The Structure and Conformation of Lys63-Linked Tetraubiquitin

Abstract

Summary Ubiquitination involves the covalent attachment of the ubiquitin C-terminus to the lysine sidechain of a substrate protein by an isopeptide bond. The modification can comprise a single ubiquitin moiety or a chain of ubiquitin molecules joined by isopeptide bonds between the C-terminus of one ubiquitin with one of the seven lysine residues in the next ubiquitin. Modification of substrate proteins with Lys63-linked polyubiquitin plays a key non-degradative signaling role in many biological processes including DNA repair and NF-κB activation, whereas substrates modified by lysine-48 (Lys48) linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked ubiquitin chains presumably stems from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetraubiquitin at a resolution of 1.96 Å and performed Small Angle X-ray scattering (SAXS) experiments and molecular dynamics (MD) simulations to probe the conformation of Lys63 tetraubiquitin in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 Ub4. Small Angle X-ray scattering (SAXS) experiments show that the tetraubiquitin chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 polyubiquitin chains.

Subunit Architecture of the Golgi Dsc E3 Ligase Required for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast

“…For example, the ubiquitin binding affinities for the Dsc2 UBA domain in isolation could be different from that of full-length Dsc2. Structurally, Lys 63 linked di-and tetra-ubiquitin adopt an open conformation with the Ile 44 hydrophobic patch exposed, whereas Lys 48 linked di-and tetra-ubiquitin adopt a closed cyclic conformation with the conserved hydrophobic patches shielded (42). Thus, structural differences could affect our ability to detect binding to Lys 48 di-ubiquitin.…”

A SnapShot of Ubiquitin Chain Elongation

“…Thus, Lys-48-Ub chains of a length greater than four appear to restrict the actions by SCF (27)(28)(29).…”

“…Lys-48-tetra-Ub can adopt closed or open conformations that are stabilized by hydrophobic or polar intrachain interactions between Ub subunits, respectively. In contrast, Lys-63-tetra-Ub exists in an open and extended conformation (29).…”

“…Previous studies have reported three high resolution structures of Lys-48 tetra-Ub, which are formed at different pH values (27)(28)(29). In one such structural model built with both crystallography and NMR data, the Lys-48 tetra-Ub chain at near physiological pH (pH 6.7) adopts a conformation consisting of a dimer of di-Ubs, with each di-Ub subunit in the closed conformation (28).…”

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage

“…To show the interaction between the UIM and the ubiquitin hydrophobic patch, we used HADDOCK to identify a favorable complex followed by energy minimization to reduce structural errors (see "Materials and Methods" for details). We also modeled the proximal ubiquitin onto the chain of AMSH using existing structures of Lys 63 -linked ubiquitin chains (42). On the basis of the biochemical data showing that the VHS domain is required for specificity toward longer chains (Fig.…”