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

Swarbrick, James D., Shaw, Daniel J., Chhabra, Sandeep, Ghai, Rajesh, Valkov, Eugene, Norwood, Suzanne, Seaman, Matthew N. J. and Collins, Brett M. (2011) VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins. PLoS One, 6 5: e20420-1-e20420-15. doi:10.1371/journal.pone.0020420


Author Swarbrick, James D.
Shaw, Daniel J.
Chhabra, Sandeep
Ghai, Rajesh
Valkov, Eugene
Norwood, Suzanne
Seaman, Matthew N. J.
Collins, Brett M.
Title VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2011-05-24
Sub-type Article (original research)
DOI 10.1371/journal.pone.0020420
Open Access Status DOI
Volume 6
Issue 5
Start page e20420-1
End page e20420-15
Total pages 15
Place of publication San Francisco, CA, United States
Publisher Public Library of Science
Collection year 2012
Language eng
Formatted 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 Mn2+ and Zn2+ 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.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Tue, 20 Sep 2011, 12:24:09 EST by Brett Collins on behalf of School of Biomedical Sciences