Caracterización estructural de chaperoninas de tipo II mediante técnicas de criomicroscopía electrónica y cristalografía de rayos X

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-03-2011Chaperonins, also known as Hsp60s, constitute an ubiquitously distributed family of molecular chaperones, found in all archaea, bacterya and eukaryotic organisms. They are macromolecular complexes composed of between 14-18 60 kDa subunits arranged in two identical rings stacked back to back. All chaperonins share several characteristics such as ATPase activity, domain structure of the subunits (with three distinct domains: equatorial, intermediate and apical), and a functional mechanism which couples ATP binding and hydrolysis to a coordinated conformational change which promotes the folding of substrate proteins. Chaperonins have been further classified in two distinct groups, I and II, based on significant structural and functional differences. Group I chaperonins, whose main representative is GroEL from E.coli, are found exclusively in all bacteria and endosymbiotic organelles such as mithochondria and chloroplasts. They are composed by two homoheptameric rings, and need the concourse of a cochaperonin, GroES in E.coli, which acts as a lid closing the cavity so that the protein could eventually fold using the information encoded in its aminoacid sequence. On the other hand, group II chaperonins are present in archaea, where they are known as thermosomes, and in the eukaryotic cytosol where a single member of this family is found, and it has been termed CCT (Chaperonin Containing TCP-1). Group II chaperonins are usually formed by two homooctameric or heterooctameric rings with a number of different subunits ranging from 1-3 in thermosomes to the 8 different subunits in the case of CCT. The main difference with group I chaperonins resides in the lack of a cochaperonin, whose function is replaced by an extended helical protrusion which closes the ring. Although there is a wide corpus of research regarding the structure and function of group I chaperonins, there is no so much information regarding group II chaperonins. In the present study we have attempted to obtain high resolution structural data of two of these chaperonins, an archaeal homo-oligomeric thermosome and the ecukaryotic cytosolic chaperonin CCT, with the aim of filling a long standing vaccum regarding the knowledge of these molecular chaperones. In the case of thermosome, we have developed a focal pairs-based cryoelectron microscopy approach aiming to determine a high resolution structure of the open conformation of an homo-oligomeric thermosome. This approach has not been completely succesful, but has served to identify several problems in the process which we believe must be useful in the future. In the case of CCT, it has been possible to obtain, using X-ray crystallography techniques, the only high resolution structure of the whole CCT chaperonin in complex with a substrate, tubulin, a breakthrough in the chaperonin research field that has allowed us to complete the structural and functional picture of this interesting molecular machine