Crystal Structure of Human Vacuolar Protein Sorting Protein 29 Reveals a Phosphodiesterase/Nuclease-like Fold and Two Protein-Protein Interaction Sites

Abstract

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a "retromer complex." In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Å resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered ␣-␤-␤-␣ sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two proteinprotein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.To function properly for the organelles in eukaryotic cells, it is necessary that specific sets of proteins and lipids are sorted and delivered to various intracellular compartments efficiently. The trafficking of proteins and lipids between intracellular organelles requires a series of proteins that are involved in the membrane transport pathways (1-3). Genetic screens in yeast have uncovered more than 50 Vps 1 genes. The products of these Vps genes are involved directly or indirectly in protein sorting or trafficking between the late Golgi and the vacuoles (4 -6). Disruption of Vps genes can cause disturbance and missorting of many proteins, such as CPY. As a vacuolar hydrolase, CPY is recognized by Vps10p in the late Golgi. Vps10p binds to CPY and conveys it via vesicle to the prevacuolar compartment where Vps10p releases it. After that, Vps10p returns to the Golgi for further rounds of transportation. Some yeast Vps gene products, Vps26p, Vps29p, Vps35p, Vps5p, and Vps17p, are necessary for the retrograde retrieval process of Vps10p from endosomes back to the trans-Golgi network (7-10). By forming a complex named "retromer complex," the five proteins are also involved in the proper sorting and transportation of dipetidyl aminopeptidase A, Kex2 protease (Kex2p), polymeric immunoglobulin receptor, and mannose 6-phosphate receptor (9 -12). This complex consists of two subcomplexes. One subcomplex, consisting of Vps35p, Vps29p, and Vps26p, is responsible for selecting cargoes retrieved from the prevacuolar compartment (10, 13). The other subcomplex of Vps5p and Vps17p provides structural components to complete the retromer complex and mechanical force for vesicle budding (10,14,15). The retromer complex is conserved and has homologs in mammalian cells. In human cells, the retromer complex comprises hVps26p, hVps29p, hVps35p, and SNX1 (the mammalian homolog of Vps5p) (16,...

Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors

“…Finally, other X-ray crystallographic analyses have shown that mammalian Vps29 has a phosphoesterase fold with a coordination site for two divalent metal cations. This fold is similar to those of a wide variety of prokaryotic and eukaryotic phosphoesterases (11,42), and indeed, recombinant Vps29 in complex with Vps26 and Vps35 has been shown to have Zn 2ϩ -dependent phosphatase activity towards a serine-phosphorylated peptide derived from the cytosolic tail of the CI-MPR (13).…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…Examples are a recently characterized phosphodiesterase from M. jannaschii 311 and a vacuolar protein sorting protein from human. 312 Another group of binuclear metalloenzymes with great pharmacological potential is the metallo--lactamase family.…”

The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)

“…The overall architecture of SNX5-PX comprises a three-stranded antiparallel ␤-sheet abutted by three ␣-helices. In addition, a one-turn 3 10 -helix that connects helices 4 and 3 and a polyproline region that leads into a long, protruding helical hairpin are present (Fig. 3A) 28 form a long, irregular strand and exhibit random coil and backbone angles.…”