A histidine-richPseudomonasmetallothionein with a disordered tail displays higher binding capacity for cadmium than zinc

Abstract

Metallothioneins (MTs) are crucial players in metal-related physiological processes. They are characterized by a high cysteine content and unique metal binding properties resulting in specific metal-thiolate clusters formation. Here we present the first NMR solution structure of a Pseudomonas MT, PflQ2 MT, using the strain P. fluorescens Q2-87. It consists of a metal binding domain and an intrinsically disordered C-terminal tail, that was not observed in other MTs so far. While not influencing the structure or function of the metal binding domain, the tail contains a potential binding motif that might be important in so far undiscovered biological interactions. Unusual is the different metal binding capacity for three ZnIIversus four CdII ions that results in two novel metal-cluster topologies. Nevertheless, the affinity for the fourth CdII ion is reduced due to transient coordination. PflQ2 MT contains an unusually large number of four histidine residues, of which only one is involved in metal ion binding. The three non-coordinating histidine residues influence neither the protein fold nor the stability in vitro. We demonstrate that reinstatement of a second coordinating histidine residue, observed for cyanobacterial MTs, in place of a non-coordinating residue in Pseudomonas MTs, decreases the kinetic lability of the cluster, while preserving the overall metal ion binding stability and the protein fold. Since high thermodynamic stability combined with high kinetic lability of metal binding are mechanistic features critical for the function of MTs, the observed replacement might be advantageous for Pseudomonas MTs with respect to metal ion handling in vivo.

Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins

“…1). 7 Our studies revealed that the long C-terminal tail is intrinsically disordered and neither influencing the metalbinding properties nor the structure of the rest of the protein. However, it might potentially serve as a biologically relevant binding site for globular proteins or other interacting molecules.…”

“…The shortened version of the protein lacking the C-terminal tail, sh_PpKT MT (residues 1-51) was constructed in the same manner. Both newly constructed plasmids, as well as previously constructed plasmids of the fulllength PflQ2 MT and its shortened version sh_PflQ2 MT, 7 were used as a DNA template for the site-directed mutagenesis to construct the C28S and S29C mutants, respectively. Oligonucleotides used for the site-directed mutagenesis are listed in Table S1 (ESI †).…”

“…All proteins were expressed and purified as previously described. 7 The identity of proteins was confirmed by electrospray ionization mass spectrometry (ESI-MS) ( Fig. S3, ESI †).…”

“…Calculations and value corrections were performed as described previously. 7 Mass spectrometry ESI-MS spectra were acquired using a Synapt G2 quadrupole time-of-flight spectrometer (Waters, UK) with a capillary voltage of 2.8 V, a cone voltage of 40 V, and a source temperature of 80 1C. The apo-forms were measured in MeOH : 0.15% aqueous formic acid (1 : 1) and the holo-forms in MeOH : 30 mM aqueous ammonium acetate (1 : 1).…”

“…Ala, Gly, Ser) in PsdMTs. Furthermore, the His content in PsdMTs is generally unusually high, some sequences contain a long C-terminal Cys-free amino acid stretch with a highly conserved sequence, 7 and in almost 90% of the sequences identified so far an additional Cys instead of a Ser residue can be found in one of the central motifs, i.e. YC À CxxC instead of YC À SxxC.…”