PDBsum entry 2grp

Ligase

PDB id: 2grp

Contents

Protein chains

157 a.a.

157 a.a.

Waters ×299

References listed in PDB file

Key reference

Title

Lysine activation and functional analysis of e2-Mediated conjugation in the sumo pathway.

Authors

A.A.Yunus, C.D.Lima.

Ref.

Nat Struct Mol Biol, 2006, 13, 491-499. [DOI no: 10.1038/nsmb1104]

PubMed id

16732283

Abstract

E2 conjugating proteins that transfer ubiquitin and ubiquitin-like modifiers to substrate lysine residues must first activate the lysine nucleophile for conjugation. Genetic complementation revealed three side chains of the E2 Ubc9 that were crucial for normal growth. Kinetic analysis revealed modest binding defects but substantially lowered catalytic rates for these mutant alleles with respect to wild-type Ubc9. X-ray structures for wild-type and mutant human Ubc9-RanGAP1 complexes showed partial loss of contacts to the substrate lysine in mutant complexes. Computational analysis predicted pK perturbations for the substrate lysine, and Ubc9 mutations weakened pK suppression through improper side chain coordination. Biochemical studies with p53, RanGAP1 and the Nup358/RanBP2 E3 were used to determine rate constants and pK values, confirming both structural and computational predictions. It seems that Ubc9 uses an indirect mechanism to activate lysine for conjugation that may be conserved among E2 family members.

Figure 1.
Figure 1. Genetic analysis of the interface between Ubc9 and RanGAP1. (a) Stereo view of the Ubc9 active site in complex with RanGAP1–SUMO^14 (PDB entry 1Z5S) in ribbon and solid-bond representation, depicting Ubc9 residues selected for genetic analysis. Residues are labeled and hydrogen-bonding interactions indicated by dashed lines. Yellow, SUMO-1; pink, RanGAP1; blue, Ubc9. Images generated with PyMOL^44. (b) Serial dilutions of S. cerevisiae ubc9 cultures bearing wild-type (WT) UBC9 or indicated ubc9 alleles containing single or double point mutations were spotted on YPAD agar and tested for growth at 23 °C (top), 30 °C (middle) and 37 °C (bottom) (Note: D127S allele was expressed under yeast endogenous promoter; see text). (c) Western blotting analysis for Smt3 conjugates (top) or Ubc9 (bottom) in strains containing indicated ubc9 alleles.

Figure 2.
Figure 2. Biochemical characterization of wild-type (WT) Ubc9 and mutants. (a) Initial rates of reaction versus p53 substrate concentration, fit to rectangular hyperbolas (Methods), are shown for wild-type Ubc9, Ubc9-N85Q, Ubc9-Y87A and Ubc9-D127A. Left and right y-axes indicate rates for wild-type and mutant enzymes, respectively. (b) Bar charts depicting the kinetic constants apparent K[d] and k[2] for wild-type and mutant Ubc9, plotted on a log scale (see Supplementary Table 1). Error bars in a and b are s.d.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2006, 13, 491-499) copyright 2006.