ABSTRACT
The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modelling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.
Footnotes
Author accepted manuscript. Manuscript expanded and resubmitted to Journal of Biological Chemistry as a full submission.
ABBREVIATIONS
- BEST
- band-selective short transient excitation
- DIPSI
- decoupling in the presence of scalar interactions
- DTT
- dithiothreitol
- EDTA
- ethylene diamine tetra-acetic acid
- EPR
- electron paramagnetic resonance
- HSQC
- heteronuclear single quantum coherence
- ICP
- inductively coupled plasma
- IPTG
- isopropyl-β-thiogalactoside
- ITC
- isothermal titration calorimetry
- MS
- mass spectrometry
- NMR
- nuclear magnetic resonance
- NTA
- nitrilotriacetic acid
- OES
- optical emission spectrometry
- SUMO
- small ubiquitin-like modifier
- TCEP
- tris(2-carboxyethyl)phosphine
- TOCSY
- total correlated spectroscopy
- TROSY
- transverse relaxation optimised spectroscopy
- UPF
- unidentified protein function
- UV
- ultraviolet