Structural Organization of Essential Iron-Sulfur Clusters in the Evolutionarily Highly Conserved ATP-binding Cassette Protein ABCE1

Dominik Barthelme,Urte Scheele,Stephanie Dinkelaker,Adam Janoschka,Fraser Macmillan,Sonja-Verena Albers,Arnold J.M Driessen,Marco Salamone Stagni,Eckhard Bill,Wolfram Meyer-Klaucke,Volker Schuönemann,Robert Tampé;

doi:10.1074/jbc.m700825200

Abstract

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.

Highlights

Iron-sulfur (Fe-S)2 clusters constitute an ancient prosthetic group, which can be found in proteins from all living organisms
The cytosolic, essential Fe-S protein ABCE1 could explain this phenomenon, because of the fact that Fe-S clusters can be transported from mitochondria to the cytosol [8, 9], where they are incorporated into the protein moiety
ABCE1 belongs to the superfamily of ATP-binding cassette (ABC) proteins with twin ABC ATPase domains, which are arranged in a headto-tail orientation via a flexible linker and hinge region [14]

Summary

Introduction

Iron-sulfur (Fe-S)2 clusters constitute an ancient prosthetic group, which can be found in proteins from all living organisms. (i) Functional studies of each cysteine in Fe-S cluster assembly of ABCE1 were performed in yeast. All ABCE1 preparations, except the C24S mutant, showed a brownish color, typical for Fe-S cluster proteins (Fig. 1C).

Results

Conclusion