Erik Walinda

Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin

Background: The autophagic receptor NBR1 is commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Results: Molecular recognition of ubiquitin and polyubiquitin by NBR1 is described. Conclusion: The ubiquitin-associated domain of NBR1 shows unexpectedly high affinity for monoubiquitin but lacks polyubiquitin linkage specificity. Significance: NBR1 may be highly efficient at forming intracellular inclusions with ubiquitylated proteins via non-linkage-specific association with ubiquitin. NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix α-3, which is tilted farther from helix α-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.

The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates

Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy.

Ubiquitylation Directly Induces Fold Destabilization of Proteins

Ubiquitin is a common post-translational modifier and its conjugation is a key signal for proteolysis by the proteasome. Because the molecular mass of ubiquitin is larger than that of other modifiers such as phosphate, acetyl, or methyl groups, ubiquitylation not only influences biochemical signaling, but also may exert physical effects on its substrate proteins by increasing molecular volume and altering shape anisotropy. Here we show that ubiquitylation destabilizes the fold of two proteins, FKBP12 and FABP4, and that elongation of the conjugated ubiquitin chains further enhances this destabilization effect. Moreover, NMR relaxation analysis shows that ubiquitylation induces characteristic structural fluctuations in the backbone of both proteins. These results suggest that the ubiquitylation-driven structural fluctuations lead to fold destabilization of its substrate proteins. Thus, physical destabilization by ubiquitylation may facilitate protein degradation by the proteasome.

Dual Function of Phosphoubiquitin in E3 Activation of Parkin.

Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR, and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis. Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL-binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2∼Ub binding triggers large scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin-E2∼Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin.

Efficient identification and analysis of chemical exchange in biomolecules by R1ρ relaxation dispersion with Amaterasu

UNLABELLED We introduce here a novel acquisition and processing methodology for cross-polarization based 1D rotating-frame relaxation dispersion NMR experiments. This easy-to-use protocol greatly facilitates the screening, acquisition, processing and model fitting of large on- and off-resonance R1ρ relaxation dispersion NMR datasets in an automated manner for the analysis of chemical exchange phenomena in biomolecules. AVAILABILITY AND IMPLEMENTATION The Amaterasu package including the spreadsheet, Bruker pulse programs and analysis software is available at www.moleng.kyoto-u.ac.jp/∼moleng_01/amaterasu CONTACT : sugase@moleng.kyoto-u.ac.jp.

Practical considerations for investigation of protein conformational dynamics by 15N R1ρ relaxation dispersion

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R1ρ relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.

F 1 F 2 -selective NMR spectroscopy

Fourier transform NMR spectroscopy has provided unprecedented insight into the structure, interaction and dynamic motion of proteins and nucleic acids. Conventional biomolecular NMR relies on the acquisition of three-dimensional and four-dimensional (4D) data matrices to establish correlations between chemical shifts in the frequency domains F1, F2, F3 and F1, F2, F3, F4 respectively. While rich in information, these datasets require a substantial amount of acquisition time, are visually highly unintuitive, require expert knowledge to process, and sample dark and bright regions of the frequency domains equally. Here, we present an alternative approach to obtain multidimensional chemical shift correlations for biomolecules. This strategy focuses on one narrow frequency range, F1F2, at a time and records the resulting F3F4 correlation spectrum by two-dimensional NMR. As a result, only regions of the frequency domain that contain signals in F1F2 (“bright regions”) are sampled. F1F2 selection is achieved by Hartmann–Hahn cross-polarization using weak radio frequency fields. This approach reveals information equivalent to that of a conventional 4D experiment, while the dimensional reduction may shorten the total acquisition time and simplifies spectral processing, interpretation and comparative analysis. Potential applicability of the F1F2-selective approach is illustrated by de novo assignment, structural and dynamics studies of ubiquitin and fatty-acid binding protein 4 (FABP4). Further extension of this concept may spawn new selective NMR experiments to aid studies of site-specific structural dynamics, protein–protein interactions and allosteric modulation of protein structure.

Cooperative Domain Formation by Homologous Motifs in HOIL-1L and SHARPIN Plays A Crucial Role in LUBAC Stabilization

SUMMARY The linear ubiquitin chain assembly complex (LUBAC) participates in inflammatory and oncogenic signaling by conjugating linear ubiquitin chains to target proteins. LUBAC consists of the catalytic HOIP subunit and two accessory subunits, HOIL-1L and SHARPIN. Interactions between the ubiquitin-associated (UBA) domains of HOIP and the ubiquitin-like (UBL) domains of two accessory subunits are involved in LUBAC stabilization, but the precise molecular mechanisms underlying the formation of stable trimeric LUBAC remain elusive. We solved the co-crystal structure of the binding regions of the trimeric LUBAC complex and found that LUBAC-tethering motifs (LTMs) located N terminally to the UBL domains of HOIL-1L and SHARPIN heterodimerize and fold into a single globular domain. This interaction is resistant to dissociation and plays a critical role in stabilizing trimeric LUBAC. Inhibition of LTM-mediated HOIL-1L/SHARPIN dimerization profoundly attenuated the function of LUBAC, suggesting LTM as a superior target of LUBAC destabilization for anticancer therapeutics.

Biological and Physicochemical Functions of Ubiquitylation Revealed by Synthetic Chemistry Approaches

Most intracellular proteins are subjected to post-translational modification by ubiquitin. Accordingly, it is of fundamental importance to investigate the biological and physicochemical effects of ubiquitylation on substrate proteins. However, preparation of ubiquitylated proteins by an enzymatic synthesis bears limitations in terms of yield and site-specificity. Recently established chemical ubiquitylation methodologies can overcome these problems and provide a new understanding of ubiquitylation. Herein we describe the recent chemical ubiquitylation procedures with a focus on the effects of ubiquitylation on target proteins revealed by the synthetic approach.

High-Sensitivity Rheo-NMR Spectroscopy for Protein Studies

Shear stress can induce structural deformation of proteins, which might result in aggregate formation. Rheo-NMR spectroscopy has the potential to monitor structural changes in proteins under shear stress at the atomic level; however, existing Rheo-NMR methodologies have insufficient sensitivity to probe protein structure and dynamics. Here we present a simple and versatile approach to Rheo-NMR, which maximizes sensitivity by using a spectrometer equipped with a cryogenic probe. As a result, the sensitivity of the instrument ranks highest among the Rheo-NMR spectrometers reported so far. We demonstrate that the newly developed Rheo-NMR instrument can acquire high-quality relaxation data for a protein under shear stress and can trace structural changes in a protein during fibril formation in real time. The described approach will facilitate rheological studies on protein structural deformation, thereby aiding a physical understanding of shear-induced amyloid fibril formation.