Molecular Mechanism Of The Self-assembly Of The Activating Immunoreceptor Complex DAP12-NKG2C

Natural killer (NK) cells, the third major lymphocyte population, are involved in the innate immune response against certain infections and Tumors. The activation status of NK cells is delicately balanced by the integration of activating and inhibitory signals at the immunological synapse between the NK cell and a target cell. The DAP12-NKG2C activating immunoreceptor complex is one of the multisubunit transmembrane (TM) protein complexes transducting activating signals in NK cell. DNAX activation protein of 12 kDa (DAP 12) consists of a short extracellular tail, a single transmembrane domain and cytoplasmic signaling module ITAMs, and forms a disulfide-bonded homodimer.DAP 12 dimer associates with the receptor-natural killer group 2C(NKG2C) through contacts between the basic and acidic residues of transmembrane domains. The NMR structure of DAP12-NKG2C complex was determined in micelles recently, providing the first structural insight into the TM contacts within an assembled DAP12-NKG2C immunoreceptor complex. Nonetheless, this complex structure shows a puzzling aspect that only one of the acidic residues is in the direct contact with the basic residue of the NKG2C, which was not consistent with the experimental data. The assembly principle of the complex DAP12-NKG2C is still not well understood and lots of structural details of its signaling mechaism are still missing.Membrane lipid rafts are highly ordered microdomains that enriched in saturated lipids, cholesterol and sphingolipid in plasma membrane.Lipid rafts recruitment of enormous transmembrane proteins involves various membrane signaling and trafficking in multiple bioprocess.Moreover, lipid rafts are also considered as a platform forphosphatidylinositol (4,5) bisphosphate (PIP2) to regulate the ionic lipid-protein interaction. Recently, contradictory conclusions arose while lots of efforts are made to clarify the T cell signaling mechanism in lipid raft, however, the role of lipid raft in NK cell is still not well explicit.In this dissertation, we adopt both experimental methods and molecular dynamics simulations to investigate the molecular mechanism of the signal transduction and self-assembly of the activating immunoreceptor complex DAP12-NKG2C. The main progress lists as follows:1. TOXCAT, as a major experimental method in vivo, along with other methods in vitro, were used to characterize the association interface of the DAP 12 TM homodimer. Instead of the common GXXXG dimerization motif, mutating either of the polar residues D50 and T54 significantly decreased the TOXCAT signal for the dimerization of DAP 12 TM domain. Therefore, D50XXXT54 is determined to be the crucial dimerization interface of DAP 12 homodimer.2. A combined coarse-grained (CG) and atomistic (AT) molecular dynamics simulation has also identified both D50 and T54 at the dimerization interface through the conformational difference between wild-type and mutant DAP 12 TM homodimers. The experimental and computational results of the DAP 12 TM dimer are in excellent agreement with the previously reported NMR structure obtained in detergent micelles.3. Both coarse grained and atomistic molecular dynamic simulation methods were applied to investigate the self-assembly dynamics of the transmembrane domains of the TM domain of DAP12-NKG2C activating immunoreceptor complex. We demonstrated that a five-polar-residue motif including 2 Asps and 2 Thrs in DAP 12 dimer, as well as 1 Lys in NKG2C TM, which plays an important role in the assembly structure of the DAP12-NKG2C TM heterotrimer, which refined its NMR structure.Furthermore, we provided clear evidences to exclude the possibility that another NKG2C could stably associate with the DAP12-NKG2C heterotrimer. Based on the simulation results, we proposed a revised assembly principle scheme for the DAP12-NKG2C activating immunoreceptor complex.4. We investigated the locations of the DAP12-NKG2C complex and PIP2 in a mixed lipid bilayer which can form lipid raft by coarse grained simulations. Our results firstly demonstrated that the TM domain of DAP12-NKG2C complex was resided in non-raft domain while the juxtamembrane (JM) domain was recruited to the raft domain/non-raft domain joints due to the ionic interaction between its polybasic residue region and the acidic lipid PIP2. Meanwhile, we showed that PIP2 waslocated on the raft domain and clustered around the JM domain of DAP12-NKG2C, and Ca2+ could regulate the signal transduction of DAP12-NKG2C by modulating the interaction between the JM domainand PIP2.