VPS29 binds Zn²⁺ in solution as determined by NMR spectroscopy.

<p>(<b>A</b>) A number of residues in the [¹H,¹⁵N]-HSQC spectra of VPS29 show specific perturbations on addition of Zn²⁺. Spectra are shown of VPS29 in the apo state (black) or in the presence of 200 µM ZnCl₂ (red). Resonances showing significant chemical shift changes are indicated. (<b>B</b>) Zn²⁺ binds to VPS29 in solution within the same major pocket identified by X-ray crystallography. Residues showing significant chemical shift upon Zn titration are highlighted on the structure in red (Δδ greater than 2 SD). Each monomer from the asymmetric unit of the Zn²⁺-bound mouse VPS29 crystal structure has been mapped, with crystallographically identified Zn²⁺ ions indicated in blue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone-0020420-g003" target="_blank"><b>Fig. 3</b></a> for details). Note however, that VPS29 is monomeric in solution, not dimeric. No significant evidence is seen for binding to the low occupancy Zn²⁺ site coordinated by Asp55, His57 and His33 (dashed circle). (<b>C</b>) Titration of VPS29 with Zn²⁺ can be followed by observing the change in intensity for the Gly133 NH resonance in bound and unbound states. (<b>D</b>) Plot of the bound and unbound intensity ratio for the Gly133 NH resonance as a function of Zn²⁺ concentration. The blue line shows the fit to the Hill equation. The estimated affinity is low with an overall <i>K</i>_d&gt;250 µM. (<b>E</b>) Binding of VPS29 to either Mn²⁺ or Zn²⁺ cannot be measured by ITC, confirming the low affinity of interaction. A weak endothermic signal is observed upon metal titration at 25°C but the binding affinity cannot be estimated. Top panels show raw data and bottom panels show integrated normalised data. No significant binding signals were observed under these conditions (0.04 mM protein, 2.5 mM metals, 25°C). Other metals including Mg²⁺, Ca²⁺ and Ni²⁺, and temperature regimes from 10–37°C produced similar negative results. (<b>F</b>) Mn²⁺ and Zn²⁺ bind to EDTA exothermically and with high affinity under identical conditions by ITC (0.2 mM EDTA, 2.5 mM metals, 25°C). The binding affinity is too high to be determined at the concentrations used.</p

VPS29 binds specifically to SNX1 but with low affinity <i>in vitro</i>.

<p>(<b>A</b>) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Harbour1" target="_blank">[32]</a>, confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association <i>in vivo</i> is relatively weak or transient. (<b>B</b>) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association <i>in vitro</i>. A selected region is shown for the ¹⁵N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (<b>C</b>) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (<b>D</b>) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (&gt;2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Hierro1" target="_blank">[24]</a>. The side-chains of the VPS29 hydrophobic surface are indicated. (<b>E</b>) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [¹H,¹⁵N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.</p

Purified HMunc18c is monomeric in solution.

<p><b>A</b>. Elution profile of purified HMunc18c on a calibrated analytical size exclusion chromatography column (S200 10/300 GL). HMunc18c eluted at a volume consistent with a ∼70 kDa protein. Peak fractions were analysed on 4–12% gradient SDS-PAGE (inset). <b>B</b>. Elution profile of HMunc18c examined by SEC-MALS. The horizontal blue line corresponds to the SEC-MALS calculated mass (right axis) plotted with the refractive index indicating the peak (left axis) of the protein in the sample (68,200 Da ±0.5%).</p

Interactions of VPS29 examined in this study.

<p>VPS29 has structural similarity to phosphatase enzymes and has the potential to bind two divalent cations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Collins3" target="_blank">[27]</a>. VPS29 binds VPS35 with an interface that incorporates the metal-binding pocket, the α3 helix and the Phe63 side-chain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Hierro1" target="_blank">[24]</a>. The α3 helix is known to adopt different conformations in previous crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Hierro1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Collins3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Wang1" target="_blank">[30]</a>, and Phe63 is known to adopt different conformations upon metal binding to VPS29 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Collins3" target="_blank">[27]</a>. A conserved hydrophobic surface lies opposite to the VPS35 interface, and is known to be required for binding to TBC1D5 in mammalian cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Harbour1" target="_blank">[32]</a>, and to the heterodimeric SNX complex in yeast <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Collins3" target="_blank">[27]</a>. Critically mutation of Leu152 to Glu is known to abolish these interactions. Note, the SNX proteins are also thought to form contacts directly with VPS35 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Rojas2" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020420#pone.0020420-Haft1" target="_blank">[23]</a>.</p

Isothermal titration calorimetry data.

<p>The raw data (upper part of each panel) and integrated normalized data (lower part of each panel) are shown from ITC experiments between HMunc18c or Munc18a-His and cognate/non-cognate Sx partners.</p

Summary of crystallographic structure determination statistics^a.

a<p>Highest resolution shell is shown in parentheses.</p

¹⁵N NMR relaxation data for VPS29 indicates a generally rigid structure with no large-scale mobility with well defined secondary structural elements.

<p>(<b>A</b>) Longitudinal T1 and transverse T2 relaxation times as well as the {¹H}¹⁵N heteronuclear NOEs are shown as a function of protein sequence. Residues within helix α3 are highlighted in grey. Six N-terminal (non-native) resides are shown and are labelled as residues 201–206. The protein secondary structure is indicated at the bottom of the figure. Data was recorded using a 600 MHz spectrometer. (<b>B</b>) The TALOS+ artificial neural network (ANN)-predicted secondary structural elements of VPS29. Length of bars corresponds to probability of a residue to be helix (black) or β-strand (grey).</p

Crystal structures of VPS29 bound to Mn²⁺ and Zn²⁺.

<p>Crystal structure of VPS29 determined by X-ray crystallography bound to either Mn²⁺ (<b>A</b>) or Zn²⁺ (<b>B</b>). Top panels show overall protein structures as ribbon diagrams with anomalous difference maps contoured at 3σ shown in red. Each monomer from the asymmetric unit is indicated in green and blue. Mn²⁺ ions are shown as magenta spheres, and Zn²⁺ ions are shown as salmon spheres. Middle panels (i) show zoomed in regions of the putative active site residues and bound metal ions. Bottom panels (ii) show enlarged regions for minor low occupancy sites distal to the major binding pocket.</p

The α3 helix of VPS29 adopts a compact conformation in solution.

<p>(<b>A</b>) Comparison of previous VPS29 crystal structures reveals differences in the orientations of the α3 helix. VPS29 adopts an extended α3 orientation in the mouse apo VPS29 crystal structure (PDB 1Z2X; green), and a compact orientation in the VPS35-bound human VPS29 structure (PDB 2R17; blue). Ala100, Gln103 and Tyr129 are shown for each structure, to show residues for which long-range NOEs are observed. In the apo mouse VPS29 structure these residues would be too far apart to observe these NOE contacts. (<b>B</b>) RDC correlation plots indicate the apo mouse VPS29 protein adopts a compact structure in solution, where the α3 helix is similar to the VPS35-bound conformation, but not the previous mouse VPS29 crystal structure. Shown are 133 ¹D_HN RDCs fitted to the apo human VPS29 structure (PDB 1W24), human VPS29 in complex with VPS35 (PDB 2R17) and the mouse VPS29 structure (PDB 1Z2X). The residues of helix α3 are highlighted in red (95–107). Residue 99 was not included due to severe overlap in the 2D ¹⁵N IPAP spectra. Single value decomposition analysis of the HN-N RDCs to the apo human, VPS35-bound human or apo mouse X-ray structures yielded the following: The largest component of the alignment tensor (Szz) were 1.31e⁻³, 1.35e⁻³, 1.09e⁻³ and Rhombicities (S_yy−S_xy/S_zz) were 0.53, 0.53 and 0.48. (<b>C</b>) 2D ¹HN-¹H strips from the 3D ¹⁵N NOESY-HSQC show the NOE connectivities along helix α3 (residues 97–107). Diagonal peaks for each strip are labelled with an asterisk and proximal protons that give rise to observable NOEs are annotated. Medium range i, i+2 ¹HN-¹HN NOES are underlined while medium range i, i+3 and i, i+4 ¹Hα-¹HN NOES are identified with arrows. Both are diagnostic of a regular alpha helix. Two long range NOEs to the Hε protons of Tyr129 from the ¹HN of Ala100 and Gln103 are labelled as well as the long-range (cross sheet) ¹HN-¹HN NOE between Y129 and F122. (<b>D</b>) ¹D_HN RDCs observed (black) compared to those calculated for each residue for mouse VPS29 structure (PDB 1Z2X, green), apo human VPS29 structure (PDB 1W24, red) and human VPS29 in complex with VPS35 (PDB 2R17, blue). (<b>E</b>) Detail comparing the close fit of the ¹D_HN RDCs to the α3 orientation in the apo human VPS29 compared to the poor fit to the mouse α3 orientation.</p

ITC derived thermodynamic parameters for Munc18:Sx-His and Munc18:ΔNSx-His interactions.

<p>Values reported are average and standard deviation from at least three experiments.</p