The X-ray Structure of the Zinc Transporter ZnuA from Salmonella enterica Discloses a Unique Triad of Zinc-Coordinating Histidines

Zinc ion coordination as a modulating factor of the ZnuA histidine-rich loop flexibility: A molecular modeling and fluorescence spectroscopy study

“…Whereas the same coordination may be likely assumed also by S. enterica ZnuA [14], the crystal structure of this protein has revealed a distinct metal coordination involving a His residue from the mobile His-loop and it has been suggested that this peculiar metal coordination may be favored by low metal occupancy [11]. These findings suggest that the two metal binding regions may be correlated and that the His-loop may have a direct role in zinc transport, either by facilitating zinc transfer from the His-loop to the primary metal binding site or from ZnuA to ZnuB.…”

“…We have chosen to use a model of the S. enterica ZnuA, rather than its available X-ray structure [11], because in that structure, due to its high mobility, the Hisloop is absent with the exception of a short segment containing an histidine coordinating the metal. This particular ligand arrangement [11] may correspond to a ZnuA transient intermediate in which the zinc is transferred from the His-loop to the primary metal binding site, but it is not suitable to explain the results of our fluorescence experiments.…”

“…We have chosen to use a model of the S. enterica ZnuA, rather than its available X-ray structure [11], because in that structure, due to its high mobility, the Hisloop is absent with the exception of a short segment containing an histidine coordinating the metal. This particular ligand arrangement [11] may correspond to a ZnuA transient intermediate in which the zinc is transferred from the His-loop to the primary metal binding site, but it is not suitable to explain the results of our fluorescence experiments. The model used to elucidate the spectroscopic results contains two metal ions in two different zinc sites: the zinc ion in the primary site maintains the ligands Glu59, His60, His147 and His211, already described in the E. coli X-ray diffraction [10], while the secondary zinc ion, easily modeled thanks to the plasticity of the histidine-rich loop, was arbitrarily bound to residues Glu132, Asp136, His141 and His228 [14], this latter conserved as a zinc ligand based on the Yatsunyk and coworkers experimental evidence [10].…”

“…In the 3D ZnuA structure from S. enterica [11] the primary zinc binding site is located in proximity of the Trp residues with a coordinating residue coming from His-loop (His140 in the place of His60, used as a ligand in the models [14]), that is very improbable as a stable ligand of the primary zinc site, since binding of the first zinc doesn't induce a reduction of the mobility of the fluorophore located in the His- loop. It is then likely that the structure detected by the X-ray diffraction represents an intermediate state, possibly favored by the low metallation of the protein sample used in crystallographic experiments [11], in the process of transferring the Zn 2+ ion from the secondary to the primary zinc site.…”

“…The crystal structures of ZnuA from Synechocystis sp. 6803 [9], Escherichia coli [10] and S. enterica [11], show a well conserved structure characterized by two (a/b)4 domains connected by a long and flexible a-helix. A primary high-affinity metal binding site is located in the cleft between the two domains.…”