Structure And Molecular Mechanism Of Leptospira Interrogans LigA,LigB Big Domain And Trypanosoma Brucei Q388A3 PDZ Domain

This dissertation focuses on the structure and molecular mechanism of Leptospira interrogans LigA,LigB Big and Trypanosoma brucei Q388A3 PDZ domain.1.Pathogenic species of Leptospira use immunoglobulin-like proteins as adhesion molecules to bind to the extracellular matrix of host cells.Lig(Leptospiral immunoglobulin-like)proteins,expressed in pathogenic Leptospira with high-molecular-mass,belong to IgSF(immunoglobulin super family).LigA(Leptospiral immunoglobulin-like protein A),a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like(Big)domains,has been proved to be involved in the pathogenisis of Leptospira.In this work,the structure of the fourth Big domain of LigA(LigA4 Big domain)from Leptospira interrogans was solved by nuclear magnetic resonance(NMR).The structure of LigA4 Big domain demonstrates a similar fold pattern compared with other Big domains,implying some common structural features of Big domain family.On the other hand,it displays some structural characteristics significantly different from classic Ig-like domain.Furthermore,Stains-all assay and NMR chemical shift perturbation revealed the Ca2+ binding property of LigA4 Big domain.LigB,another outer membrane protein also containing tandem repeats of bacterial Ig-like(Big)domains and a non-repeat C-terminal tail,has been identified as a virulence factor participated in the interaction between Leptospira and host cells.A Big domain of LigB,LigBCen2R,was reported previously to bind the GBD domain of fibronectin,suggesting its important role in leptospiral infections.Here we determined the solution structure of LigBCen2R by NMR spectroscopy.LigBCen2R adopts a canonical Ig-like fold which contains a ?-sandwich of ten strands distributed in three sheets.We indicated that LigBCen2R is able to bind to Ca2+ with a high affinity by isothermal titration calorimetry experiment.NMR perturbation experiment identified a number of residues involved in Ca2+ binding.Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern,but vary in some structural characters.Since Lig proteins play a important role in the infection to host cells,our study will provide a structural basis to understand the interactions between Leptospira and host cells.2.PDZ domains are a group of protein interaction modules which bind short C-terminal motifs of target proteins and always found in scaffold proteins.To our knowledge,no PDZ domain in Trypanosoma brucei,an eukaryotic parasite causing sleeping sickness,has been studied.Q388A3,a conserved protein in the kinetoplastid parasites,is a 1634-residue protein containing a PDZ domain at its C-terminus.In this work,the three-dimensional structure of Q388A3 PDZ domain was determined by NMR spectroscopy.Q388A3 PDZ domain adopts a PDZ-like fold composed by a five-stranded ?-sheet capped by two a-helices,which is similar to the PDZ domains from HtrA family members.Meanwhile,Q388A3 PDZ domain shows some structural features quite different from HtrA PDZ domain.In addition,surface plasmon resonance and NMR chemical shift perturbation demonstrate the interactions between Q388A3 PDZ domain and OMP-like peptide,which implies a similar function of Q388A3 PDZ domain with HtrA PDZ domains.We identified the potential residues critical for ligand binding of Q388A3 PDZ domain by site-directed mutagenesis.Interestingly,different peptide binding capacities of PDZ domains were influenced by distinct critical residues and structure within the peptide-binding site.