Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase

Abstract

C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase. Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain. In contrast to nearly all homodimeric proteins, CHIP is asymmetric. To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly. We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box. We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization. This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues. Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion. The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites. Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function. This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly.Generally, the lowest energy state of protein assembly is symmetrical, whereas asymmetry is associated with energy frustration and structural instability 1 . However, symmetry is not evolutionarily constrained, and many oligomeric enzymes are known to be asymmetric with only half-of-sites active 2-5 . In the majority of such oligomers, asymmetry results from conformational changes triggered by ligand binding 2,6,7 . Another mechanism to fold and activate asymmetric dimers requires that one of the ligand-binding sites is deformed 3,6,7 . However, very few of the vast number of known homo-multimeric proteins assemble into asymmetric structures 6 . Among the exceptions are the C-terminus of Hsc/p70-Interacting Protein (CHIP) 8 , an E3 ubiquitin ligase that associates with cyoplasmic Hsp70 and Hsp90 chaperones, and Hikeshi 9 , a nuclear import protein that also binds Hsp70. Unveiling the mechanism of symmetry breaking in homo-oligomers will shed light on new principles of folding and assembly for this important class of proteins.The CHIP homodimer consists of three domains: tetratricopeptide repeat (TPR), helix-turn-helix (HH), and U-box 8 (Fig. 1). CHIP targets misfolded, chaperone-bound substrates for proteasomal degradation by transferring ubiquitin from a compatible E2 ubiquitin conjugating-enzyme, which associates with the U-box domain, to a lysine residue on the target protein 10, 11 . The crystal structure of murine CHIP (CHIP, PDB: 2C2L), which differs from human CHIP by one residue at the N-terminus, showcases an asymmetric homodimer in which only one U-box domain has an accessible E2-binding surface. The E2 binding site/U-box in t...

E3 ligase activity of Carboxyl terminus of Hsc70 interacting protein (CHIP) in Wharton's jelly derived mesenchymal stem cells improves their persistence under hyperglycemic stress and promotes the prophylactic effects against diabetic cardiac damages

“…The Carboxyl terminus of Hsc70 interacting protein (CHIP) cDNA encodes a 34.5 kDa wellconserved protein that has around 98% sequence similarity with the mouse and ∼60% similarity with the fruit fly 31 . CHIP contains a conserved U-box domain at their C terminus having E3 ligase activity and an N terminal tetratricopeptide repeat (TPR) domain responsible for interaction with Hsc/p70 and HSP90 clients 32,33 . An earlier study demonstrated the protective effect of CHIP against myocardial injury induced apoptosis and oxidative stress in the CHIPsufficient animal model 34 .…”

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism

“…(1) Each monomer is composed of a TPR domain (pink cube), a U-box domain (green sphere), and a dimerization domain (blue cylinders). Molecular dynamics studies 50 have shown that the monomer in solution is stabilized through TPR-Ubox interactions which block the E2 binding site (X). (2) In the dimer, the two helix-coils come together such that one bends and generates two smaller, kinked helices.…”

“…This apparent discrepancy has been explained by a model of conformational flexibility 49 , in which the CHIP dimer switches between two opposed asymmetric conformations (Figs 2 , 4 and 8c ) through a short-lived, symmetric conformation (Figs 3 and 8c ). A recent molecular dynamics simulation with the CHIP monomer and dimer 50 calculated that a monomer in solution would be most stable when the TPR and U-box domains interact, which would occlude the E2 binding site (Figs 1 and 8c ). Such a monomer would thus be non-functional, and CHIP E3 ligase activity could only be activated after dimer formation, when one of the two E2 binding sites would become accessible to the enzyme.…”

Chaperone-directed ubiquitylation maintains proteostasis at the expense of longevity

“… 47 The structure of dimeric mouse CHIP reveals an unusual asymmetry in which the protomers adopt radically different conformations 48 and breaking of symmetry during homodimeric assembly induces E3 ubiquitin ligase activity. 49 As a consequence of this asymmetric arrangement, one of the 2 catalytical U-box domains is sterically hindered and not accessible for E2 enzyme binding. Thus, the asymmetric structure of the CHIP dimer provides an elegant means for coupling a dimeric chaperone to a single ubiquitylation system, supporting the formation of a monotonic polyubiquitin chain.…”