We are requesting funding for acquiring a 700-MHz solution NMR spectrometer to satisfy the rapidly rising need of high-field NMR access at Harvard Medical School (HMS) for structural studies of challenging biological systems, in particular, membrane proteins. Recently, the Chou and Wagner labs spearheaded the effort to obtain atomic resolution structures of membrane proteins using solution NMR, e.g., the structures of M2 H+ channel from influenza virus (Schnell &Chou, Nature 2008) and the voltage-gated anion channel (VDAC-1) of mitochondrion (Hiller et al, Science 2008). The success has spurred a wave of collaborations that involves structural characterization of membrane proteins by NMR. The ongoing projects are of high academic and medical interests, but their progress is slow due to the aging NMR facility at HMS. The new spectrometer, which would significantly accelerate the projects, will be largely operated and maintained by James Chou (PI) and major users including Gerhard Wagner, Ellis Reinherz, Christopher Walsh, Stephen Harrison, Bing Chen, and Kai Wucherpfennig. The following projects would immediately benefit from the instrument. (1) In their continued focus on proton translocators, the Chou lab will determine the structure of the BM2 H+ channel of influenza B virus, for providing structural details for designing new anti-flu compounds. They will also complete the structure of the 32 kDa protein in the mitochondrial inner membrane, UCP2, which uncouples the H+ electrochemical potential for heat generation. UCP2 inhibitors could restore pancreatic 2-cell glucose sensing, for the treatment of type-2 diabetes. (2) The Wagner lab will determine the structural basis of recognition between VDAC-2 and the pro-apoptotic protein Bak. Disrupting the VDAC-2/Bak interaction with small molecules may have anti-tumor activity. In another project, they will study the macromolecular complexes involved in the RNA translation initiation. (3) The Reinherz group will study the structures of the HIV-1 MPER domain with transmembrane segment attached to the lipid bicelles. Structural details will guide HIV vaccine design. (4) Also in an effort to guide HIV vaccine design, the Chen and Harrison labs will determine by NMR the structure of the fusion intermediate state of the HIV gp41 fusion protein, which is difficult to crystallize. (5) The Wucherpfennig lab will continue their study of the membrane regions of immunoreceptors, by solving the structure of an activating immune receptor complex from the Natural Killer cells, consisting of the transmembrane domains of the dimeric DAP12 signaling module and the NKG2C receptor chain. (6) The Walsh lab has a long-term interest in the mechanism of the enterobactin non-ribosomal peptide synthetase, a mega enzyme consisting of multiple functional domains. After determining the structure of the 37 kDa EntF domain (Frueh et al., Nature 2008), they will solve the structure of the 50 kDa C-domain and characterize its interactions with EntF and EntB. Structural details may lead to the design of new antibiotics. Users who are not NMR experts will carry out the experiments in collaboration with James Chou and Gerhard Wagner.
Studies of the viral proton channels by the Chou lab will inspire new therapeutic approaches for fighting flu infections, whereas the HIV related projects from the labs of Reinherz, Harrison, and Chen will guide HIV vaccine design. The mitochondrial voltage-gated anion channel is one of the centerpieces in the programmed cell-death, and thus understanding its interaction with the pro- and anti-apoptotic factors can potentially identify new cancer therapeutic targets. The proposed 700 MHz NMR spectrometer would provide us sufficient instrumentation to move the above studies forward, while maintaining the postdoctoral fellow positions involved in these projects;it has no environmental impact.
