bioRxiv Science⌕ Search
Home/Archive/bioRxiv

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.
View original source ↗Export metadata ↓

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.

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

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

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗
bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗
bioRxiv2026

Interventional breath analysis reveals oral microbial conversion of 1,2-Propanediol into Propanal and 1-Propanol

Background: Orally exhaled breath contains volatile organic compounds (VOCs) that may reflect local microbial metabolism as well as systemic processes. Consequently, the breath sampling route can influence VOC profiles and complicate biomarker interpretation. Previous studies have shown that 1-propanol is substantially enriched in orally compared with nasally exhaled breath. Objective: To determine whether a controlled oral challenge with 1,2-propanediol (1,2-PD) induces sampling route-dependent changes in breath 1-propanol concentrations. Methods: Ten healthy volunteers received 0.1 mL 1,2-PD diluted in approximately 50 mL water. The solution was used as an oral rinse for approximately 30 s and then expectorated. Matched oral and nasal end-tidal breath samples were collected before administration, immediately afterwards, and at 15-min intervals. Propanal, 1-propanol, acetol, acetone, and isoprene were analysed using gas chromatograph-ion mobility spectrometer (GC-IMS). Results: Oral 1,2-PD exposure produced a rapid and transient increase in exhaled propanal and 1-propanol signals, with the largest responses immediately after administration and at the second post-intervention measurement. In contrast, nasal signals remained comparatively stable. Acetone and isoprene showed no consistent intervention-related changes, regardless of the sampling route. No reproducible increase in acetol was detected, although interpretation was limited by analytical interference. Conclusion: A controlled oral 1,2-PD challenge elicited a route-specific propanal and 1-propanol response, consistent with local microbial metabolism of 1,2-PD within the oral cavity. Both fucose and 1,2-PD have been previously reported in human saliva, including elevated concentrations in selected disease-associated contexts. Bacteria can degrade salivary mucin glycans, releasing sugars including fucose. Together, these observations motivate investigation of a proposed pathway linking mucin-derived fucose to 1,2-PD and subsequent propanal and 1-propanol formation in the human oral cavity.

biochemistry↗