Capturing the catalytic intermediates of parkin ubiquitination.

Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified α-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity.

Distinct phosphorylation signals drive acceptor versus free ubiquitin chain targeting by parkin.

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of the Ubl domain in parkin, both mediated by the OMM kinase, PINK1. How an OMM protein is selected for ubiquitination is unclear. Parkin targeted OMM proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we used chimeric proteins, tagged with ubiquitin (Ub), to evaluate parkin ubiquitination of mitochondrial acceptor proteins pre-ligated to Ub. We find that pUb tethered to the mitochondrial target proteins, Miro1 or CISD1, is necessary for parkin recruitment and essential for target protein ubiquitination. Surprisingly, phosphorylation of parkin is not necessary for the ubiquitination of either Miro1 or CISD1. Thus, parkin lacking its Ubl domain efficiently ubiquitinates a substrate tethered to pUb. Instead, phosphorylated parkin appears to stimulate free Ub chain formation. We also demonstrate that parkin ubiquitination of pUb-tethered substrates occurs on the substrate, rather than the pUb modification. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb on the OMM protein and subsequent parkin phosphorylation triggers free Ub chain formation. This finding accounts for the broad spectrum of OMM proteins ubiquitinated by parkin and has implications on target design for therapeutics.

A mechanistic review of Parkin activation.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.

The first junior European Calcium Society meeting: calcium research across scales, Kingdoms and countries.

The first junior European Calcium Society online meeting, held October 20-21, 2020, aimed to promote junior researchers in the Ca2+ community. The meeting included four scientific sessions, covering Ca2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

Monitoring Interactions Between S100B and the Dopamine D2 Receptor Using NMR Spectroscopy.

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and interacts with a wide range of proteins to modulate their functions. The dopamine D2 receptor is one potential S100B binding partner that may play a key role in neurological processing. In this chapter, we describe the use of NMR spectroscopy to examine the interaction between calcium-bound S100B and the third intracellular loop (IC3) from the dopamine D2 receptor. We provide details that allow the strength of the interaction (K d) between the two proteins to be determined and the IC3 site of interaction on the structure of S100B to be identified. Both these characteristics can be identified from a single series of nondestructive experiments.

Structural basis of the specificity of USP18 toward ISG15.

Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin proteases and Ubl proteases, collectively termed DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity toward ubiquitin but specifically deconjugates the interferon-induced Ubl ISG15. To identify the molecular determinants of this specificity, we solved the crystal structures of mouse USP18 alone and in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation, thus revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15, thus providing evidence that USP18's ISG15 specificity is mediated by a small interaction interface. Our results may provide a structural basis for the development of new drugs modulating ISG15 linkage.

Glucose and fluoxetine induce fine structural change in human serum albumin.

Human serum albumin has been used as a model protein for protein folding and ligand binding studies over many decades. Due to its long life period and high concentration in plasma, HSA is highly sensitive to glycation. It is reported that 175 mg/dL glucose concentration is a threshold of kidney activity for the beginning of excretion of glucose. pH denaturation of HSA in absence and presence of different concentrations of glucose is studied and based on the Pace two-state model, the findings are analyzed. In addition, florescence emission data of albumin range in the period of 300-500 nm was depicted. The amounts of free energy change and [D]1/2 parameters of unfolding in correspond to florescence date indicate that glucose induces fine structural change in human serum albumin. Results showed that 175 mg/dL glucose concentration is a critical point for albumin structural and functional alteration.

Fluoxetine competes with cortisol for binding to human serum albumin.

Human serum albumin (HSA) is an important protein that carries variety of substances like some hormones and drugs in blood. Pharmacological studies of the interaction of many drugs and HSA are reported during several decades, specially recently years. Interaction of cortisol and fluoxetine hydrochloride (FLX) (as a common anti-stress drug) with HSA (as their carrier in blood) has been studied separately by using different spectroscopic techniques. Here, considering the increment of anti-stress drugs consumption, conformational change of HSA in presence of cortisol and FLX in 50 mM tris buffer, at pH = 7.5 and 37°C, is investigated via pH meter, UV absorption and fluorescence spectroscopy and circular dichroism methods. pH meter findings indicate that the acid denaturation of HSA in the presence of drug and cortisol occurs in the similar manner and this pattern is different relative to the denaturation of HSA in the absence of two reagents. The results of the other techniques consistent with the pH meter findings show that FLX effects on the physiochemical properties of HSA are as that of Cortisol. In-vivo study in Rats confirms in-vitro findings which means blood cortisol level increased in the presence of FLX. Experimental results indicate that FLX and cortisol alter the structural aspects of HSA in similar manner, so, this findings lead to the following reasonable conclusion: "FLX is a competitive ligand for the binding of cortisol to HSA. Binding of FLX to HSA interferes to the interaction of cortisol-HSA."