Abstract
Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC p ~ 199.8 kcal K−1 mol−1, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.
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Abbreviations
- 1,5-IAEDANS:
-
5-((((2-Iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid
- 5-BMF:
-
5-(Bromomethyl)fluorescein
- DTT:
-
Dithiothreitol
- FRET:
-
Fluorescence resonance energy transfer
- IPTG:
-
Isopropyl β-d-thiogalactopyranoside
- ITC:
-
Isothermal titration calorimetry
- PDB:
-
Protein Data Bank
- PLP:
-
Pyridoxyl 5′-phosphate
- PMSF:
-
Phenylmethylsulfonyl fluoride
- TCEP:
-
Tris(2-carboxyethyl)phosphine hydrochloride
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Acknowledgment
This work was supported by a grant the National Science Foundation, CHE-0111161 (J.A.C.).
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Nuth, M., Cowan, J.A. Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site. J Biol Inorg Chem 14, 829–839 (2009). https://doi.org/10.1007/s00775-009-0495-7
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DOI: https://doi.org/10.1007/s00775-009-0495-7