Bullitt E, Jones C H, Striker R, Soto G, Jacob-Dubuisson F, Pinkner J, Wick M J, Makowski L, Hultgren S J
Department of Biophysics, Boston University School of Medicine, MA 02118, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12890-5. doi: 10.1073/pnas.93.23.12890.
The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.
与毒力相关的P菌毛的主要亚组件，菌毛杆（由PapA组成）和顶端纤维（由PapE组成），在体外由纯化的伴侣 - 亚基复合物重构而成。在亚基组装成成熟菌毛之前，它们在伴侣 - 亚基复合物中保持具有组装能力的构象。PapD伴侣部分地通过β拉链相互作用与亚基C末端存在的保守基序结合。还发现该保守基序中的氨基酸残基对于菌毛形成所必需的亚基 - 亚基相互作用至关重要，从而揭示了一种分子机制，通过该机制PapD伴侣可以防止周质中亚基 - 亚基的过早相互作用。通过冻融技术在体外模拟了PapA和PapE的伴侣保护的C末端的解封，分别导致菌毛杆和顶端纤维的形成。PapA的β拉链前沿的突变产生了具有改变的螺旋对称性和方位无序的菌毛杆。螺旋每圈亚基数量的这种变化最有可能反映了β拉链前沿在形成右手螺旋圆柱体中的作用。细胞器发育是所有活细胞中的一个基本过程，这些研究为免疫球蛋白样伴侣如何控制致病细菌中毒力相关细胞器的形成提供了新的线索。
