Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2

Abstract

Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum-associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48-linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin-tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95-107 and 130-135) flanking the active site cysteine are poorly defined. (15)N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester-linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side-chains of Asp98 or Asp99.

Keywords: ERAD; NMR spectroscopy; UBC7; Ube2g2; spin relaxation; ubiquitin conjugating enzyme.

Figure 1

A) Ribbon diagram of the closest-to-mean solution structure of Ube2g2 determined by NMR with the side-chain of catalytic residue C89 depicted as sticks. B) Backbone diagram of 10 lowest energy NMR structures of Ube2g2. C) Overlay of the closest-to-mean solution structure (blue) and the closest-to-mean crystal structure (PDB ID: 2cyx) (magenta) of Ube2g2.

Figure 2

Distribution of observed NOE restraints utilized for the solution structure determination of Ube2g2. gray: Short/medium range NOEs (i-j <= 4); black: long range NOE's (i-j >= 5). Relatively few NOE restraints were observed for the 13-residue insertion loop (residues 96-107) and the loop encompassing residues 130-135.

Figure 3

Residue specific ¹⁵N spin relaxation rates measured for Ube2g2 at 800 MHz: A) R₁ values. B) R₂ values and C) Steady state heteronuclear NOE values. Shown in D) are the squared generalized order parameters (S²) as a function of residue resulting from a model free ¹⁵N spin relaxation analysis of Ube2g2.

Figure 4

Residual dipolar coupling (RDC) analysis of Ube2g2. A) Observed RDCs for Ube2g2 aligned in 8 mg/ml bacteriophage Pf1 (top) and in 4.5% stretched acrylamide gel (bottom). B) Best fit of the amide ¹⁵N-¹H RDC data (using the 4.5% stretched polyacrylamide gel medium) to a typical member of the NMR structural ensemble. Annotations refer to specific residue numbers. C) Same as B except the best fit is to the average internuclear vector coordinates of the 10 member NMR structural ensemble assuming that each structural member is equally populated.

Figure 5

Structural features observed for the active site of the Ube2g2 solution structure. A) Overlay of the crystal structures (PDB ID: 2cyx) (in magenta) and the 10 member NMR structural ensemble (in black) of Ube2g2. The side-chains of residue N81, Y83, C89 and S134 are shown as sticks. Compared to the crystal structure, the side-chains of N81 and S134 in the NMR structures are in closer proximity to the active site cysteine residue, C89. B) Selected member of Ube2g2 solution structural ensemble in which the side-chains of residues H94 and D98 are within hydrogen bonding distance. C) Selected member of Ube2g2 solution structural ensemble in which the side-chain of residue H94 is within hydrogen bonding distance of both C89 and D99. D) Selected member of the Ube2g2 NMR structural ensemble in which N81, Y83 and S134 are all in close proximity to the active site cysteine, C89.

Figure 6

pH dependence of imidazole ring chemical shifts for His94 (circles) and His79 (diamonds). A) pH dependence of ¹⁵Nδ1 and ¹⁵Nε2 ring chemical shifts. B) pH dependence of ¹Hε1 ring chemical shifts. The solid line denotes the resulting best fit of the corresponding data points to the modified Henderson-Hasselbalch equation.