Crystal structures of frataxin from P. ingrahamii in complex with Co(II) and Eu(III): insights into the metal binding properties and conformational flexibility.

NOGUERA, ME; ROMANEA; COUSIDO-SIAH A; MITSCHLER A; PODJARNY A; SANTOS J

Cordoba

Congreso; I Reunión Latinoamericana de Cristalografía y IX Reunión Anual de la Asociación Argentina de Cristalografía; 2013

Asociación Argentina de Cristalografía

P005 Crystal structures of frataxin from P. ingrahamii in complex with Co(II) and Eu(III): insights into the metal binding properties and conformational flexibility M.E. Noguera1,*, E.A. Roman1,*, A. Cousido-Siah2, A. Mitschler2, A. Podjarny2, J. Santos1 1Instituto de Química y Físico-Química Biológicas, Universidad de Buenos Aires, Junín 956, (1113AAD), Buenos Aires, Argentina. 2Department of Integrative Biology, IGBMC, CNRS, INSERM, Université de Strasbourg, Illkirch, France. mnoguera.unq@gmail.com *These authors contributed equally to this work. Frataxin is a highly conserved protein among different organisms and plays an essential role in iron homeostasis, participating in multiple cellular processes. Several of them seem to involve binding of iron and other proteins to a large negatively-charged patch located on the protein surface. Frataxin deficiency causes Friedreich?s ataxia. Whereas a large number of biophysical studies reported binding of iron to frataxin, the precise number, affinity and specificity of these binding sites is still unclear. In this work, we address these questions through the crystallographic study of the frataxin from the psychrophilic bacterium Psychromonas ingrahamii (pFXN). This is an interesting model since, in addition to this negative patch on the protein surface, stability is highly influenced by pH, and thus can be studied the relation between binding affinity and global stability. Crystals of pFXN were obtained as described early [1], and then soaked with CoCl2 or EuCl3. X-ray diffraction data were acquired at 1.3 and 1.8 Å for Co and Eu containing crystals, respectively. Both crystals belong to the space group P 1211, and have two protein molecules per asymmetric unit. The structures were solved by molecular replacement, using the coordinates of the same protein in the apo form, solved recently by our group [1], and refined to R/Rfree values of 0.184/0.211and 0.214/0.260 for the Co and Eu crystals, respectively. Six binding sites were observed for Co and nine for Eu, some of them conserved with the mesophilic homolog fromE. coli[2]. Most of these sites have not been reported in other PDB crystal structures. Here, we describedthese metal binding sites in detailand explored their impact in the conformational flexibility of this variant through a combined analysis of the crystallographic B-factors and molecular dynamics simulations. [1] Roman, E.A. et al., Biochim Biophys Acta, 1834, 1168-80 (2013). [2] Pastore, C. et al., Febs J, 274, 4199-210 (2007).