Where Honey Bee Vitellogenin may Bind Zn²⁺-Ions

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 (ICP-MS) 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 illustrate the capacity of honey bee Vg to 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.

Identification of 121 variants of honey bee Vitellogenin protein sequences with structural differences at functional sites

“…Except for p.Gly146Ser, all of the nsSNPs cluster at one side of the structure (Figure 4a). Gly146 is buried in the subdomain, close to a set of predicted Zn 2+ -coordinating residues and a proposed DNA binding region (Leipart et al in manuscript 44 ). 37 The remaining nsSNPs increase the polarity of buried residues or increase the hydrophobicity at the surface, except for p.Ile132Met, which maintains the hydrophobic core (Figure 4b,c).…”

“…Honey bee Vg has been demonstrated to sense oxidative stress 75 and suggested protecting honey bees from reactive oxidative species. Our earlier study shows that two disulfide bridges are conserved in the C‐terminal region, which is proposed to coordinate Zn 2+ (Leipart et al 2021 in manuscript 44 ) (Figure 7a). Proteins with a positive surface charge and disulfide bridges on neighboring residues, sometimes including Zn 2+ , are shown to protect against oxidative stress 76,77 .…”

“…11 (Figure 7b ). The two remaining nsSNPs, not introducing serine, are rare and drastic substitutions (Figure 3d ), one increasing the hydrophobicity of the buried structural elements, and the other introducing a large aromatic residue close to a predicted Zn 2+ ‐binding site (Leipart et al in manuscript, 44 ). The positive surface charge of the C‐terminal is not altered by any of the six nsSNPs (Figure 7c ).…”

How Honey Bee Vitellogenin Holds Lipid Cargo: A Role for the C-Terminal

“…The vWF domain is packed tightly in the lipid binding site, while the C-terminal is a separate solvent-exposed region ( Figures 2C,E ). We proposed a possible zinc-coordination site that resides between the two adjacent disulfide bridges in the C-terminal region in manuscript, Leipart et al (2022b) . Similar coordination sites of four cysteine residues are often found in redox switches ( Ilbert et al, 2006 ; Pace and Weerapana, 2014 ) that can cause conformational changes: During oxidative stress, zinc is released, resulting in oxidative folding and creation of disulfide bridges ( Ruddock and Klappa, 1999 ).…”

“… (A) Lamprey Vg has a disulfide bridge in the α-helical subdomain (yellow sticks, black arrow), while (B) MTP has two disulfide bridges (yellow sticks, black arrows) in the same subdomain. (C) The alignment is derived from in the manuscript, Leipart et al (2022b) , and shows two regions in the α-helical subdomain. The taxa, UniProt ID, and protein type are included on the sequences left.…”