Oligomerization state determination of Atg5~Atg16L1 complexes

During autophagosome formation, multimerization of the Atg12-Atg5~Atg16L1 complex via homo-oligomerization of the coiled coil domain of Atg16L1 is essential for isolation membrane expansion (Mizushima 1998, 2003). Further characterization of the multimerization state of the mammalian Atg5~Atg16L1 complex was carried out using analytical gel filtration and multiple angle laser light scattering (MALLS) experiments.

The constructs used are summarized in Figure 3.10.

Figure 3.10: Constructs of mammalian Atg16L1 co-purified with Atg5 that were used to determine the oligomerization state of the complex. * was only used in the MALLS experiments, ** was purified on its own without Atg5.

Analytical size exclusion chromatography was first used to confirm oligomerization of the Atg5~Atg16L1 complex. The Superdex 200 10/300 GLcolumn was calibrated with proteins of known molecular weight to generate a standard curve (Figure 3.11 panel B). As seen in Figure 3.11 panel A, the Atg5~Atg16L1 complexes and Atg16L1 alone eluted at volumes corresponding to species larger than the monomeric proteins. Elution volumes were used to calculate the molecular weights of the proteins using the standard curve (Table 3.1).

Figure 3.11: Analytical gel filtration of the Atg5~Atg16L1 complexes. (A) Overlaid chromatograms of multimerized Atg5~Atg16L1 complexes or Atg16L1 on its own. (B) Calibration curve generated using

molecular weight protein standards (Pharmacia) and a Superdex 200 10/300 GL column.

Kav=(Ve - Vo)/(Vt - Vo) where Kav=gel phase distribution coefficient, Ve =elution volume of the protein, Vo = void volume of the column, Vt =total column bed volume.

Table 3.1: Analytical gel filtration of Atg16L1(53-168) and Atg5~Atg16L1 complexes

Atg5(1-275)

multimerized complex (kDa)

monomeric complex (kDa)

ratio oligomer:

monomer

mAtg16L1 (53-168) 91.2 13.6 6.7

His-hAtg5~

His-mAtg16L1 (1-70) 108.9 44.5 2.4

His-mAtg5~

His-mAtg16L1 (1-106) 153.9 50.5 3

mAtg5~ mAtg16L1 (1-113) 146.9 45.9 3.2

mAtg5~ mAtg16L1 (1-168) 232.3 52.4 4.4

His-hAtg5~ mAtg16L1 (1-231) 369.1 63.3 5,8

His-mAtg5~ mAtg16L1 (1-265) 440.9 68.8 6.4

h=human (no thrombin cleavage site), m=murine (with thrombin cleavage site)

While Atg16L1(53-168) and the Atg5~Atg16L1 complexes clearly multimerized, they did not display a consistency in their oligomerization stoichiometry. In fact, as the length of Atg16L1 in complex with Atg5 increased, so too did the oligomerization ratio of the complexes from a ratio of 2 for 70) to 6 for the Atg5~Atg16L1(1-265) complex (Table 3.1). However, the molecular weight calculated from the elution volume greatly depends on the shape of the molecule. For a non-globular protein, an apparent higher molecular weight would be observed. Here, the extended shape of the coiled coil domain made it appear as though the Atg5~Atg16L1 complexes had a higher molecular weight.

One way to offset the bias introduced by the non-globular shape of the coiled coil domain of Atg16L1 was to determine the molecular mass by MALLS (Figure 3.12).

Figure 3.12: MALLS of Atg16L1(53-168) and Atg5~Atg16L1 complexes. The peaks correspond to their elution volumes. The horizontal line of the same color corresponds to the molar mass (y-axis).

Table 3.2: MALLS of Atg16L1(53-168) and Atg5~Atg16L1 complexes

Atg5(1-275) Atg16L1 ( ) multimerized

complex (kDa) monomeric

complex (kDa) multimerized:

monomeric

mAtg16L1 (53-168) 22.8 13.6 1.7

His-hAtg5~ His- mAtg16L1 (1-70) 79.8 44.5 1.8

His-mAtg5~ His- mAtg16L1 (1-106) 83.2 50.5 1.6

mAtg5~ mAtg16L1 (1-113) 81.4 45.9 1.8

mAtg5~ mAtg16L1 (1-168) 110 52.4 2.1

His-hAtg5~ mAtg16L1 (1-231) 106 63.3 1.7

His-mAtg5~ mAtg16L1 (1-265) 112 68.8 1.6

h=human (no thrombin cleavage site), m=murine (with thrombin cleavage site)

Analysis of the determined molecular weights (Table 3.2) of Atg16L1(53-168) and the Atg5~Atg16L1 complexes shows that Atg16L1(53-168) is present as a dimer.

Furthermore the Atg5~Atg16L1 complexes all form dimers consisting of two copies of Atg16L1 and Atg5 each. Of note, the Atg5~Atg16L1(1-70) complex which consisted of only the N-terminal Atg5-binding region of Atg16L1 was also present as a dimer. The Atg16L1 coiled coil domain is predicted by the program COILS (Lupas 1991) to begin at approximately residue 80. Dimerization of the minimal Atg5~Atg16L1 complex could be mediated by electrostatic interactions. If this is so, using high salt concentrations would inhibit dimer formation.

3.1.3 Stability test and limited proteolysis experiments of Atg5~Atg16L1(1-231) Crystallization can take days, even months or longer, therefore the stability of the mammalian Atg5~Atg16L1(1-231) complex was tested. The complex was kept at room temperature and samples were taken at regular intervals. These samples were analyzed on an SDS-PAGE gel and showed that the complex remained intact over a period of nine days (Figure 3.13) and should be stable during crystallization trials.

Figure 3.13: Stability test of the Atg5~Atg16L1(1-231) complex at room temperature. 15% SDS-PAGE gel showing the protein complex after incubation at RT over nine days with samples taken at the intervals indicated.

However, for crystallization it is often useful to identify shorter fragments of the protein of interest (Dale 2003). These fragments most likely are more compact and contain fewer disordered and flexible regions which might hinder crystallization. The goal was to identify stable fragments of Atg16L1 which interact with Atg5.

Limited proteolysis experiments of the Atg5~Atg16L1(1-231) complex using trypsin and chymotrypsin were performed. Digestions yielded several shorter, stable bands (Figure 3.14 panel A). To determine if the cleaveage products could still interact

with Atg5, the digested samples were loaded onto a 1 mL HisTrap FF column. An ~18 kDa fragment co-eluted with Atg5 (Figure 3.14 panel B). A sample containing this fragment was blotted onto PVDF membrane for N-terminal sequencing (Seqlab, Göttingen). The identified residues were L-Q-A-E which corresponds to residues 56-59 of Atg16L1. According to the size of the fragment (~20 kDa), given that it began at amino acid residue 56, it most likely included the complete coiled coil domain up to residue 231.

Figure 3.14: Limited proteolysis experiments of Atg5~Atg16L1(1-231). (A) 15% SDS-PAGE gel of trypsin and chymotrypsin digestion time courses (in min). Proteases were added in a ratio of 1:200 (w/w) and incubated at RT for the time indicated. (B) 15% SDS-PAGE gel of Atg5~Atg16L1(1-231) after 45 min digestion with trypsin. The reaction was stopped with 4 mM Pefabloc and loaded onto a 1 mL HisTrap FF column. The box indicates the fragment sent for N-terminal sequencing.

The identification of this stable Atg16L1 fragment led to the cloning of the Atg16L1(53-168) construct. Residue 168 was chosen as the ending residue because this comprised just over half of the coiled coil domain and had the lowest predicted disorder compared to the remaining coiled coil domain according to the DisEMBL protein disorder predictor 1.5 (Linding 2003). However, despite the co-elution of the sequenced fragment with Atg5 (Figure 3.14), Atg16L1(53-168) was seen to completely dissociate from Atg5 during purification when both proteins were co-expressed (3.7 panel A). Atg16L1(53-168) did not yield crystals but was used for oligomerization studies (see Chapter 3.1.2).