Homeodomain proteins are transcription factors present in all eukaryotes and play key roles in cellular differentiation during development. In fact, mutations in homeodomain-encoding genes give rise to several inherited diseases in humans. The homeodomain consists of a 60-residue DNA binding domain composed of disordered N- and C-terminal regions and three helical segments. These structural features, together with the highly charged nature of the polypeptide, determine a typically low stability, property also shared by other DNA-binding proteins. Conversely, homeodomains usually present a large DNA-binding affinity. From a molecular recognition perspective, the association process is sequence-specific and represents an example of the “induced fit” mechanism. Thus, it has been shown that disordered regions of the protein become structured upon complex formation, which highlights the relevance of the protein flexibility in this process. Herein we have investigated the relations between stability and DNA-binding in the human HESX-1 homeodomain. The sequence information available for homeodomains reveals that salt bridges connecting pairs 19-30, 31-42, and 17-52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA-binding properties of human HESX-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19-30 and 31-42. This stabilizing influence results from the more favourable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast, only polar side chains seem to be tolerated at positions 17-52. Interestingly, despite the significant influence of pairs 19-30 and 31-42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. In other words, this analysis reveals an intriguing lack of correlation between binding strength and the homeodomain conformational stability. Previous studies have shown that the induced fit of the protein disordered regions plays a key role in the molecular recognition process. On the contrary, according to our data, the potential adjustment of the structured core of the protein seems to have little influence on the complex stability. This features might allow and independent modulation of stability and DNA-binding by evolution.

La presente tesis ha sido realizada en el Departamento de Química Bio-Orgánica del Instituto de Química Orgánica General del Consejo Superior de Investigaciones Científicas, bajo la dirección del Dr. Juan Luis Asensio. Para ello, Mario Torrado ha contado con la ayuda de una beca/contrato de Formación de Profesorado Universitario (FPU), que le fue concedida por el Ministerio de Educación, Cultura y Deporte. El trabajo ha sido financiado por el “Plan Nacional de I+D+i” (Ministerio de Economía y Competitividad) a través de los proyectos CTQ2007-67403/BQU y CTQ2004-04994/BQU.

Parte de los resultados obtenidos en este trabajo se recogen en el siguiente artículo: Torrado, M., Revuelta, J., Gonzalez, C., Corzana, F., Bastida, A., Asensio, J.L., 2009. Role of conserved salt bridges in homeodomain stability and DNA binding. The Journal of Biological Chemistry 284, 23765-23779.

Peer reviewed