| Our work focuses on the cloning,expression,purification and determination of the solution structure of human MEKK3 PB1 domain by NMR method.In the process of structure determination,we have found an interesting proline isomerization in MEKK3 PB1.Based on the structure of MEKK3 PB1,we studied interaction between MEKK3 PB1 and PB1 domain of its downstream MAPK kinase MEK5.Another work we have conducted is,solving the solution structure of a human protein Mogl, and further studied its interaction with nucleocytoplasmic transport protein Ran.Chapter 1 is a brief review of the MAPK signaling pathway superfamily and PB1 domain.MAPK signaling pathway has been found involved in various biological processes as well as pathological processes.ERK5 signaling pathway is unique among the MAPK signaling pathways in that it is PB1-dependent,PB1 domain is a recently found module mediating protein-protein interaction.PB1 participates in many signaling pathways.In chapter 2,we have solved the solution structure of human MEKK3 PB1 domain. MEKK3 PB1 was expressed in E.coli,purified with a Ni-chelating column,then used for NMR experiments.MEKK3 PB1 adopts a typical ubiquitin-like fold,includes fiveβstrands and twoαhelices.The fiveβstrands are arranged in a twisted parallel and anti-parallelβ-sheet.The twoαhelices are nearly orthogonal to each other and lie at the opposite side of theβ-sheet.The structure is stabilized by a hydrophobic core consisting of a large number of residues.To our interest,the spectra exhibits a set of crosspeaks with relatively low-intensity in addition to the major ones,an intramolecular conformation exchange is the only possible reason for the lo(?)v-intensity signals in the spectra.The chemical shift differences for backbone amides between the minor and the major conformations become larger as the residues get more close to residue Pro39.Taking these into consideration,we speculated that cis/trans isomerization around Gln38-Pro39 imide bond may be the source of the conformational heterogeneity.Consistent with this hypothesis,characteristic NOEs and chemical shift difference between the 13Cβand the 13Cγnuclei for the cis and trans conformations of Pro39 supported our hypothesis.The major backbone differences between the cis and trans conformations lie in the G37-L40 turn connectingα1 andβ3, the region where Pro-39 is located.In cis form,it adopts a type Vlbβturn,whereas in trans conformation,this region does not belong to any typical type ofβturn. Backbone dynamics studies have unraveled internal motions inβ3/β4-turn on microsecond-millisecond timescale.Further we have investigated its binding property with MEK5 PB1.ITC and SPR experiments have demonstrated that MEKK3 PB1 binds MEK5 PB1 tightly with a Kd of about 10-8M.Mutagenesis analysis revealed that residues in the basic cluster of MEKK3 PB1 contributes differently to the PB1-PB1 interaction.As a highly conserved residue,Lys7 plays an important role in the interaction between MEKK3 PB1 and MEK5 PB1.The interaction was disrupted by the mutation K7A.Similarly,R5A attenuated interaction dramatically.In contrast, the interaction exhibited much weaker decrease upon R14A or R76A mutations.So we proposed that Lys7 and Arg5 are critical to the interaction between MEKK3 PB1 and MEK5 PB1.In chapter 3,we have solved the solution structure of human Mog1 protein.Mog1 is a 186-residue protein relating to the nucleocytoplasmic transportation.Mog1 was expressed in E.coli,purified with a Ni-chelating column,then used for NMR experiments.Mog1 contains 9βstrands,2αhelices and 2 310helices,of which 7βstrands are arranged in parallel and anti-parallelβ-sheets with twoαhelices lying at the opposite side of theβ-sheet.A major difference between hMog1 and ScMog1 is that,in hMog1,residues 146-164 form a big loop.while in the corresponding position in SeMog1,it adopts anαhelix.Gel filtration chromatogram demonstrated that Mog1 and Ran could form a heterodimer.However,further work is needed to determine the interface between Mog1 and Ran.
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