The Structure And Function Of Human Mog1 And Ran Protein Complex By NMR Method

The small GTPase Ran,plays important roles in multiple cellular processes,e.g.,nucleus-cytoplasm transportion,mitosis spindle assembly and nuclear envelope assembly.Mogl(Myelin Oligodendrocyte Glycoprotein)has been recently identified as a Ran binding protein,where in vitro assays demonstrated that Mogl can promote the relase of GTP or GDP from Ran.It,however,remains elusive regarding the underlying molecular mechanism and biological roles of the regulation of Ran function by Mogl.Although the crystal structure and solution structures of Ran and Mogl have been solved,respectively,the structural characterization of the Mogl/Ran complex remains a challenge due to the dynamic nature and the multiple flexible regions(? and? of Ran and several long loops of Mogl)in the complex.NMR is particularly suited to elucidate the structure and dynamics of protein complexes even in the presence of conformational flexibility.Several years ago,Dr.Qi Hu and Ying Liu solved the solution structure of hMogl,and thus paved an avenue for us to exploit the structure and functions of the 47 kDa Mogl/Ran complex,using the state-of-the-art NMR techniques.The chemical shift perturbation experiment demonstrated that the apo and holo forms were in slow exchange,with gradual disappearance of the old peaks and appearance of new resonance.The re-assignment of the complex peaks was therefore is required,which was difficult to obtain using the standard sequential assignment strategy,because of the perdeuterated sample instability during unfolding and refolding.To overcome the NMR size limitation,we recruited the methyl-TROSY based NMR approaches,First,the methyl chemical shifts were assigned using the combination of site-directed mutations and recognition of intra-molecular methyl NOE patterns.Secondly,the interfacial methyl groups were identified by the 13C edited NOESY-HMQC experiments with and without 15N decoupling during 1H evolution in the indirect dimension,where one protein was 15N and methyl isotopic selective labelled while the other protein was perdeuterated.Thirdly,fine structural information was retrieved from the intermolecular methyl-methyl NOEs.Finally,we build a complex structure model using the above restraints,as well as the Paramagnetic relaxation enhancement and Residual dipolar couplings.Our structural model revealed interfacial residues crucial for the GTP/GDP releasing from Ran catalyzed by Mogl.Such loss-of-function mutations were thereafter validated by binding and enzymatic assay,demonstating hMogl was the guanine release factor of Ran in vitro.Our structure model brought out a possible mechanism of gunine releasing.Collaborated with Heng Liu,we concluded that hMogl regulated RanGTP at an appropriate level to assure mitotic chromosome segregation(See Heng Liu's thesis for more details regarding the studies).