Table 1.
Statistics of crystallographic data collection and refinement.
Figure 1.
Yeast two-hybrid analysis of the interaction of μ4 with the cytosolic tail of APP.
(A) Superposition of the surface and ribbon representations of human wild-type μ4 C-terminal domain (pdb entry 3L81). The insets show an enlargement of the μ4-binding site and the putative μ2-binding site with residues chosen for the yeast two-hybrid (Y2H) analysis. The APP peptide (TYKFFEQ; stick model) bound to the μ4-binding site is in yellow, and the EGFR peptide (FYRALM; stick model; pdb entry 1BW8) superposed to the putative μ2-binding site is in magenta. (B) Sequence alignment of the C-terminal domain of the μ subunits of known crystal structure depicting critical residues at the corresponding μ2- and μ4-binding sites. Disordered loops are in yellow letters, and arrows and cylinders represent β-strands and α-helices, respectively. M.m., Mus musculus; R.n., Rattus norvegicus; H.s., Homo sapiens. (C) and (D) Yeast were co-transformed with plasmids encoding Gal4bd fused to the cytosolic tail of the amyloid precursor protein (APP) indicated on the left, and Gal4ad fused to wild-type or mutant μ4 constructs indicated on top of each panel. (C) Y2H analysis of μ4 with mutations on the YKFFE binding site (μ4-binding site). (D) Y2H analysis of μ4 with mutations on a putative YXXØ binding site (μ2-binding site). Mouse p53 fused to Gal4bd and SV40 large T antigen (T Ag) fused to Gal4ad were used as controls. Co-transformed cells were spotted onto His-deficient (-His) or His-containing (+His) plates and incubated at 30°C. Growth is indicative of interactions. Some mutations on either of the two sites affect the interaction of μ4 with the cytosolic tail of APP.
Figure 2.
Isotermal titration calorimetry analysis of the interaction of μ4 with the APP sorting signal.
Isothermal titration calorimetry of the APP ENPTYKFFEQ peptide with recombinant C-terminal domain of wild-type μ4 (A), μ4-D190A (B), or μ4-R283D (C). The stoichiometry (N) and Kd for the interaction of the ENPTYKFFEQ peptide with either μ4-WT or μ4-D190A are expressed as the mean ± SEM (n = 3). Because the interaction of the ENPTYKFFEQ peptide with μ4-R283D is undetectable the stoichiometry and Kd were not determined (N/D).
Figure 3.
Crystal structure of the μ4-D190A C-terminal domain bound to the APP sorting signal.
Ribbon representation of human μ4-D190A C-terminal domain with subdomain A colored red, subdomain B colored orange, and the APP peptide (TYKFFEQ; stick model) colored yellow. The position of the N- and C-termini are indicated. The inset shows the orientation of the APP peptide side chains on the binding site, with atoms of the peptide colored yellow (carbon), red (oxygen), or blue (nitrogen). The crystal structure of μ4-D190A C-terminal domain bound to the APP peptide is very similar to that of wild-type μ4.
Figure 4.
Interaction of the APP peptide with binding site residues on μ4-D190A.
(A–G). Hydrogen-bonds are indicated by dashed lines. (A) Direct and water-mediated hydrogen bonding between backbone-residues of β4 (μ4-D190A) and residues 688–690 of the APP peptide. Side-chains of the APP peptide are omitted for clarity. (B) The hydroxyl group and the aromatic ring of Tyr-687 in the APP peptide hydrogen-binds Glu-265, and forms a hydrophobic interaction with Leu-261 of μ4-D190A, respectively. (C) Glu-691 in the peptide forms hydrogen bonds with His-256 and Ser-257 via its main-chain carbonyl and side chain carboxylate, respectively. (D) Phe-689 of APP binds into a hydrophobic groove, formed by the side chains of Phe-255, Val-259, and Leu-261 of μ4-D190A. (E) Phe-690 is deeply buried in a cavity formed by the hydrocarbon portions of His-256, Thr-280, and Arg-283 of μ4-D190A. (F) The aromatic ring of Phe-690 in the peptide participates in a cation-π interaction with the side-chain of Arg-283 in μ4-D190A. Arg-283 also forms a bidentate salt bridge with the C-terminal carboxylate of the peptide. (G) Two-dimensional, schematic representation of the interactions shown in A-F using LigPlot+ [51], showing peptide-protein hydrogen bonds in green, and hydrophobic contacts in grey. The numbers of the APP peptide residues are as in APP695.
Figure 5.
Thermal stability analysis of the C-terminal domain of μ4.
The thermal unfolding of the recombinant C-terminal domain of wild-type μ4, μ4-D190A, or μ4-R283D was analyzed by differential scanning fluorimetry following fluorescence changes in the presence of SYPRO Orange. Representative melting curves of each μ4 variant are shown. The calculated Tm value, defined as the maximum of the first derivative of the raw data, is expressed as the mean ± SD (n = 3).
Figure 6.
Limited proteolysis analysis of the C-terminal domain of wild-type μ4.
Recombinant C-terminal domain of wild-type μ4 (A), μ4-D190A (B), or μ4-R283D (C) were incubated with proteinase K at 25°C at an enzyme:substrate ratio of 1∶100, and after the times indicated on top of the panel the digestion was stopped by addition of PMSF. The reaction products were analyzed by SDS-PAGE and gels stained with Coomassie Brilliant Blue. In this condition similar stable fragments are produced from all μ4 variants. Samples from a similar gel shown in (A) were electroblotted onto a PVDF membrane. The three bands shown in lane 7 were excised and processed for N-terminal sequencing by Edman degradation, and the resulting amino acid sequences are shown on the right. (D) Amino acid sequence of the recombinant C-terminal domain of human μ4 (residues 160-453; accession number in parenthesis), with the N-terminal sequence of the fragments shown in (A) highlighted in different colors. (E) Surface model of the μ4 C-terminal domain with amino acids of the proteolytic fragments colored as in A and D. The regions digested are colored in grey, corresponding to a structured loop (S–L), an unstructured loop (U–L), and unstructured N-terminal residues (N–T) (represented as grey lines). (F) The same μ4 variants were processed as in A to C, but incubated with proteinase K at 50°C at the indicated times on top of the panel. In this case the three μ4 variants have different levels of sensitivity to proteinase K. The position of molecular mass markers is indicated on the left.
Figure 7.
HA-epitope-tagged μ4 variants incorporate into endogenous AP-4 complex.
H4 neuroglioma cells were transfected with a plasmid encoding either of the indicated HA-epitope-tagged variants of μ4. After 16-h, cell lysates were prepared and samples were subjected to SDS-PAGE followed by immunoblot with mouse anti-HA-epitope antibody (A). Samples of cell lysates were also subjected to immunoprecipitation using mouse antibody to the ε subunit of AP-4 followed by SDS-PAGE and immunoblotting with horseradish peroxidase-conjugated anti-HA-epitope antibody (B), or immunoprecipitation using rabbit anti-HA-epitope antibody followed by SDS-PAGE and immunoblotting with mouse antibody to the ε subunit of AP-4 (C). The position of molecular mass markers is indicated on the left.
Figure 8.
APP redistributes from endosomes to the TGN upon overexpression of μ4-F255A-HA.
MD-MB-231 cells were cotransfected with a plasmid encoding either of the indicated HA-epitope-tagged variants of μ4, and with a plasmid encoding APP-GFP carrying the double mutation F615P/D664A. After 24-h cells were fixed, permeabilized, stained for EEA1 and TGN46, and examined by fluorescence microscopy. Merging green, red, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. Insets show 2× magnifications. Bar, 10 µm.