NMR studies of polyubiquitin chains: insights into structural basis of functional diversity

Signaling by polyubiquitin (polyUb) chains mediates numerous cellular processes. All these processes involve the covalent modification of the substrate protein with a polyubiquitin chain that is itself formed by isopeptide linkages between the C-terminus of one Ub and a specific Lys residue of the next Ub. Remarkably, the outcome of the signaling event depends on the specific Lys residue that is involved in the formation of the polyUb signal. In the current model of Ub-mediated signaling, diversity in signaling arises from the ability of differently linked polyUb chains to act as functionally distinct signals. Such a model predicts that the distinct structures adopted by alternatively linked polyUb chains modulate their recognition by various effector proteins. However, direct structural evidence in support of this view has been lacking. This work is aimed at elucidating structural differences (if any) between Lys48- and Lys63-linked polyUb chains, and to investigate the structural basis of the specific recognition of Lys48-linked polyUb chains by UBA domains. Using a combination of NMR methods, Lys48-linked Ub2 chains are shown to adopt a 'closed' conformation in solution under physiological conditions. A switch in the conformation of these chains, from 'closed' to 'open' states with decreasing pH is described. The Ub2 interface in the 'closed' conformation is shown to be dynamic, allowing functionally important hydrophobic residues sequestered at the interface to be accessible to Ub-recognition factors. In contrast, Lys63-linked Ub2 chains are shown to be characterized by an extended conformation, with no definitive interface between the Ub units. Such an extended conformation allows each Ub moiety to independently bind a UBA molecule, in a manner similar to the monoUb-UBA interaction. The results presented in this study suggest that the specific recognition of Lys48-linked Ub2, however, may not involve such a simple 'one-UBA-per-Ub' interaction. The interaction of UBA with Lys48-linked Ub2 appears to involve the primary association of the UBA domain with the proximal Ub in Ub2. The relative positioning of the distal Ub in the chain allows its simultaneous association with the same UBA domain, leading to a higher affinity UBA-Ub2 interaction. The results provide the first experimental evidence that alternately linked polyUb chains adopt distinct conformations, and suggest that specific recognition of these chains might indeed depend on differences in their structures

Ubistatins Inhibit Proteasome-Dependent Degradation by Binding the Ubiquitin Chain

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys^(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system

Are Zinc-Finger Domains of Protein Kinase C Dynamic Structures That Unfold by Lipid or Redox Activation?

Protein kinase C (PKC) is activated by lipid second messengers or redox action, raising the question whether these activation modes involve the same or alternate mechanisms. Here we show that both lipid activators and oxidation target the zinc-finger domains of PKC, suggesting a unifying activation mechanism. We found that lipid agonist-binding or redox action leads to zinc release and disassembly of zinc fingers, thus triggering large-scale unfolding that underlies conversion to the active enzyme. These results suggest that PKC zinc fingers, originally considered purely structural devices, are in fact redox-sensitive flexible hinges, whose conformation is controlled both by redox conditions and lipid agonists. Antioxid. Redox Signal. 14, 757-766.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90473/1/ars-2E2010-2E3773.pd

Effects of cyclization on conformational dynamics and binding properties of Lys48-linked di-ubiquitin

In solution, Lys48-linked di-ubiquitin exists in dynamic equilibrium between closed and open conformations. To understand the effect of interdomain motion in polyubiquitin chains on their ability to bind ligands, we cyclized di-ubiquitin by cross-linking the free C terminus of the proximal ubiquitin with the side chain of residue 48 in the distal ubiquitin, using a chemical cross-linker, 1,6-Hexane-bis-vinylsulfone. Our NMR studies confirm that the cyclization affects conformational dynamics in di-ubiquitin by restricting opening of the interface and shifting the conformational equilibrium toward closed conformations. The cyclization, however, did not rigidly lock di-ubiquitin in a single closed conformation: The chain undergoes slow exchange between at least two closed conformations, characterized by interdomain contacts involving the same hydrophobic patch residues (Leu8-Ile44-Val70) as in the uncyclized di-ubiquitin. Lowering the pH changes the relative populations of these conformations, but in contrast with the uncyclized di-ubiquitin, does not lead to opening of the interface. This restriction of domain motions inhibits direct access of protein molecules to the hydrophobic patch residues located at the very center of the interdomain interface in di-ubiquitin, although the residual motions are sufficient to allow access of small molecules to the interface. This renders di-ubiquitin unable to bind protein molecules (e.g., UBA2 domain) in the normal manner, and thus could interfere with Ub2 recognition by various downstream effectors. These results emphasize the importance of the opening/closing domain motions for the recognition and function of di-ubiquitin and possibly longer polyubiquitin chains