Abstract
Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.
Highlights
Eukaryotic cells use an interconnected system of membranebound compartments to partition intracellular space, allowing a multitude of biological reactions to proceed simultaneously in distinct chemical environments
The C-terminal region of human VPS39 contains a putative zinc finger domain with four predicted zinc-binding residues (Cys[841], Cys[844], His[863], Cys[866]). These residues are predicted to coordinate a single zinc ion based on homology to the zinc finger domain of S. cerevisiae protein Pcf[11] (Figure 1B–D)
Analysis of the eluted fractions by SDS-PAGE showed a single predominant band that migrated as would be expected for the VPS39 zinc finger domain (5.1 kDa; Figure 1E), with a much less intense band at higher apparent molecular mass that was presumed to be a small amount of SDS-resistant VPS39 dimer
Summary
Eukaryotic cells use an interconnected system of membranebound compartments to partition intracellular space, allowing a multitude of biological reactions to proceed simultaneously in distinct chemical environments. SNARE activity is tightly regulated by both Sec1/Munc[18] family proteins, which bind directly to SNAREs, and by multi-protein ‘tethering’ complexes that bring vesicles into close apposition to allow the physical contact of SNARE proteins on opposing membranes[2]. The conserved multi-subunit tethering complexes CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) combine both of these activities by incorporating the Sec1/Munc[18] family protein VPS33A3–5. The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. The non-native structure we observe may have implications for rational protein design
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