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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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Related in: MedlinePlus

NMR assignement of VPS29.VPS29 [1H,15N]-HSQC spectrum. Assigned peaks are labelled by residue number.
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pone-0020420-g002: NMR assignement of VPS29.VPS29 [1H,15N]-HSQC spectrum. Assigned peaks are labelled by residue number.

Mentions: In this study we use NMR spectroscopy and X-ray crystallography to examine the functional interactions of VPS29, their affinities, and potential conformational changes in VPS29 upon complex formation in order to distinguish between the above possibilities. As a prerequisite for ligand binding and structural experiments we performed 15N and 13C triple resonance experiments to assign the backbone 1H, 15N and 13C resonances of the mouse VPS29 protein (Fig. 2). VPS29 displays excellent NMR spectral properties, and we have been able to obtain 97% of the backbone assignments for the protein. Amide assignments are missing for Asn16, Ala20, Trp93, Gly94, Glu125 and Ile130. All backbone NOEs are consistent with the secondary structure observed in the X-ray structures of VPS29 (data not shown).


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)

NMR assignement of VPS29.VPS29 [1H,15N]-HSQC spectrum. Assigned peaks are labelled by residue number.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101248&req=5

pone-0020420-g002: NMR assignement of VPS29.VPS29 [1H,15N]-HSQC spectrum. Assigned peaks are labelled by residue number.
Mentions: In this study we use NMR spectroscopy and X-ray crystallography to examine the functional interactions of VPS29, their affinities, and potential conformational changes in VPS29 upon complex formation in order to distinguish between the above possibilities. As a prerequisite for ligand binding and structural experiments we performed 15N and 13C triple resonance experiments to assign the backbone 1H, 15N and 13C resonances of the mouse VPS29 protein (Fig. 2). VPS29 displays excellent NMR spectral properties, and we have been able to obtain 97% of the backbone assignments for the protein. Amide assignments are missing for Asn16, Ala20, Trp93, Gly94, Glu125 and Ile130. All backbone NOEs are consistent with the secondary structure observed in the X-ray structures of VPS29 (data not shown).

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