| Gram-negative bacteria have evolved several secretion systems to secrete a huge variety of proteins to maintain their physiological functions, including bacterial communication, nutrient uptake, immunity, pathogenicity, etc. Currently, gram-negative bacteria are divided into eight different secretion systems, referred to as type I-VIII, or T1SS-T8SS. This thesis mainly focuses on the structure and function of T5SS and T6SS related proteins.Type V secretion system (T5SS) widely exists in the pathogenic bacteria to deliver some outer membrane proteins (OMPs), such as protease, adhesion, pathogenic factor, etc. These precursor proteins were transported across the inner membrane with SecYEG complex, and the unfolded proteins were delivered to the BAM complex, which assemble and insert the proteins into the outer membrane.The BAM complex includes five proteins, referred to as BamA-E, BamA is an integral β-barrel outer membrane protein, and BamB/C/D/E are four accessory lipoproteins. This paper cloned, expressed and purified BamB/C/D/E, and determined the structures of BamB and BamD, provided structural basis for the transport mechanism of BAM complex.The crystal of BamB diffracted to2.0A, the overall structure is composed of eight-bladed β-propeller motifs. Pull-down and western blotting assays indicate that BamB interacts directly with POTRA1-3domain of BamA, and the C-terminal region of POTRA1-3domain play an important role in the interaction.The structure of BamD at2.6A resolution shows that this lipoprotein is composed of ten a-helices that form five tetratricopeptide-repeat (TPR) motifs. The a10has been shown to be important for the assembly of the BAM complex. The N-terminus is likely to play an important role in the insertion of other outer membrane proteins. The C-terminus serves as a platform for the interactions with other component of the BAM complex. T6SS apparatus is a dynamic macromolecular machine, which was recently discovered in gram-negative bacteria. T6SS plays an essential role in interspecific competition. Recent studies have shown that donor cells use T6SS to inject toxic effectors into receptor cell, analogous to a contractile bacteriophage tail. These effectors destined for the periplasm would attack the prey cell wall and membrane to kill the competitor cells. To protect the donor cells, the corresponding immunity proteins are exported into the periplasmic space to inhibit the effectors catalytic activities.This thesis determined the crystal structures of type Ⅵ amidase effector3(Tae3) with type Ⅵ amidase immunity3(Tai3) and glycoside hydrolase VgrG3C with its immunity protein TsaB.The crystal structure and small angle X-ray scattering (SAXS) indicate that Tae3-Tai3complex exists as a stable heterohexamer in solution, which is composed of two Tae3molecules and four Tai3molecules. The residues Cys23and His81of Tae3form a catalytic dyad, and their mutations C23A, H81A abolished the amidase activity when compared with the wild-type activity. The inhibition of Tae3is attributed to the insertion of the loop of α3-α4of Tai3into the catalytic groove. Furthermore, the conserved motif Gln91-Asp92-Tyr93in Tai3may play a key role in the inhibition process.The VgrG3C-TsaB complex behaves as a heterotetramer in solution, included two VgrG3C-TsaB heterodimer. VgrG3C has inverting glycosyl hydrolase features, adopting T4-lysozyme-like fold. Cell viability assay revealed that two invariant amino acids Glu827and Asp842constitute the active dyad residues of VgrG3C. TsaB acts as a molecular mimic of the VgrG3C substrate, inserting into the active pocket of VgrG3C. As a result, the active cleft is blocked by TsaB and the catalytic activity of VgrG3C is abolished.Structural and biochemical analysis of Tae3-Tai3and VgrG3C-TsaB complexes provided insights into the inhibition mechanism of the effector by its immunity protein. These studies should be helpful in designing new anti-pathogen targets. |
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