Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.     

Structural insights into anaphase-promoting complex function and mechanism

The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

Evidence for Bidentate Substrate Binding as the Basis for the K48 Linkage Specificity of Otubain 1

Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination.     

The Folding of a Family of Three-Helix Bundle Proteins: Spectrin R15 Has a Robust Folding Nucleus,Unlike Its Homologous Neighbours

Three homologous spectrin domains have remarkably different folding characteristics. We have previously shown that the slow-folding R16 and R17 spectrin domains can be altered to resemble the fast folding R15, in terms of speed of folding (and unfolding), landscape roughness and folding mechanism, simply by substituting five residues in the core. Here we show that, by contrast, R15 cannot be engineered to resemble R16 and R17. It is possible to engineer a slow-folding version of R15, but our analysis shows that this protein neither has a rougher energy landscape nor does change its folding mechanism. Quite remarkably, R15 appears to be a rare example of a protein with a folding nucleus that does not change in position or in size when its folding nucleus is disrupted. Thus, while two members of this protein family are remarkably plastic, the third has apparently a restricted folding landscape.     

Endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development

Postembryonic development of plant organs requires a constant interplay between the cell cycle and the developmental programs. Upon endo- and exogenous signals, plant cells can enter, exit or modify the cell cycle. Alteration of mitotic cycles to endoreduplication cycles, where the genome is duplicated without mitosis, is common in plants and may play a role in cell differentiation. The switch from the mitotic to endocycles is regulated by Ccs52A, a plant orthologue of the yeast and animal Cdhl proteins, acting as substrate-specific activator of the anaphase-promoting complex E3 ubiquitin ligase. Here, several aspects of endoreduplication are discussed with special attention on nitrogen-fixing nodule development where endoreduplication is an integral part of symbiotic cell differentiation.     

Subunits and substrates of the anaphase-promoting complex

The initiation of anaphase and exit from mitosis depend on a ubiquitination complex called the anaphase-promoting complex (APC) or cyclosome. The APC is composed of more than 10 constitutive subunits and associates with additional regulatory factors in mitosis and during the G1 phase of the cell cycle. At the metaphase-anaphase transition the APC ubiquitinates proteins such as Pds1 in budding yeast and Cut2 in fission yeast whose subsequent degradation by the 26S proteasome is essential for the initiation of sister chromatid separation. Later in anaphase and telophase the APC promotes the inactivation of the mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating subunit cyclin B. The APC also mediates the ubiquitin-dependent proteolysis of several other mitotic regulators, including other protein kinases, APC activators, spindle-associated proteins, and inhibitors of DNA replication.     

The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms

Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topology but altered regional stability, using a protein engineering approach. Our data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. Our results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.     

Thermodynamic analysis of an antagonistic folding-unfolding equilibrium between two protein domains

A simple model is formulated for analyzing the coupled folding-unfolding equilibrium present in a unique class of molecular switch proteins. We previously fused two single-domain proteins, barnase and ubiquitin, such that the free energy stored in the folded structure of one subunit is used to drive unfolding of the other. Here, we present a thermodynamic test of that mechanism. The antagonistic interaction is represented by a coupling free energy term DeltaGX. DeltaGX is the penalty imposed on folding of one domain by the native structure of the other. If DeltaGX=0, then neither domain senses the other and they fold and unfold independently. If DeltaGX>0, then destabilizing one domain will stabilize the other, and vice versa. In the limit where DeltaGX is greater than the intrinsic stability of either protein, then only one domain can be folded at any given time. We estimate DeltaGX by measuring stability parameters for a series of mutants that destabilize either the barnase or ubiquitin domains. Fitting the data to the model leads to a DeltaGX value of approximately 4 kcal mol(-1). DeltaGX is proposed to depend on both the length of the linker peptides used to join the two proteins, and on the inherent structural plasticity of each domain. We predict that shortening the linkers from their current lengths of two and three amino acid residues will increase structural and thermodynamic coupling.     

The SV40 Capsid Is Stabilized by a Conserved Pentapeptide Hinge of the Major Capsid Protein VP1

The simian virus 40 (SV40) outer shell is composed of 72 pentamers of VP1. The core of the VP1 monomer is a β-barrel with jelly-roll topology and extending N- and C-terminal arms. A pentapeptide hinge, KNPYP, tethers the C-arm to the VP1 β-barrel core. The five C-arms that extend from each pentamer insert into the neighbouring pentamers, tying them together through different types of interactions. In the mature virion, this element adopts either of six conformations according to their location in the capsid. We found that the hinge is conserved among 16 members of the Polyomaviridae, attesting to its importance in capsid assembly and/or structure. We have used site-directed mutagenesis to gain an understanding into the structural requirements of this element: Y299 was changed to A, F, and T, and P300 to A and G. The mutants showed reduction in viability to varying degrees. Unexpectedly, assembly was reduced only to a small extent. However, the data showed that the mutants were highly unstable. The largest effect was observed for mutations of P300, indicating a role of the proline in the virion structure. P300G was more unstable than P300A, indicating a requirement for rigidity of the pentapeptide hinge. Y299T and Y299A were more defective in viability than Y299F, highlighting the importance of an aromatic ring at this position. Structural inspection showed that this aromatic ring contacts C-arms of neighbouring pentamers. Computational modelling predicted loss of stability of the Y mutants in concordance with the experimental results. This study provides insights into the structural details of the pentapeptide hinge that are responsible for capsid stability.     

Mutations in the hydrophobic core of ubiquitin differentially affect its recognition by receptor proteins

Ubiquitin (Ub) is one of the most highly conserved signaling proteins in eukaryotes. In carrying out its myriad functions, Ub conjugated to substrate proteins interacts with dozens of receptor proteins that link the Ub signal to various biological outcomes. Here we report mutations in conserved residues of Ub's hydrophobic core that have surprisingly potent and specific effects on molecular recognition. Mutant Ubs bind tightly to the Ub-associated domain of the receptor proteins Rad23 and hHR23A but fail to bind the Ub-interacting motif present in the receptors Rpn10 and S5a. Moreover, chains assembled on target substrates with mutant Ubs are unable to support substrate degradation by the proteasome in vitro or sustain viability of yeast cells. The mutations have relatively little effect on Ub's overall structure but reduce its rigidity and cause a slight displacement of the C-terminal β-sheet, thereby compromising association with Ub-interacting motif but not with Ub-associated domains. These studies emphasize an unexpected role for Ub's core in molecular recognition and suggest that the diversity of protein-protein interactions in which Ub engages placed enormous constraints on its evolvability.     

A theoretical interpretation of the transient sialic acid toxicity of a nanR mutant of Escherichia coli

This article reports on experimental evidence that an Escherichia coli nanR mutant shows inhibited growth in N-acetylneuraminic acid. This effect is prevented when inocula are grown in an excess of glucose, but not in an excess of glycerol. The nanATEK operon is controlled by catabolite repression, suggesting that diminished expression of the nanATEK operon in the presence of glucose explains the inocula effects. Neither double nanR-nagC nor nanR dam mutants show growth inhibition in the presence of N-acetylneuraminic acid. A theoretical model of N-acetylneuraminic acid metabolism (i.e., in particular of the nanATEK and nagBACD operons) is presented; the model suggests an interpretation of this effect as being due to transient high accumulations of GlcNAc-6P in the cell. This accumulation would lead to suppression of central metabolic functions of the cell, thus causing inhibited growth. Based on the theoretical model and experimental data, it is hypothesised that the nanATEK operon is induced in a two-step mechanism. The first step is likely to be repressor displacement by N-acetylneuraminic acid. The second stage is hypothesised to involve Dam methylation to achieve full induction.     

Identification of a Mechanical Rheostat in the Hydrophobic Core of Protein L

The ability of proteins and their complexes to withstand or respond to mechanical stimuli is vital for cells to maintain their structural organisation, to relay external signals and to facilitate unfolding and remodelling. Force spectroscopy using the atomic force microscope allows the behaviour of single protein molecules under an applied extension to be investigated and their mechanical strength to be quantified. protein L, a simple model protein, displays moderate mechanical strength and is thought to unfold by the shearing of two mechanical sub-domains. Here, we investigate the importance of side-chain packing for the mechanical strength of protein L by measuring the mechanical strength of a series of protein L variants containing single conservative hydrophobic volume deletion mutants. Of the five thermodynamically destabilised variants characterised, only one residue (I60V) close to the interface between two mechanical sub-domains was found to differ in mechanical properties to wild type (ΔF_I60V-WT = − 36 pN at 447 nm s^− 1, Δx_uI60V-WT = 0.2 nm). Φ-value analysis of the unfolding data revealed a highly native transition state. To test whether the number of hydrophobic contacts across the mechanical interface does affect the mechanical strength of protein L, we measured the mechanical properties of two further variants. protein L L10F, which increases core packing but does not enhance interfacial contacts, increased mechanical strength by 13 ± 11 pN at 447 nm s^− 1. By contrast, protein L I60F, which increases both core and cross-interface contacts, increased mechanical strength by 72 ± 13 pN at 447 nm s^− 1. These data suggest a method by which nature can evolve a varied mechanical response from a limited number of topologies and demonstrate a generic but facile method by which the mechanical strength of proteins can be rationally modified.