Characterization and Design of Nickel-binding Proteins towards Metal Recovery Applications

Nickel is an important metal in the adoption of greener technologies needed to transition to cleaner economies, and mines face pressure to source it more sustainably. Nature has evolved nickel-specific ATP-binding cassette (ABC) importers that can transport nickel across a cell’s membrane from the environment into the cytoplasm. Specifically, their solute-binding component known as the nickel-binding protein (NiBP) scavenges the cell’s periplasmic space to deliver metals to the cognate permease complex for transport. Existing methods for metal-binding characterization are not amenable to protein engineering efforts that rely on screening libraries of variants with different substrates. Using natural changes in protein fluorescence during a metal-binding event, we first optimized an assay previously reported in literature to measure the binding affinity (K¬D) of NiBP complexes with nickel and other metals. This assay was validated by determining the KD of the well-characterized NiBP CjNikZ from Campylobacter jejuni, which was comparable to reported values, so it was then used to demonstrate the presence of Ni(II)-binding activity in the uncharacterized NiBP called CcNikZ-II from Clostridium carboxidivorans. Next, we determined the crystal structure of CcNikZ-II, which revealed the potential Ni(II)-binding site located close to a short variable loop. Mutagenesis identified the CcNikZ-II residues involved in Ni(II) binding, but the role of the variable (v-)loop of this protein (TEDKYT) remained unclear. We purified nine CcNikZ-II homologues and determined the nickel-binding affinity of these proteins using an intrinsic fluorescence quenching assay, which showed all these proteins have higher KD for Ni(II) than CcNikZ-II. Furthermore, we replaced the CcNikZ-II v-loop sequence (TEDKYT) with those from the other homologues with higher affinity and found that the engineered CcNikZ-II variants have a higher binding affinity to Ni(II). Metal promiscuity screening further demonstrated the importance of the secondary coordination sphere in controlling affinity and specificity. Finally, an engineered E. coli strain (Ni_v.1) was created and tested in 10 ppm NiCl2 solution matching environmental conditions and demonstrated 7-fold improvement in nickel bioaccumulation performance compared to controls. Thus, both wildtype and engineering microbial NiBPs can be engineered for improved metal binding and selectivity and used for developing bio-based technologies for metal recovery applications.

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