Expression, purification, and crystal structure of N-terminal domains of human ubiquitin-activating enzyme (E1)

Figures & data

Fig. 1. Domain architectures of human and S. cerevisiae ubiquitin E1.

Notes: A, adenylation domain; CC, cysteine catalytic domain; UFD, ubiquitin-fold domain. C632 and C600 are the active site cysteine residues for human and S. cerevisiae E1, respectively.

Fig. 2. Cloning of the N-terminal domains of human ubiquitin E1.

Notes: The N-terminal domains of human ubiquitin E1 were amplified and inserted into the pETDuet-1 vector with an N-terminal 6 × His tag for recombinant expression.

Fig. 3. SDS-PAGE of the purified N-terminal domains of human ubiquitin E1.

Notes: The purified proteins were resolved via 12% SDS-PAGE, stained with Coomassie Brilliant Blue R-250, and then de-stained with an ethanol: acetic acid: water (2:1:7) mixture. The left lane is the protein marker, and the right lane contains 1.5 μL of the purified recombinant proteins.

Fig. 4. Crystallization of N-terminal domains of human ubiquitin E1.

Notes: (A) Crystals of N-terminal domains of human ubiquitin E1 after three days at room temperature with the reservoir buffer containing 0.1 M Na₃Citrate pH 5.6 and 3.2 M NH₄Ac. (B) Optimized crystals of N-terminal domains of human ubiquitin E1 by microseeding, after two days growth at room temperature with the reservoir buffer containing 0.1 M Na₃Citrate pH 5.6 and 3.0 M NH₄Ac.

Fig. 5. Diffraction patterns of the crystal of the N-terminal domains of human ubiquitin E1.

Notes: The crystal diffracted to 2.75 Å resolution. B is the partial enlargement of region in A surrounded by rectangle.

Table 1. Data collection and refinement statistics.

Data collection	N-terminal domains of human ubiquitin E1
Space group	C2221
Cell dimensions
a, b, c (Ǻ)	94.13, 186.81, 135.22
α, β, γ (°)	90, 90, 90
Wavelength (Ǻ)	0.975
Resolution (Ǻ)	30.0–2.75(2.80–2.75)^a
Completeness (%)	98.4 (84.0)^a
Average I/σ(I)	11.3 (1.8)^a
Redundancy	7.7 (4.9)^a
^bRmerge	0.175 (0.563)^a
Total number of observations	239125
Total number unique	30890
Refinement
Resolution (Ǻ)	30–2.75
Number of reflections	30860
^cRwork/Rfree	0.205/0.264
Number of atoms/residues
Protein	5736/731
Water	116
R.m.s.d. in bond length (Ǻ)	0.010
R.m.s.d. in bond angles (°)	1.370
Ramachandran-plot analysis^d
Favored regions (%)	94.80 (693)
Allowed regions (%)	5.06 (37)
Disallowed regions (%)	0.14 (1)

^a Numbers in parentheses donate highest resolution shell.

^b R_merge = Σ_hΣ_l | I_ih−  < I_h>| / Σ_hΣ_I < I_h>, where < I_h > is the mean of the observations I_ih of reflection h.

^c R_work = Σ(||F_p(obs)| − |F_p(calc)||) / Σ|F_p(obs)|; R_free = R factor for a selected subset (5%) of the reflections that were not included in prior refinement calculations.

^d PROCHECK was used for Ramachandran-plot analysis.

Fig. 6. Sequence alignment of the N-terminal domains of human ubiquitin E1 (1-449) and S. cerevisiae E1 (1-424).

Notes: Secondary structures of human ubiquitin E1 (1-439) and S. cerevisiae E1 (1-424) were labeled above and below the amino acid sequence based on the crystal structure (PDB ID: 4P22 and 3CMM).

Fig. 7. Overall Structure of N-terminal domains of human ubiquitin E1.

Notes: Schematic diagram of the structure of the N-terminal domains of human E1, and the right figure is the view of the left structure by a 90° rotation around a horizontal axis. A and B are two molecules in the same crystallographic asymmetric unit.

Fig. 8. Structural Comparison of the N-terminal domains of human ubiquitin E1 with Its Yeast Homologs.

Notes: (A and B) Superimposed N-terminal domains of human ubiquitinE1 (colored blue) with S. cerevisiae Uba1 1-424 (PDB ID: 3CMM, colored pink) and S. pombe Uba1 1-392 (PDB ID: 4II2, colored cyans). (C) Superimposed FCCH domains from human ubiquitin E1 and yeast Uba1 (the same color with A and B). (D, E, and F) The relative position of human, S. cerevisiae and S. pombe ubiquitin E1 FCCH domains with the IAD, the encircled region is the ubiquitin-binding surface.

Fig. 9. Comparison of the dimerization pattern of human ubiquitin E1 IAD with Its homologs in yeast uba1 and non-canonical E1s.

Notes: (A) Cartoon diagram of human E1 IAD dimer in the same crystallographic asymmetric unit. (B) Pseudo-symmetrical dimerization of S. cerevisiae Uba1 IAD and AAD (PDB ID: 3CMM). (C and D) Dimerization of the Atg7 and Uba5 AADs, respectively (PDB ID: 3VH1 and 3H8 V).