Non-native fold of the putative VPS39 zinc finger domain

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.

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VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

Biochemical Journal ◽

10.1042/bcj20170588 ◽

2017 ◽

Vol 474 (21) ◽

pp. 3615-3626 ◽

Cited By ~ 11

Author(s):

Morag R. Hunter ◽

Edward J. Scourfield ◽

Edward Emmott ◽

Stephen C. Graham

Keyword(s):

Zinc Finger ◽

Terminal Segment ◽

Ring Domain ◽

Core Complex ◽

Endosomal Membrane ◽

Late Endosomes ◽

Early Endosomes ◽

Membrane Tethering ◽

New Gene ◽

Ring Interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

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VPS18 recruits VPS41 to the human HOPS complex via a RING-RING interaction

10.1101/169185 ◽

2017 ◽

Cited By ~ 1

Author(s):

Morag R. Hunter ◽

Edward J. Scourfield ◽

Edward Emmott ◽

Stephen C. Graham

Keyword(s):

Zinc Finger ◽

Terminal Segment ◽

Ring Domain ◽

Core Complex ◽

Endosomal Membrane ◽

Late Endosomes ◽

Early Endosomes ◽

Ring Domains ◽

Membrane Tethering ◽

Ring Interaction

ABSTRACTEukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET and HOPS, to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

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Faculty Opinions recommendation of BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1006519.81856 ◽

2002 ◽

Author(s):

Anjana Rao

Keyword(s):

Zinc Finger ◽

Germ Line ◽

Specific Protein ◽

Epigenetic Reprogramming ◽

Zinc Finger Domain ◽

Male Germ Line ◽

Insulator Protein

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Hsp70 Escort Protein: More Than a Regulator of Mitochondrial Hsp70

Current Proteomics ◽

10.2174/1570164615666180713104919 ◽

2018 ◽

Vol 16 (1) ◽

pp. 64-73 ◽

Cited By ~ 1

Author(s):

David O. Nyakundi ◽

Stephen J. Bentley ◽

Aileen Boshoff

Keyword(s):

Zinc Finger ◽

Zinc Finger Domain ◽

C Terminus ◽

Cellular Localisation ◽

The Matrix ◽

Mitochondrial Hsp70 ◽

Cellular Compartments ◽

Intramolecular Communication ◽

Self Aggregation ◽

J Protein

Hsp70 members occupy a central role in proteostasis and are found in different eukaryotic cellular compartments. The mitochondrial Hsp70/J-protein machinery performs multiple functions vital for the proper functioning of the mitochondria, including forming part of the import motor that transports proteins from the cytosol into the matrix and inner membrane, and subsequently folds these proteins in the mitochondria. However, unlike other Hsp70s, mitochondrial Hsp70 (mtHsp70) has the propensity to self-aggregate, accumulating as insoluble aggregates. The self-aggregation of mtHsp70 is caused by both interdomain and intramolecular communication within the ATPase and linker domains. Since mtHsp70 is unable to fold itself into an active conformation, it requires an Hsp70 escort protein (Hep) to both inhibit self-aggregation and promote the correct folding. Hep1 orthologues are present in the mitochondria of many eukaryotic cells but are absent in prokaryotes. Hep1 proteins are relatively small and contain a highly conserved zinc-finger domain with one tetracysteine motif that is essential for binding zinc ions and maintaining the function and solubility of the protein. The zinc-finger domain lies towards the C-terminus of Hep1 proteins, with very little conservation outside of this domain. Other than maintaining mtHsp70 in a functional state, Hep1 proteins play a variety of other roles in the cell and have been proposed to function as both chaperones and co-chaperones. The cellular localisation and some of the functions are often speculative and are not common to all Hep1 proteins analysed to date.

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