can be divided into three stages:aFG, aE and βF. We also characterized the DPC-binding state and the famesylated state of Ykt6 by steered molecular dynamics simulations of constant velocity. We found the external force of unlipidated state is least. This suggests that farneysl side chain and DPC stabled the structure of Ykt6, making the combination of SNARE core and longin domain stronger.Our work shows the dynamics-function relationship is greatly important to Ykt6. SNARE Ykt6 can response to lipid binding or lipidation with its hydrophobic groove and shift the population of conformational states. Besides, our work reveals that molecular dynamics simulation and steered molecular dynamics are very powerful tools for the studies on large-scale motion of proteins and other biological macromolecules. We anticipate that computational simulation will be more powerful with the development of computer technology and play a more important role in the exploration of structure-dynamies-function relationship of biological macromolecules.