ABSTRACT
The ATPase SecA is required for translocation of most proteins across the cytoplasmic membrane in bacteria. In Escherichia coli, SecA contains a small metal binding domain (MBD) at its extreme C-terminus that is widely conserved in other bacterial species and is required for its interaction with SecB. The MBD is thought to coordinate Zn2+ via a conserved cysteine-containing motif. Here, we investigated the metal binding properties of two E. coli proteins that contain SecA-like MBDs: YecA and YchJ. Both proteins copurify with metal, predominantly zinc. However, both proteins also copurify with significant amounts of iron. In YecA, iron binding is mediated by the MBD. Re-evaluation of the metal-binding properties of SecA indicate that: (i) SecA copurifies with stoichiometric amounts of iron; (ii) binding is mediated by the MBD; (iii) the MBD binds to iron with equal or greater affinity than to zinc; and (iv) the affinity for iron (but not for zinc) is mediated by a highly conserved serine in the metal-binding motif. Taken together, our results suggest that iron is a physiological ligand of SecA-like MBDs.
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