Friedreich Ataxia:A Computational Dynamic Model of the KeyProteins Involved in the Yeast Fe-S Cluster

BiogenesisAmela I. 1, Cedano J. 1, Delicado P. 2, Querol E. 1

1.- Institut de Biotecnologia i de Biomedicina and Depa rtament de Bioquímica iBiologia Molecular. Universitat Autònoma de Barcelona. 08193-Bellaterra, Barcelona. Spain.2.- Departament d’Estadística i Investigació Operativ a. Universitat Politècnica de Catalunya. 08034-Barce lona. Spain.

METHODS:The sequence, structure and function of the proteins Yfh1, Isu1, Isu2 and Nfs1 were studied in detail applying different bioinformatics tools.

These information was contrasted with the current literature as well as several interactomics databases. Dockings were made for the

couples of proteins Yfh1/Isu1, Isu1/Isu2 and Isu1/Nfs1. The Escher NG protein-protein automatic docking system of the Vega ZZ project

was used as the basic method for the docking experiments. BiGGER-Chemera 3.0 and Hex 5.1 were employed to validate the docking

results. To extract the significant solutions from the docking output-datafile, an unsupervised and automatic clustering program, which is

now under submission and is called DockAnalyse, was developed with the R software environment. DockAnalyse was applied to choose

the best docking solutions and, therefore, to model the dynamic protein-interaction mechanism among these four proteins. Tridimensional

structure studies and representations were made using UCSF Chimera, PyMOL and RasMol.

RESULTS:A detailed bioinformatics study of the key proteins involved in the yeast ISC biogenesis process has been carried out. These proteins

are: the yeast frataxin Yfh1 (PDB code: 2GA5), the scaffolding proteins Isu1/Isu2 and the cysteine desulfurase Nfs1. The 3D structures

of the proteins Isu1, Isu2 and Nfs1 have been theoretically modeled because its real three-dimensional structures have not been solved

yet. Iron and sulfur atoms were added to Yfh1 and Nfs1 respectively. The Autodock force field and the Gasteiger method was used to

compute the molecule charge in VEGA ZZ. To eliminate the wrong added ions, the molecular modeling program ArgusLab was

employed. Docking experiments, together with an exhaustive analysis of the docking output-datafile, were made for the protein pairs

which have been shown to physically and functionally interact. These protein pairs are: Isu1/Yfh1, Isu1/Isu2 and Isu1/Nfs1. The

developed program DockAnalyse was applied to each of the docking output-datafiles and the obtained representative solutions are

shown in Figure 2. Owing to all the studies mentioned above, a dynamic interaction mechanism with which the four analysed proteins

generate the ISCs inside the yeast mitochondria has been postulated. For the scaffolding proteins Isu1/Isu2, iron binding pockets, which

pressumably are the ISC links, were identified in a suitable position and interaction sites were also localized. Isu1 and Isu2 tails seem to

enable the ISC scaffolding function as they have been predicted as interaction regions. In the case of Yfh1, negatively charged residues

implicated in the iron handling, which are high evolutionary conserved, were analysed and seem to be very close to the Isu1/Isu2 iron

binding pockets after the docking experiment. This suggests that Yhf1 can act as the iron donor for the ISC biogenesis process. The

function of these group of electro-negative residues, together with the identified interaction region that is also high evolutionary

conserved, fit properly with the current literature and the proposed model Regarding Nfs1, it is the protein that seems to act as the

Sulfur donor. These iron and sulfur atoms are very close to the Isu1 iron binding pocket. The Isu2 iron biniding pocket might help that of

Isu1 to form the entire ISC (see Figures 2 and 3).

CONCLUSION:• The sequence, structure and function of the proteins

Yfh1, Isu1, Isu2 and Nfs1 have been bioinformatically

studied.

• Protein-protein docking experiments have been

applied to these proteins and a new clustering method

has been developed to analyse the docking results.

• A model by which these proteins dynamically interact

together to generate the ISCs inside the yeast

mitochondria has been suggested.

• This hypothesis would be helpful to better

understand the function and molecular properties of

Frataxin and be useful for a future treatment of FRDA.

Acknowledgments:

Many thanks to my thesis directors for giving me the opportunity to apply their resources

to the study of the disease that affects me. This work is supported by the grant

200400041119 (UAB-SCH 2005) from the Universitat Autònoma de Barcelona.

Further information:

• E-mail address: iamela@bioinf.uab.es

• Web site address: http://ibb.uab.es/ibb/ � search for Staff “Amela, Isaac”

• PDF version of the poster is available at: http://bioinf.uab.es/rker/poster/symbiosis.pdf

References :

1. Lill, R. and U. Muhlenhoff (2006). "Iron-sulfur protein biogenesis in eukaryotes:

components and mechanisms." Annu Rev Cell Dev Biol 22: 457-86.

2. Ausiello, G., G. Cesareni, et al. (1997). "ESCHER: a new docking procedure applied

to the reconstruction of protein tertiary structure." Proteins 28(4): 556-67.

3. Palma, P.N., Krippahl, L., Wampler, J.E. and Moura, J.J.G. (2000). "BiGGER: A new

(soft) docking algorithm for predicting protein interactions." Proteins: Structure,

Function, and Genetics 39:372-384.

4. Ritchie, D.W. & G.J.L. Kemp (2000). “Protein Docking Using Spherical Polar Fourier

Correlations.” Proteins: Structur, Function, and Genetics 39, 178-194. WORK IN PROGRESS

Figure 1. Schematic representation of the proteins involved in the ISC biogenesis process inside the yeast mitochondria. As can be seen, Isu1 andIsu2 act as scaffolding proteins for the ISC formation. The protein Yfh1 donatesthe iron, whereas the protein Nfs1 contributes with the sulfur. The alreadygenerated ISCs are employed to supply both intra and extra mitochondrial Fe-S proteins. The ISC transporter might be the mitochondrial membrane proteinAtm1.

a

b

c

INTRODUCTION:Friedreich ataxia (FRDA) is a neuro-degenerative and hereditary disease which affects the equilibrium, movements coordination,

muscles and heart. It is the most common autosomal recessive ataxia and it is associated with a pronounced lack of a protein

named Frataxin. This protein has been associated with iron inside the mitochondria and seems to play an important role in the

mitochondrial Fe-S clusters (ISCs) assembly/maturation. There are supposed high similarities between the human and yeast

molecular mechanism that involve Frataxin. Moreover, in yeast it has been experimentally demonstrated that the yeast Frataxin

(Yfh1, PDB code: 2GA5) interacts with the protein Isu1, while Isu1 interacts both with the proteins Isu2 and Nfs1. These four

proteins together generate the central platform for the ISC biogenesis, where Isu1/Isu2 are the scaffold proteins and Yfh1 and

Nfs1 are the iron and sulfur donors respectively (see Figure 1).

Figure 2. The representative solutions for each of the studied dockings are shown: (a):isu1/yfh1 [green/blue], (b):isu1/isu2 [green/yellow], (c):isu1/nfs1 [green/red]. In all the cases, the predicted interaction regions are coloured in magenta. Moreover: in (a), the Yfh1 residues implicated in the iron handling are also coloured in magenta but displayed in the “wire” format. The iron atoms are coloured in red; in (b), the three crucial cysteine residues of Isu1 and Isu2 that form the iron binding pocket in each of the proteins are coloured in magenta and displayed in the “ball and stick” format; in (c), the sulfur atom donated from Nfs1 to Isu1 is coloured in yellow.

Figure 3. Schematic representation of the postulated dynamic mechanism ofinteraction for the ISC biogenesis process inside the yeast mitochondria. Theproteins are coloured as follows: Isu1 (green), Isu2 (yellow), Yfh1 (blue) andNfs1 (red). Isu1 and isu2 act as scaffolding proteins and as the central plattformfor the ISC biogenesis process. Yfh1 and Nfs1 interact alternatively with theIsu1/Isu2 complex to donate iron and sulfur respectively.