A conserved asparagine in a ubiquitin-conjugating enzyme positions the substrate for nucleophilic attack

The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born–Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step—the formation of a tetrahedral, zwitterionic intermediate—is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.     

Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein‐activity‐based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α‐globin probe, which was used for the enrichment and proteomic identification of α‐globin‐modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α‐globin, an excess of which aggravates β‐thalassemia symptoms. This development opens new opportunities for activity‐based‐probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.     

Determination of Backbone Angle ψ in Proteins Using a TROSY-Based α/β-HN(CO)CA-J Experiment

Transverse relaxation-optimized NMR experiment (TROSY) for the measurement of three-bond scalar coupling constant between ¹H^α_i−1 and ¹⁵N_i defining the dihedral angle ψ is described. The triple-spin-state-selective experiment allows measurement of ³J_H^αN from ¹³C^α, ¹⁵N, and ¹H^N correlation spectra H₂O with minimum resonance overlap. Transverse relaxation of ¹³C^α spin is minimized by using spin-state-selective filtering and by acquiring a signal longer in ¹⁵N-dimension in a manner of semi-constant-time TROSY evolution. The ³J_H^αN values obtained with the proposed α/β-HN(CO)CA-J TROSY scheme are in good agreement with the values measured earlier from ubiquitin in D₂O using the HCACO[N] experiment.     

The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family

The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.     

Microsecond Protein Dynamics from Combined Bloch-McConnell and Near-Rotary-Resonance R1p Relaxation-Dispersion MAS NMR

Studying protein dynamics on microsecond-to-millisecond (μs-ms) time scales can provide important insight into protein function. In magic-angle-spinning (MAS) NMR, μs dynamics can be visualized by rotating-frame relaxation dispersion experiments in different regimes of radio-frequency field strengths: at low RF field strength, isotropic-chemical-shift fluctuation leads to “Bloch-McConnell-type” relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased rate constants (“Near-Rotary-Resonance Relaxation Dispersion”, NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemical-shift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.     

Evaluation of protein 15N relaxation times by inverse Laplace transformation

Relaxation times (T1, T2, T1rho) are usually evaluated from exponential decay data by least-squares fitting methods. For this procedure, the integrals or amplitudes of signals must be determined, which can be laborious with large data sets. Moreover, the fitting requires a priori knowledge of the number of exponential components responsible for the decay. We have adapted inverse Laplace transformation (ILT) for the analysis of relaxation data. Exponential components are resolved with ILT to reciprocal space on their corresponding relaxation rate values. The ILT approach was applied to 3D linewidth-resolved 15N HSQC experiments to evaluate 15N T1 and T2 relaxation times of ubiquitin. The resulting spectrum is a true 3D spectrum, where the signals are separated by their 1H and 15N chemical shifts (HSQC correlations) and by their relaxation rate values (R1 or R2). From this spectrum, the relaxation times can be obtained directly with a simple peak-picking procedure.