VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins James D. Swarbrick 1 , Daniel J. Shaw 2 , Sandeep Chhabra 1 , Rajesh Ghai 2 , Eugene Valkov 3¤ , Suzanne J. Norwood 2 , Matthew N. J. Seaman 4 , Brett M. Collins 2 * 1 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia, 2 Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia, 3 School of Chemistry and Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia, 4 Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge University, Cambridge, United Kingdom 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. Citation: Swarbrick JD, Shaw DJ, Chhabra S, Ghai R, Valkov E, et al. (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. doi:10.1371/journal.pone.0020420 Editor: Ludger Johannes, Institut Curie, France Received December 21, 2010; Accepted May 2, 2011; Published May 24, 2011 Copyright: ß 2011 Swarbrick et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work is supported by funds from the Australian Research Council (DP0878608), and NHMRC (511072). BMC is supported by an ARC Future Fellowship (FT100100027). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: b.collins@imb.uq.edu.au ¤ Current address: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom Introduction Retromer is a protein complex peripherally associated with endosomal organelles, and controls trafficking of a number of critical cargo molecules within tubulovesicular carriers to the trans Golgi network (TGN) [1,2,3,4]. Biochemical and genetic studies in yeast and higher eukaryotes have identified two distinct retromer sub-complexes; a core trimer composed of VPS35-VPS29-VPS26 (VPS: vacuolar protein sorting) and an associated homo or hetero- dimer of sorting nexin (SNX) proteins, containing combinations of SNX1, SNX2, SNX5 and SNX6. The current model postulates that the core complex is a cargo loading assembly that binds to the cytoplasmic tails of trafficking receptors such as the cation independent mannose-6-phosphate receptor (CI-MPR), Wntless, sortilin and DMT1 via the large VPS35 subunit [5,6,7,8, 9,10,11,12,13,14,15]. The SNX proteins drive the membrane remodelling required to form the tubulovesicular transport structures [16,17,18,19], and along with the small GTPase Rab7 may regulate recruitment of retromer to endosomal membranes through binding to phosphatidylinositol-3-phosphate (PtdIns(3)P) [18,19,20,21]. The exact roles of the individual subunits of the core retromer complex remain unclear. The function of the large VPS35 subunit is perhaps the best defined, as it forms the central scaffold for assembly with VPS29 and VPS26 [22,23,24,25], it binds directly to transmembrane cargo molecules [5,7,12], and also associates with SNX proteins [19,23,26]. It can therefore be thought of as the primary hub for the spatiotemporal assembly of functional transport intermediates. The roles of VPS29 and VPS26 are not as well understood. Each of these proteins is required for the stability of the core trimer in vivo [22,24,27,28,29], and based on structural similarity to arrestin molecules it has been suggested that VPS26 may play an ancillary role in recruiting cargo molecules or accessory proteins [22,29], although there is currently no experimental evidence for this. VPS29 is the smallest subunit of retromer and its structure reveals a striking similarity to Ser/Thr phosphatase enzymes [24,27,30]. This discovery led to the hypothesis that with VPS35 and VPS26, VPS29 may be the catalytic subunit of a trimeric phosphatase (core retromer), with some similarity to the PP2A holoenzyme [2]. Support for this came from the observation that VPS29 displayed weak phosphatase activity against phosphorylat- PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20420