Resolving the zinc binding capacity of honey bee vitellogenin and locating its putative binding sites

Abstract The protein vitellogenin (Vg) plays a central role in lipid transportation in most egg‐laying animals. High Vg levels correlate with stress resistance and lifespan potential in honey bees (Apis mellifera). Vg is the primary circulating zinc‐carrying protein in honey bees. Zinc is an essential metal ion in numerous biological processes, including the function and structure of many proteins. Measurements of Zn2+ suggest a variable number of ions per Vg molecule in different animal species, but the molecular implications of zinc‐binding by this protein are not well‐understood. We used inductively coupled plasma mass spectrometry to determine that, on average, each honey bee Vg molecule binds 3 Zn2+‐ions. Our full‐length protein structure and sequence analysis revealed seven potential zinc‐binding sites. These are located in the β‐barrel and α‐helical subdomains of the N‐terminal domain, the lipid binding site, and the cysteine‐rich C‐terminal region of unknown function. Interestingly, two potential zinc‐binding sites in the β‐barrel can support a proposed role for this structure in DNA‐binding. Overall, our findings suggest that honey bee Vg bind zinc at several functional regions, indicating that Zn2+‐ions are important for many of the activities of this protein. In addition to being potentially relevant for other egg‐laying species, these insights provide a platform for studies of metal ions in bee health, which is of global interest due to recent declines in pollinator numbers.


ICP-MS continued
We attempted to control for Zn 2+ interacting with the SUMO-tag by incubating the SUMO-tag with Zn 2+ before ICP-MS. Incubation resulted in significantly higher Zn 2+ levels in SUMO-tag samples compared to the non-incubated tagged β-barrel subdomain (Mann-Whitney U test: w = 0, p-value = 0.0114). The difference in concentration indicated that the incubation caused the association of Zn 2+ with the SUMO-tag. However, we could not rule out more specific Zn 2+-binding to SUMO. The net determined Zn 2+ concentration for the β-barrel subdomain was much lower compared to the concentration for native full-length Vg. Figure 1B shows a mean of 1524.0000 µg/L (SD ± 261.6562), while Figure S7 shows the mean as 64.2000 µg/L (SD ± 13.4350), and the significance suggested 0.01 bound Zn 2+ per β-barrel molecule. We attempted to examine this result with an independent approach and expressed the β-barrel subdomain in the presence of Co 2+, attempting to displace Zn 2+ with this cation. Co 2+ is considered a good structural and functional model for studying Zn 2+ -binding sites as the coordination is similar and exchange for Zn 2+ to Co 2+ occurs in nature (Lane andMorel, 2000, Shumilina et al., 2014). In contrast to Zn 2+ , Co 2+ coordination causes a readily detected change in the protein's UV-Vis spectrum (Bertini and Luchinat, 1984, Shumilina et al., 2014, Sivo et al., 2017. The conducted experiments are presented below.

UV-Vis Spectroscopy
SUMO fusion proteins were expressed and purified as described in the main manuscript (i.e., Initially, in a storage buffer consisting of PBS at pH 7.4, 10% glycerol, and 0.5 M L-arginine, they were centrifuged at 17000xg for 10 min. Supernatants were concentrated using Amicon filters with 10 kDa cut-off at 3250xg for 30 min, producing approximately 4 mg/mL protein samples. These were transferred to the UV-VIS cuvette (path length 10 mm, 500 µL capacity, Hellma item number 108-002-10-40). A UV-Vis spectra in the range of 200-800nm at room temperature were acquired. If Co 2+ successfully replaced Zn 2+ , we would have expected to see a distinct Co 2+ -specific peak pattern near the 500-750 nm range (Sivo et al., 2017, Lane andMorel, 2000). However, we did not observe this (negative results are presented in Figure S8A).
The samples were then used for NMR and intrinsic tryptophan fluorescence spectroscopy (see below).

NMR Spectroscopy
Co 2+ can create a paramagnetic shift in protein NMR spectra (Lane and Morel, 2000). Therefore, we transferred stocks of SUMO-fusion proteins exposed to Zn 2+ , Zn 2+ Co 2+ , and Co 2+ (as described above) to NMR tubes. D2O up to 5% v/v was added. We then acquired 1D proton spectra (25°C, water suppression using Watergate, 512 scans, processed using exponential multiplication with a line broadening of 0.3 Hz), and inspected the spectra without finding any significant differences between the samples ( Figure S8B).

Intrinsic Tryptophan Fluorescence Spectroscopy
We also looked for fold changes in response to divalent cation(s) present during expression.
To do this, we transferred 5 µL of the NMR samples (described above) into 300 PBS buffer at pH 6.5 in a quartz cuvette (path length 5 mm) and conducted an emission scan (excitation wavelength 295 nm, slit widths 5 nm, 310-500 nm). The spectra, which primarily provide information on the microenvironment of the tryptophans in the protein (Knappskog andHaavik, 1995, Takita et al., 2003) did not show any significant difference.     are colored according to this scale. A) The buried residues in the β-barrel subdomain are well conserved, including the residues in cluster βb.1, βb.2, and the DNA binding motif β-sheet.
The regions closer to the surface are less conserved. B) The α-helices in the α-helical subdomain are well conserved. The residues in Cluster αh.1 and αh.2 are also conserved. C) One of the β-sheet in the DUF1943 domain includes cluster Duf.1, Duf.2, and the zinc motif identified by MotifScan. The conservation of the residues in the β-sheet are variable, but the clusters and zinc motif are conserved. As shown in the MSA, residue H926 is not conserved.
D) The C-terminal is generally not conserved, except the four residues presented as cluster Ct and the third disulfide bridge (labeled).   (Schneider andStephens, 1990, Crooks et al., 2004). C) Residue 140 to 233 in the β-barrel subdomain, using the same species as in the full-length MSA ( Figure S1), aligned to CTCF proteins. The conserved residues from the β-barrel subdomain, identified in the CTCF proteins, are in bold.