Structural and functional studies of bacterial outer membrane lipopolysaccharide insertionand Schmallenberg virus replication

Endotoxins;Bunyaviruses

Dong, Haohao ; Naismith, Jim ; Naismith, Jim

University:University of St Andrews

Department:Chemistry (School of)

关键词: Endotoxins;    Bunyaviruses;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/7338/HaohaoDongPhDThesis.pdf?sequence=3&isAllowed=y

来源: DR-NTU

【 摘 要 】

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) ofGram-negative bacteria and plays a fundamental role in protecting the bacteria fromharsh environments and toxic compounds. The LPS transport system is responsiblefor transporting LPS from the periplasmic side of the inner membrane (IM) to theOM, in a process involving seven LptA-LptG proteins. The current model forlipopolysaccharide transport (Lpt) suggests that LPS is initially extracted by a four-protein complex, LptBCFG, from the inner membrane to the periplasm, where LptAmediates further transport to the OM. Another two protein complex, LptD/E,catalyses the assembly of LPS at the OM cell surface. However, the details of thistransport mechanism have remained unknown, mainly due to a lack of structuralinformation.In chapter 1 and 2 of this thesis, I report materials and methods for all LptD/E, andSchmallenberg virus (SBV) nucleoprotein (NP) experiments and the theories andsoftware that were used in determining structures of LptD/E, SBV NP and the SBVNP/RNA complex.In chapter 3 of this thesis, I report the first crystal structure of the outer membraneprotein LptD/E complex. LptD forms a 26-strand ß-barrel in a closed form and LptEis a roll-like structure located inside LptD to form “barrel and plug” architecture.Through structural analysis, function assay and molecular dynamics simulation, weproposed a mechanism in which the hydrophilic head of LPS molecule, including theoligosaccharide core and the O antigen, directly penetrates through the hydrophilic ß-barrel whilst the hydrophobic lipid A tail is inserted into an intramembrane hole, witha lateral opening between strand ß1 and ß26 of the LptD. LptE may assist thisprocess.In chapter 4, I report the crystal structure of the SBV NP in two conformations:tetrameric when the protein was purified under native conditions, and trimeric whendenatured and refolded during purification. The SBV NP has a novel fold and wehave also identified that the N-terminal arm is crucial for RNA binding, and the N- and the C-terminal arm is essential for RNA multimerisation with adjacent protomersand for viral RNA encapsidation.Chapter 5 describes the crystal structure of SBV NP in complex with a 42 nucleotidelong RNA (polyU). This ribonucleoprotein (RNP) complex was crystallized as a ring-like tetramer with each protomer bound to 11 ribonucleotides. Eight of thesenucleotides are bound in a positively charged cleft between N- and C- terminaldomains and three are bound in the N-terminal arm. I also compared the structure tothat of other NPs from negative-sense RNA viruses, and found that SBV NPsequesters RNA using a different mechanism. Furthermore, the structure suggests thatwhen RNA binds the protein, there are conformational changes in the RNA-bindingcleft, and in the N- and C-terminal arms. Thus our results reveal a novel mechanismof RNA encapsidation by orthobunyaviruses NP.