Figure 1.
(A) Domain organisation of yeast ATG16 and human ATG16L1. ATG5 binding motifs are coloured green (yeast) and pink (human); the coiled-coil domain (CCD) is cyan (yeast) and blue (human); the human WD40 repeats are yellow. The position of the Crohn's Disease susceptibility polymorphism T300A is marked on human ATG16L1. (B) Structure of the yeast and human ATG5 binding motifs and the yeast ATG16 CCD. Colours as in panel (A). (C) Expression constructs used in this study outlining the boundaries of human ATG16L1. FL – full length ATG16L1, CCD1 – coiled-coil domain construct 1, CCD2 – coiled-coil domain construct 2.
Table 1.
Summary of human ATG16L1 construct expression in E. coli Rosetta™ 2 cells.
Figure 2.
Purification of recombinant human ATG16L1 coiled-coil.
(A) GST-CCD1-FLAG-6His expression produces a truncated product (orange arrowhead). (B) Glutathione sepharose purification of GST-CCD2-FLAG-6His. 1 – total cell lysate, 2 – soluble extract, 3 – unbound flow through, 4–7 successive elution fractions. (C) CCD2-FLAG-6His following removal of the GST tag by TEV cleavage (lane 1). (D) Elution fractions of CCD2-FLAG-6His after anion exchange. The position of the truncated protein is marked by an orange arrowhead. (E) CCD3-FLAG-6His (lane 1) is highly pure and shows no evidence of truncation following glutathione sepharose affinity purification, TEV cleavage and hydrophobic interaction chromatography. In all panels the black arrowhead marks the bands representing the expected size of the ATG16L1 construct; M denotes PageRuler™ Plus Prestained protein standards (Thermo Scientific).
Figure 3.
The coiled-coil of human ATG16L1 is alpha helical.
(A) PSIPRED prediction of the secondary structure of the minimal coiled-coil domain (residues M126 – A207; CCD3) of human ATG16L1. Sequencing numbering corresponds to the human protein, alpha helices are marked as pink cylinders and the confidence level of the prediction for each residue is provided by the blue bars. (B) Circular dichroism analysis of CCD3. Mean residual ellipicity (θ) plotted against wavelength (nm) indicates a helical protein.
Figure 4.
Characterisation of the oligomeric status of the human ATG16L1 coiled-coil domain.
(A) Size exclusion chromatography of CCD3. CCD3 – red trace; Blue Dextran – blue trace; Molecular size markers – black trace (mass labelled in kDa). (B) Native-PAGE analysis of CCD3. 1 µg of CCD3 was analysed by Native-PAGE both before (lane 1) and after (lane 2) freeze/thawing at −80°C. M – NativeMark™ protein standards (Life Tehnologies). (C) Nanospray Electrospray Ionisation Mass Spectroscopy (ESI-MS) analysis of CCD3 under conditions that preserve non-covalent interactions. CCD3 dimers are detected in the low m/z range corresponding to charge states of +11 to +7. There is no evidence of any additional species of CCD3. (D) Analytical ultracentrifugation sedimentation velocity data. Interference optical signal distributions of CCD3FH at time intervals of 320 s at a rotor speed of 50,000 rpm and a temperature of 20°C, with systematic noise subtracted. The residuals are from the fit with the hybrid discrete/continuous model. Component sedimentation coefficient distributions showed a dominant population of the dimeric species, with a uniform frictional ratio of Fk,w = 1.73. The final r.m.s.d. was 0.006.
Figure 5.
The coiled-coil of ATG16L1 is highly conserved across vertebrates.
(A) Cross-species alignment of the coiled-coil (human residues M126 – A207) of ATG16L1 reveals levels of sequence identity with the human sequence of between 73% and 100%. The percentage identity listed is in comparison with the human sequence. The common names and database identifies for each sequence are listed in Materials and Methods. (B) The syntenic position of Atg16L1 is well conserved across species. The three adjacent upstream and downstream genese to Atg16L1 are displayed. Genes are denoted by individual blocks; yellow indicates a position on the forward strand and green a position on the reverse strand. Gene identities are as follows: ATG16L1 – autophagy related protein 16 isoform 1; SAG – S-antigen, retina and pineal gland; DGKD – diacylglycerol kinase delta; USP40 – ubiquitin specific peptidase 40; INPP5D – inositol polyphosphate-5-phosphatase; NEU2 – sialidase 2; NGEF - neuronal guanine exchange factor; TRPV2 – transient receptor potential cation channel, subfamily V, member 2; UBB – ubiquitin B; AC106876.2 – uncharacterised; 00570 (ENSMODG00000000570) – uncharacterised; 23152 (ENSMODG00000023152) – uncharacterised, BLASTp indicates a possible ortholog of glyceraldehydes 3-phosphate dehydrogenase.