Insights into structure and dynamics of the AAA+ motor of magnesium chelatase

Sammanfattning: The insertion of Mg2+ into protoporphyrin IX represents the first committed step in the chlorophyll and bacteriochlorophyll biosynthetic pathways. The reaction is catalyzed by the multisubunit enzyme Mg-chelatase, which consists of three subunits, known as I (molecular weight ~40 kDa), D (~70 kDa), and H (~140 kDa). To fully understand this first step in chlorophyll biosynthesis each protein component of Mg-chelatase needs to be characterized and be coupled into a context of its macromolecular ensemble. Thus it is vital to study the 3D structure of individual modules, but to gain full functional insight into the system the structure and dynamics of the catalytic assemblies needs to be determined. The results presented in this thesis are mainly derived from electron microscopy (EM) single-particle techniques. In combination with several other established methods in protein characterization, e.g. X-ray crystallography, bioinformatics, biochemical assays and mass-spectrometry it has provided a poweful tool to obtain quasi-atomic information of the structure and dynamics of R. capsulatus Mg-chelatase. It is presented here that the I- and D-subunits form bipartite chaperone-like complex (ID-complex) with a C3 point-group symmetry and the single-particle cryo-EM 3D reconstruction of the ID-complex in presence of ADP is solved to 7.5 Å resolution. The largest subunit H, carries the protoporphyrin IX, and it is known to associate with a preformed ID-complex. The results presented here give the first insights into the structure of an H-subunit. Single-particle EM 3D-reconstruction was used to solve the structure in apo and substrate bound conformations at resolution of 25 Å, and revealed a conformational change upon substrate binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The presented quasi-atomic model of the ADP-induced ID-complex reveals the complex structure of the D-ring, which belongs to a unique clade of ATPase inactive AAA+ proteins that has not yet been characterized structurally, and how it assembles with the ATPase active I-ring. Furthermore, the ID-complex is an ATP-fuelled AAA+ motor that is most likely dependent upon ATP-hydrolysis for the conformational rearrangement that is required before H-subunit association may take place and the metal insertion can be completed. An autoinhibitory mechanism for the ATP-fuelled motions of the ID-complex is presented, that may be of general importance for the mechanistic understanding of all bipartite AAA+ family members.