Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis

Kei Hirabayashi,Eiki Yuda,Naoyuki Tanaka,Sumie Katayama,Kenji Iwasaki,Takashi Matsumoto,Genji Kurisu,F.Wayne Outten,Keiichi Fukuyama,Yasuhiro Takahashi,Kei Wada

doi:10.1074/jbc.m115.680934

Abstract

ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.

Highlights

The ATP-binding cassette (ABC)3 is a ubiquitous, universally conserved ATPase domain/subunit historically defined as the nucleotide-binding domain of an ABC transporter
With the availability of complete genomes and the refinement of bioinformatic tools, it has become apparent that ABC type ATPase domains are present in ABC transporters and in a variety of nontransporter proteins, the most well known examples of which are the structural maintenance of chromosome (SMC) proteins involved in chromosome segregation/condensation and DNA repair (6 – 8)
It is likely that structural changes in the SufB and SufD subunits are driven by SufC ATPase activity in the SufBCD complex and that the dynamic motion of the complex should provide important clues regarding the molecular mechanism of Fe-S cluster biogenesis

Summary

Experimental Procedures

Expression and Purification of the E. coli SufBCD Complex— To purify the SufBCD complex, the entire suf operon was expressed simultaneously. Crystals of the SufBCD complex were obtained at 4 °C using a reservoir solution containing 31% (v/v) pentaerythritol propoxylate (5/4 PO/OH), 100 mM sodium citrate (pH 5.5), and 200 mM KCl. The protein concentration was 35 mg/ml in 50 mM MES (pH 7.0). Mercury and platinum derivatives were obtained by soaking native crystals for 2 h in mother liquor containing 1 mM methylmercury(II) acetate, 5 mM methylmercury(II) chloride, or 10 mM potassium tetranitro platinate(II). The EM structure of the SufBCD complex has been deposited in the Electron Microscopy Data Bank under accession number EMD-3163. Solution Scattering Data Collection and Analysis—The SufBCD complex (2–18 mg/ml) for small angle x-ray scattering (SAXS) experiments was prepared in 50 mM Tris-HCl (pH 7.8) and 150 mM NaCl. SAXS experiments were performed at room temperature on a Rigaku BioSAXS-1000, using CuK␣ radiation from the Rigaku FR-X rotating anode x-ray generator. A Hg, methylmercury(II) acetate. b Hg, methylmercury(II) chloride. c Pt, potassium tetranitro platinate(II)

Structural parametersa

Results

Discussion