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bioRxiv · 10.1101/2022.10.03.510666

Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies

Diep, P.·Stogios, P. J.·Evdokimova, E.·Savchenko, A.·Mahadevan, R.·Yakunin, A. F.
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Abstract

Extracytoplasmic solute binding proteins (SBPs) are molecular shuttles involved in the cellular uptake of various small molecules and metal ions including Ni(II). Our previous study with the Ni(II) binding proteins (NiBPs) CjNikZ from Campylobacter jejuni and CcNikZ-II from Clostridium carboxidivorans demonstrated they were able to bind Ni(II) at low micromolar affinity without the need for additional chelators. Here, we determined the crystal structure of apo CcNikZ-II, which revealed a Ni(II) binding site comprised of the highly conserved double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Alanine scanning mutagenesis of the CcNikZ-II Ni(II) binding site identified Glu60 and His511 as essential for high affinity binding to Ni(II). Phylogenetic analysis of >4000 SBP sequences demonstrated the presence of two clusters of proteins containing the HH-prong with CcNikZ-II and CjNikZ. To provide insights into the role of the double His-prong and v-loop sequence in Ni(II) binding of NiBPs, nine purified CcNikZ-II homologues containing the HH-prong and v-loop were screened using an automated screening workflow. Metal binding assays with purified homologous NiBPs revealed high Ni(II) binding affinity without requirement for chelators indicating that the double His prong represents a signature motif for the presence of Ni(II) binding activity in SBPs. The engineered CcNikZ-II variants with the wild type v-loop (TEDKYT) replaced with v-loops from NiBPs with higher affinity showed up to an order of magnitude higher affinity for Ni(II). In addition, the v-loop appears to play a role in metal ion specificity as purified wild type and engineered NiBPs with different v-loop sequences showed distinct metal profiles. This work paves way for metalloprotein engineering of NiBPs towards biocatalytic and metal recovery applications.

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Diep, P., Stogios, P. J., Evdokimova, E., Savchenko, A., Mahadevan, R., Yakunin, A. F.. 2022-10-03. Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies. https://doi.org/10.1101/2022.10.03.510666

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