Structure and dynamics of the iron-sulfur cluster assembly scaffold protein IscU and its interaction with the cochaperone HscB.
IscU is a scaffold protein that functions in iron-sulfur cluster assembly and transfer. Its critical importance has been recently underscored by the finding that a single intronic mutation in the human iscu gene is associated with a myopathy resulting from deficient succinate dehydrogenase and aconitase [Mochel, F., Knight, M. A., Tong, W. H., Hernandez, D., Ayyad, K., Taivassalo, T., Andersen, P. M., Singleton, A., Rouault, T. A., Fischbeck, K. H., and Haller, R. G. (2008) Am. J. Hum. Genet. 82, 652-660]. IscU functions through interactions with a chaperone protein HscA and a cochaperone protein HscB. To probe the molecular basis for these interactions, we have used NMR spectroscopy to investigate the solution structure of IscU from Escherichia coli and its interaction with HscB from the same organism. We found that wild-type apo-IscU in solution exists as two distinct conformations: one largely disordered and one largely ordered except for the metal binding residues. The two states interconvert on the millisecond time scale. The ordered conformation is stabilized by the addition of zinc or by the single-site IscU mutation, D39A. We used apo-IscU(D39A) as a surrogate for the folded state of wild-type IscU and assigned its NMR spectrum. These assignments made it possible to identify the region of IscU with the largest structural differences in the two conformational states. Subsequently, by following the NMR signals of apo-IscU(D39A) upon addition of HscB, we identified the most perturbed regions as the two N-terminal beta-strands and the C-terminal alpha-helix. On the basis of these results and analysis of IscU sequences from multiple species, we have identified the surface region of IscU that interacts with HscB. We conclude that the IscU-HscB complex exists as two (or more) distinct states that interconvert at a rate much faster than the rate of dissociation of the complex and that HscB binds to and stabilizes the ordered state of apo-IscU.
Kim
Jin Hae
JH
Biophysics Graduate Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
Füzéry
Anna K
AK
Tonelli
Marco
M
Ta
Dennis T
DT
Westler
William M
WM
Vickery
Larry E
LE
Markley
John L
JL
D019943:Amino Acid Substitution; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D001059:Apoproteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C095453:HscB protein, E coli; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D011994:Recombinant Proteins; D015032:Zinc