Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan;1861(1 Pt A):3154-3163.
doi: 10.1016/j.bbagen.2016.07.020. Epub 2016 Jul 26.

Understanding the role of dynamics in the iron sulfur cluster molecular machine

Danilo di Maio  1 , Balasubramanian Chandramouli  1 , Robert Yan  2 , Giuseppe Brancato  3 , Annalisa Pastore  4
Affiliations

Understanding the role of dynamics in the iron sulfur cluster molecular machine

Danilo di Maio et al. Biochim Biophys Acta Gen Subj. 2017 Jan.

Abstract

Background: The bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic FeS cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear.

Methods: We carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface.

Results: We show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow FeS cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation.

Conclusions: We conclude that the observed 'fluid' IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements.

General significance: Our study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex.

Keywords: CyaY; Frataxin; Iron-sulfur cluster biogenesis; Molecular dynamics; Structure.

PubMed Disclaimer

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
The IscS-IscU complex. (A) The complex structure, as formed by the IscS dimer (orange) and two IscU protomers (pink) as obtained starting from the 3LVL coordinates. The catalytic loop of IscS is colored in blue and the PLP cofactor is displayed as spheres. (B) Zoomed view of the interface between IscS and IscU. Side chains of the main interacting residues are shown as sticks, while the rest of the protein is depicted as cartoons.
Fig. 2
Fig. 2
Backbone RMSF and IscUβ-turn opening motion. (A) Average structure of the IscS-IscU tetramer and E. coli IscS-IscU crystal structure (PDB ID: 3LVL) from the binary complex simulation, depicted as ribbons and colored according to backbone RMSF values, from lowest (blue) to highest (red). Ribbon thickness is proportional to the local RMSF value. Detailed view of the IscU active site in the binary complex showing a (B) closed form of its β-turn from the E. coli IscS-IscU crystal structure (ID PDB: 3LVL) and (C) an open form of the same turn from the average IscS-IscU complex structure from our simulation. In both B and C panels, IscS (orange) and IscU (pink) are displayed as surfaces.
Fig. 3
Fig. 3
Conformational transition of the IscS catalytic loop. Time evolution of the conformational change of the IscS catalytic loop, as monitored by (A) secondary structure, (B) radius of gyration (only backbone atoms) and (C) distance between C328 and the IscU active site (i.e. C63 and C106). (D) Structural superposition of a representative configuration of the IscS (orange)-IscU (pink) complex (catalytic loop in blue), extracted from the MD trajectory at ca. 225 ns, and the A. fulgidus IscS (green)-IscU (purple) crystal structure (only IscS was used for the fitting). C328 and the conserved cysteines on IscU are shown as sticks, as well as the [Fe—S] cluster in the A. fulgidus structure. Note the structural transition at about 125 ns (panel A–B) triggering a progressive approach of C328 towards the active site of IscU (panel C).
Fig. 4
Fig. 4
IscU orientation and dynamics within the binary complex. (A) Main collective motions of the IscS-IscU complex from PCA (Cα atoms only), showing the IscU pivotal movement around the IscS dimer and the motion of the IscS catalytic loop. Vectors display the amount and direction of the residue motion along the first eigenvector. Minor residue contributions (i.e. arrow length < 2Å) were omitted. (B) Motion of the IscU protomers around the IscS dimer. A number of uncorrelated IscU configurations (one every 10 ns) are superposed upon fitting onto the IscS dimer structure (orange). A representative α-helix of IscU is depicted as cartoons and colored as a function of simulation time, while the rest of the protein is depicted as pink ribbons. Angle formed between IscU and IscS from (C) the average IscS-IscU complex structure from our MD simulation, and (D) the A. fulgidus crystal structure.
Fig. 5
Fig. 5
The IscS-IscU-CyaY complex. (A) IscS-IscU-CyaY complex structure, where two CyaY proteins (blue) are bound in the cleft formed between IscS dimer (orange) and IscU (pink). (B) Electrostatic potential surfaces for the IscS-IscU complex (right) and CyaY (left). Structure orientation is chosen to better visualize the mutual interacting surfaces on both systems. (C) Steric clashes between CyaY and [Fe—S]-loaded IscU (purple) in a hypothetical ternary complex structure built from the superposition of A. fulgidus IscU onto the average ternary structure (with IscU omitted) from our simulation superposed on the IscS dimer only. (D) Zoomed view of the interface between the IscS dimer and CyaY. The more persistent intermolecular salt bridges (see Table S1) are depicted as sticks connecting Cα atoms (spheres) belonging to the corresponding charged residues.
Fig. 6
Fig. 6
Backbone RMSF and IscU motion within the ternary complex. (A) Average structure of the IscS-IscU hetero-tetramer from the ternary complex simulation, depicted as ribbons and colored according to backbone RMSF values, from lowest (blue) to highest (red). Ribbon thickness is proportional to RMSF value. (B) IscU protomer motions around the IscS dimer from the ternary complex simulation. Details described in the caption of Fig. 4D.
Fig. 7
Fig. 7
Essential dynamics analysis of the binary and ternary complexes. Projection of the simulated trajectories of the IscS-IscU tetramer (Cα atoms only) for both binary and ternary complexes onto the 2D ED plane described by the first two eigenvectors of the binary complex. The analysis highlights the more restricted conformational subspace spanned by the IscS-IscU assembly within the ternary complex with respect to the binary system.
Fig. 8
Fig. 8
NMR TROSY spectra and Ile side-chain conformational distributions. (A) Methyl TROSY spectra of 100 μM U-[15N, 2H], ILVCH3 IscU-Zn (black spectrum) was titrated with 2 molar equivalences of U-[2H] IscS (red spectrum) and then with 2 molar equivalences of U-[2H] CyaY (green spectrum). The assignments of the Ileδ1, Leuδ1/2 and Valγ1/2 for free IscU-Zn are shown. (B) Distributions of the Cα-Cβ dihedral angle of selected IscU isoleucines from the binary system simulation. Each plot reflects the conformation of both isoleucines of the two protomers.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Roche B., Aussel L., Ezraty B., Mandin P., Py B., Barras F. Iron/sulfur proteins biogenesis in prokaryotes: formation, regulation and diversity. Biochim. Biophys. Acta Biomembr. Bioener. 2013;1827:455–469. - PubMed
    1. Roche B., Huguenot A., Barras F., Py B. The iron-binding CyaY and IscX proteins assist the ISC-catalyzed Fe—S biogenesis in Escherichia coli. Mol. Microbiol. 2015;95:605–623. - PubMed
    1. Paris Z., Changmai P., Rubio M.A.T., Zikova A., Stuart K.D., Alfonzo J.D., Lukes J. The Fe/S cluster assembly protein Isd11 is essential for tRNA thiolation in Trypanosoma brucei. J. Biol. Chem. 2010;285:22394–22402. - PMC - PubMed
    1. Marelja Z., Stocklein W., Nimtz M., Leimkuhler S. A novel role for human Nfs1 in the cytoplasm - Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis. J. Biol. Chem. 2008;283:25178–25185. - PubMed
    1. Stehling O., Wilbrecht C., Lill R. Mitochondrial iron-sulfur protein biogenesis and human disease. Biochimie. 2014;100:61–77. - PubMed

Publication types

MeSH terms

LinkOut - more resources