Structure determination of membrane-located complexes: Aquaporin 8 and YscC secretin

A biological membrane is key to the life of a cell. In all living cells a biological membrane separates a inner
life and an outer environment. Plasma membrane has many functions such as responding to external signals and transmitting them into the cell, providing a barrier to the water soluble molecules, transporting molecules via channels, cell-to-cell communication, separating cellular reactions by compartments and creating ion gradients between them, which are used for converting an energy and signal transduction. Most of the membrane functions are performed by embedded proteins, which are
constantly in motion.
The homeostasis of water, the most abundant molecule in living organisms, is crucial for physiology of all cells. The main interest in structure determination of the water channel aquaporin 8 (AQP8) is connected with its unique position within the aquaporin family. The human AQP8 cDNA has been cloned in 1998 and since then the intensive studies concerning its intravesicular localization and activity have been preformed. Expressed in the inner mitochondrial membrane of several mammalian tissues, found in liver, gastrointestinal tract, testis, airways and kidney cells, the ammonia-permeable AQP8 could be
essential for the organism metabolism. By sequence alignment it is evident that AQP8 creates a separate subfamily, which is apart from all the other mammalian aquaporins. The special constriction region of the pore, which determines the solute permeability, is unique in AQP8 and makes it permeable to both ammonia and hydrogen peroxide in addition to water. For mammalian aquaporins, the structures solved up to now, all belong to the water-permeable subfamily. To better understand
AQP8 selectivity and gating mechanism the high-resolution structure is necessary. To assess the structure, human AQP8 was overexpressed in methylotrophic yeast Pichia pastoris as a His-tagged protein. A wide screen of different detergents and detergent-lipid combinations for optimal protein purification and 2D crystallization was essential to obtain well-ordered AQP8 crystal arrays. Removal of amino acids constituting affinity tags was necessary to achieve highly ordered crystals diffracting up
to 3 Å. Atomic force microscopy, electron microscopy and gold labeling experiments revealed the double-layered nature of 2D crystals, with tetrameric organization of AQP8, which had termini exposed outwards of the 2D crystal. In parallel, alignments to AQP4 revealed a similar, extraordinary long N-terminal of AQP8. In analogy to AQP4, where only the short isoform is able to
crystallize, 2D crystallization of the shorter AQP8 construct, with removed N-terminus, was initiated.
The translocation of proteins across the biological membrane is an essential part of cellular life. The type III secretion system (T3SS) is a major factor for the virulence or symbiosis of many Gram-negative bacteria that infect plants and animals. Bacterial effector proteins are delivered via T3SS injectisome from the pathogen cytoplasm into the eukaryotic host cells, in which they modulate the host innate immune response. The outer membrane-localized YscC oligomer belongs to the secretin family and is one of the main components of the injectisome. In this study, the YscC complex was expressed in the avirulent
strain of Yesinia enterocolitica and purified in order to construct a 3D model from cryo Electron Microscopy single particle images. The 12 Å-resolution 3D structure of the closed YscC complex was calculated from 30000 projections of vitrified YscC.
Various approaches like rotary metal shadowing of trypsin digested oligomers, mass spectrometry and Scanning Transmission Electron Microscopy were used for oligomer symmetry evaluation. The generated 3D structure of the YscC complex disclosed the N-terminal flexible domain, which forms the part of a large chamber between the bacterial outer and inner membranes, the conical shape periplasmic domain, and two differently sized ring-shaped domains linked by fine density connectors corresponding to the outer membrane spanning regions. The sample trypsinization revealed the protease-resistant core of the protein. Comparison of the sequence and structure of YscC to other, close or more distant related secretins made it possible to define homology regions located both on the N- and C-termini of the protein.