Characterization of a pre-export enzyme–chaperone complex on the twin-arginine transport pathway

Abstract

The Tat (twin-arginine translocation) system is a protein targeting pathway utilized by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli, Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. The present study demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterization suggested that the C-terminal ‘Domain IV’ of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

“…As such, domains II and III bind the guanine moieties of each pterin which extend close to the protein surface. At one extremity of the Moco molecule, domain IV constitutes a cap whose rotation would allow access to a wide funnel for Moco insertion as recently suggested1528. Notably, our comparative SAXS analysis of both the apoNarGH and holoNarGH revealed that cofactor-dependent conformational changes are restricted to the catalytic subunit NarG.…”

“…Whilst preliminary, a SAXS study performed on a chaperone-molybdoenzyme complex, apoTorAD showed a largely folded catalytic subunit15. Herein, Moco is the sole prosthetic group of TorA, catalytic subunit of the trimethylamine N -oxide reductase and member of the Mo/W- bis PGD family42.…”

“…First, the flexibility of domain IV of NarG (residues Lys1078 to Ile1184) (Supplementary Fig. 2) was assessed as it was shown to be mobile in a related member of the Mo/W- bis PGD family, and proposed to serve as a lid after cofactor insertion15. The domain IV of NarG was given as an independent structure in SASREF allowing free movement with respect to the rest of NarGH with only distance restraints of 4 Å maximum between Gln1077 and Lys1078 and also between Ile1184 and His1185 of NarG, consistent with formation of peptide bonds between these residues.…”

Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

“…This strongly suggests that NapA is present in a more ‘open’ conformation in the complex, which would be consistent with a lack of MoCo in the NapA precursor. The shape of the complex is not dissimilar in overall architecture to the SAXS‐derived model for the TorAD complex . However, unlike the TorAD complex where the component high‐resolution structures of TorA and TorD could be well fitted into the SAXS envelope by rigid body modelling, the rigid body models created for the NapDA SAXS data had less volume than the corresponding ab initio models, even when integrating flexibility into the model by allowing flexibility in the orientation of domain IV of NapA (which is the only domain of Nap/DMSO reductase family enzymes that is formed from a contiguous stretch of polypeptide chain and which is known to be flexible in the TorA precursor ).…”

“…In addition to their Tat‐targeting role, they also act as binding sites for biosynthetic chaperones . A well‐studied example of a signal‐peptide‐binding biosynthetic chaperone is TorD, which binds to the twin‐arginine signal peptide of its cognate Tat substrate trimethylamine‐ N ‐oxide reductase, TorA, and at a second site close to the mature N‐terminus . TorD binding facilitates molybdenum cofactor insertion into TorA by maintaining the apoprotein in an open conformation, simultaneously shielding the substrate from interacting with the Tat machinery while allowing the cofactor to insert.…”