Caracterización estructural y funcional del complejo formado por dnak y grp de e.Coli

Resumen

Molecular chaperones are essential for the appropriate folding of proteins in the cell. One of the main families of molecular chaperones are the so called Hsp70s (Heat shock protein 70kDa), a group of proteins involved not only in protein folding but also in other cellular processes such as apoptosis or DNA replication. This versatility is achieved in part through the interaction with co-chaperones like the Hsp40s and the nucleotide exchange factors (NEFs), which regulate the activity of Hsp70s. All Hsp70s are composed of two main domains, the nucleotide binding domain (NBD), responsible of the structural and functional changes of the chaperone, and the substrate binding domain (SBD), involved in the interaction with the substrate. DnaK, the main Hsp70 (Heat shock protein 70kDa) protein in bacteria, functions like the rest of Hsp70s through successive rounds of unfolded protein binding and release, controlled by the nucleotide-state of the NBD, in turn controlled by its interaction with Hsp40s like DnaJ, which induces ATP hydrolysis, or with NEFs like GrpE, responsible for accelerating the ADP/ATP nucleotide exchange. The goal of this thesis is to characterize the interaction between DnaK and GrpE, whose atomic structures are known. A crystallographic structure of a complex between GrpE and DnaKNBD has already been published, which nevertheless has not been able to show in full the interaction between the chaperone and its cofactor. The work shown here reveals the structure, determined by electron microscopy and image processing, of the complex between full-length DnaK and GrpE. The structure suggests a more complex interaction between the two proteins since it reveals that both DnaK domains embrace the head of GrpE. Kinetics experiments with a peptide substrate confirm the interaction of the unstructured region of the GrpE tail with the DnaKSBD acting as a pseudosubstrate, as it has been already described. These experiments also show an increase of the affinity for the substrate when DnaK is complexed with GrpE. The interaction of DnaKSBD with other GrpE regions such as the disordered N-terminal domain, already established, could explain these results. All this suggests that GrpE, besides being a nucleotide exchange factor, has a role in DnaK-peptide binding.