Crystal Structure Of Escherichia Colimembrane Protein Apolipoprotein N-acyltransferase And Phosphatidyl Ethanolamine Complex

Apolipoprotein N-acyltransferase(Lnt) is a inner membrane protein which existsonly in Gram-neagtive bacteria. It catalyses the last reaction of lipoprotein posttranslational modification. In this step an acyl chain from phospholipids will betransferred to the α-amino group of the N-terminal diacylglyceryl-modified cystein ofapolipoprotein. This enzyme is of great importance to growth and viability ofGram-negative bacteria but lacks homologues in human so that it is a good target fordeveloping new antibiotics against Gram-negative bacteria.The overexpression of Lnt using Escherichia coli have been succeed and the highresolution three-dimensional structure of Lnt have been solved by the former memberin our group (not published yet). In this study, we modified Lnt molecular with ainserted sequence to improve the repetitiveness of crystal and finally solved thethree-dimensional structure of modified Lnt and phosphatidyl ethanolamine complexusing molecular replacement method at3.5angstrom resolution.Apolipoprotein N-acyltransferase is formed by a hydrophobic transmembranedomain and a hydrophilic nitrilase catalytic domain. The transmembrane domaincontains eight α-helix, in which the second and the third formed coiled coil structureand extended to catalytic domain and then formed part of the substrate binding pocket.The overall structure of catalytic domain resembles other members in nitrilasesuperfamily, but possesses a unique α-helix positioned outward, forming another partof substrate binding pocket. After X-ray diffraction data collection we frond a strongf0-fcdensity in a pocket at the catalytic domain in the density map. According to theanalys of the active residue site and surface charge distribution of the enzyme, wemade conclusion that this strong f0-fcdensity is caused by phosphatidyl ethanolamineand this pocket is the binding site of phospholipid headgroup.Before the chapter presenting the structure of Lnt and phosphatidyl ethanolaminecomplex, the author first made a brief introduction to the background of Gram-negative bacteria and apolipoprotein N-acyltransferase. Then in the followingchapter the author summarized methods of membrane protein study. The third chapteris the experiment part, in which the author presents the modification, overexpression,purification, crystal optimization, X-ray diffraction data collection and how thestructure of this complex solved in details, as well as the author`s own experience inpurification and crystallization of membrane.