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VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

Swarbrick JD, Shaw DJ, Chhabra S, Ghai R, Valkov E, Norwood SJ, Seaman MN, Collins BM - PLoS ONE (2011)

Bottom Line: VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins.There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case.Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo.

View Article: PubMed Central - PubMed

Affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

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Metals do not affect VPS29 phosphatase activity or interaction with VPS35.(A) SDS-PAGE gel showing purified VPS29 and trimeric retromer proteins used for phosphatase assays stained with Coomassie Blue. (B) No detectable phosphatase activity was measured for VPS29 alone or in complex with VPS35 and VPS26. Phosphatase assays used the CI-MPR peptide CSSTKLVSFHDD(pS)DEDLLHI. The release of phosphate was measured using Biomol Green reagent and colorimetric assay at 620 nm. Calf intestinal alkaline phosphatase (CIAP) is shown for comparison. (C) When VPS29 has bound metal, the conformation of Phe63 is altered such that it may clash with VPS35 and inhibit binding. The diagram shows a close up of the interaction between VPS29 and VPS35 [24]. The Mn2+-bound VPS29 structure (green ribbon, and yellow side-chains) is overlayed with VPS35-bound VPS29 (blue ribbon and cyan side-chain). VPS35 is shown in surface representation. (D) No significant difference is observed in binding to VPS35 in the presence of EDTA or MnCl2 indicating that metals do not influence complex formation. VPS29 interaction with VPS35 was analysed by ITC; top panels show raw data and bottom panels show integrated normalised data.
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pone-0020420-g005: Metals do not affect VPS29 phosphatase activity or interaction with VPS35.(A) SDS-PAGE gel showing purified VPS29 and trimeric retromer proteins used for phosphatase assays stained with Coomassie Blue. (B) No detectable phosphatase activity was measured for VPS29 alone or in complex with VPS35 and VPS26. Phosphatase assays used the CI-MPR peptide CSSTKLVSFHDD(pS)DEDLLHI. The release of phosphate was measured using Biomol Green reagent and colorimetric assay at 620 nm. Calf intestinal alkaline phosphatase (CIAP) is shown for comparison. (C) When VPS29 has bound metal, the conformation of Phe63 is altered such that it may clash with VPS35 and inhibit binding. The diagram shows a close up of the interaction between VPS29 and VPS35 [24]. The Mn2+-bound VPS29 structure (green ribbon, and yellow side-chains) is overlayed with VPS35-bound VPS29 (blue ribbon and cyan side-chain). VPS35 is shown in surface representation. (D) No significant difference is observed in binding to VPS35 in the presence of EDTA or MnCl2 indicating that metals do not influence complex formation. VPS29 interaction with VPS35 was analysed by ITC; top panels show raw data and bottom panels show integrated normalised data.

Mentions: Our NMR and crystallography experiments confirm that VPS29 can associate with different divalent metal cations, but the very low affinity of these metals calls into question the functional importance of their binding. As PPP phosphatase enzymes have an absolute requirement for divalent metal cations for activity, the key question is whether VPS29 is able to function as a metallophosphatase. As outlined above we have not detected activity using small molecule substrates [27], but it has been reported that VPS29 has activity against a phosphorylated peptide derived from the cargo protein CI-MPR, and this can be enhanced by the presence of other retromer subunits [31]. However, like Hierro and colleagues [24], when we perform similar experiments with purified proteins we do not observe any phosphatase activity towards the phosphorylated CI-MPR peptide (Fig. 5A, 5B). Data is shown only for experiments incorporating Zn2+ and Mn2+. Other ions, different pH and higher protein concentrations yield similar results. Our data, and that of Hierro and colleagues [24], strongly argues therefore that VPS29 is not a metal-dependent phosphatase enzyme.


VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins.

Swarbrick JD, Shaw DJ, Chhabra S, Ghai R, Valkov E, Norwood SJ, Seaman MN, Collins BM - PLoS ONE (2011)

Metals do not affect VPS29 phosphatase activity or interaction with VPS35.(A) SDS-PAGE gel showing purified VPS29 and trimeric retromer proteins used for phosphatase assays stained with Coomassie Blue. (B) No detectable phosphatase activity was measured for VPS29 alone or in complex with VPS35 and VPS26. Phosphatase assays used the CI-MPR peptide CSSTKLVSFHDD(pS)DEDLLHI. The release of phosphate was measured using Biomol Green reagent and colorimetric assay at 620 nm. Calf intestinal alkaline phosphatase (CIAP) is shown for comparison. (C) When VPS29 has bound metal, the conformation of Phe63 is altered such that it may clash with VPS35 and inhibit binding. The diagram shows a close up of the interaction between VPS29 and VPS35 [24]. The Mn2+-bound VPS29 structure (green ribbon, and yellow side-chains) is overlayed with VPS35-bound VPS29 (blue ribbon and cyan side-chain). VPS35 is shown in surface representation. (D) No significant difference is observed in binding to VPS35 in the presence of EDTA or MnCl2 indicating that metals do not influence complex formation. VPS29 interaction with VPS35 was analysed by ITC; top panels show raw data and bottom panels show integrated normalised data.
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Related In: Results  -  Collection

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pone-0020420-g005: Metals do not affect VPS29 phosphatase activity or interaction with VPS35.(A) SDS-PAGE gel showing purified VPS29 and trimeric retromer proteins used for phosphatase assays stained with Coomassie Blue. (B) No detectable phosphatase activity was measured for VPS29 alone or in complex with VPS35 and VPS26. Phosphatase assays used the CI-MPR peptide CSSTKLVSFHDD(pS)DEDLLHI. The release of phosphate was measured using Biomol Green reagent and colorimetric assay at 620 nm. Calf intestinal alkaline phosphatase (CIAP) is shown for comparison. (C) When VPS29 has bound metal, the conformation of Phe63 is altered such that it may clash with VPS35 and inhibit binding. The diagram shows a close up of the interaction between VPS29 and VPS35 [24]. The Mn2+-bound VPS29 structure (green ribbon, and yellow side-chains) is overlayed with VPS35-bound VPS29 (blue ribbon and cyan side-chain). VPS35 is shown in surface representation. (D) No significant difference is observed in binding to VPS35 in the presence of EDTA or MnCl2 indicating that metals do not influence complex formation. VPS29 interaction with VPS35 was analysed by ITC; top panels show raw data and bottom panels show integrated normalised data.
Mentions: Our NMR and crystallography experiments confirm that VPS29 can associate with different divalent metal cations, but the very low affinity of these metals calls into question the functional importance of their binding. As PPP phosphatase enzymes have an absolute requirement for divalent metal cations for activity, the key question is whether VPS29 is able to function as a metallophosphatase. As outlined above we have not detected activity using small molecule substrates [27], but it has been reported that VPS29 has activity against a phosphorylated peptide derived from the cargo protein CI-MPR, and this can be enhanced by the presence of other retromer subunits [31]. However, like Hierro and colleagues [24], when we perform similar experiments with purified proteins we do not observe any phosphatase activity towards the phosphorylated CI-MPR peptide (Fig. 5A, 5B). Data is shown only for experiments incorporating Zn2+ and Mn2+. Other ions, different pH and higher protein concentrations yield similar results. Our data, and that of Hierro and colleagues [24], strongly argues therefore that VPS29 is not a metal-dependent phosphatase enzyme.

Bottom Line: VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins.There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case.Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo.

View Article: PubMed Central - PubMed

Affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

ABSTRACT
VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.

Show MeSH
Related in: MedlinePlus