Frataxin from Psychromonas ingrahamii as a Model to Study Stability Modulation within the CyaY Protein Family

ERNESTO A. ROMAN; SANTIAGO E. FARAJ; ALEXANDRA COUSIDO-SIAH; ANDRÉ MITSCHLER; ALBERTO PODJARNY; JAVIER SANTOS

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2013 vol. 1834 p. 1168 - 1180

1570-9639

Life adaptation to low temperatures tunes both protein stability and flexibility. This makes psychrophilic proteins excellent models to study the relation between these properties. Here we focused on frataxin (FXN), a highly conserved protein that plays a key role in iron homeostasis as an iron chaperone, from Psychromonas ingrahamii, an extreme psychrophile sea-ice bacterium which can grow up to -12 °C. The conformation of pFXN was studied by X-ray crystallography, far- and near-UV CD, and by tryptophan fluorescence providing information about its native state. Both chemical- and temperature-induced unfolding showed that pFXN behaves as a two-state folder, and its thermodynamic stability is significantly reduced and highly modulated by pH, in comparison to other frataxin homologs. pFXN was crystallized and its X-ray structure solved at 1.45 Å. Protein flexibility was investigated by analyzing B-factor profiles and molecular dynamics simulations. Although the general structural features are shared between E. coli and P. ingrahamii homolog proteins, the patches with the highest B-factor values differ between them. This indicates that the distribution of flexibility in the protein chain might also be different. More importantly, in pFXN the stretch spanning residues 62-66 shows the highest relative B-factor values highlighting this part as the most mobile region which may be implicated in binding phenomena. Interestingly, the C-terminal region (CTR) contains a cluster of negative charged residues, and also shows, in the case of pFXN, significantly higher B-factor values compared to the E. coli homolog. We suggest that the C-terminal mobility might alter the local and global dynamics, because this region is implicated in thermodynamic stabilization of FXN fold. Our analysis also proposes that there is a coupling between dynamics and electrostatics interactions within a delimited cluster of residues. In this context, the presence of metal ions would affect the flexibility of the protein.