Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

ABSTRACT␣B-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in ␣B-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92-95]. By using ␣-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G ␣B-crystallin had reduced or completely lost chaperone-like function. The addition of R120G ␣B-crystallin to unfolding ␣-lactalbumin enhanced the kinetics and extent of its aggregation. R120G ␣B-crystallin became entangled with unfolding ␣-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G ␣B-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G ␣B-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G ␣B-crystallin has decreased ␤-sheet secondary structure and an altered aromatic residue environment compared with wild-type ␣B-crystallin. The apparent molecular mass of R120G ␣B-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type ␣B-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G ␣B-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of ␣B-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

␣-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each ␣-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant ␣-crystallin subunits. We labeled ␣A-crystallin with stilbene iodoacetamide (4-acetamido-4-((iodoacetyl)amino)stilbene-2,2-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled ␣A-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent ␣A-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min ؊1 at 37°C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several ␣A-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of ␣A-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.␣-Crystallin, the major lens protein of the mammalian eye, is a member of the small heat shock protein family (1, 2). Like other small heat shock proteins, ␣-crystallin is a high molecular mass complex consisting of a large number of subunits. The two polypeptides of ␣-crystallin found in the lens of the mammalian eye, ␣A and ␣B, are encoded by evolutionarily related genes and share more than 50% identity in amino acid sequence (3, 4). For many years, ␣-crystallin was thought to be lens-specific. However, recent advances in detection methods have revealed much wider non-lenticular tissue distributions in heart, thymus, skin, lung, retina, and brain (5-8).

␣A-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30 -40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between ␣A-crystallin and other proteins. We found that Hsp27 and ␣B-crystallin readily exchanged with fluorescence-labeled ␣A-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of ␣A-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts ␣A-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20 -56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of ␣A-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of ␣A-crystallin, consisting of small multimers of ␣A-crystallin subunits in a dynamic equilibrium with the oligomeric complex.

No abstract

Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease and non-selective inhibitors are protective in rodent disease models. Due to their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.

No abstract

alpha-Crystallin is a major lens protein, comprising up to 40% of total lens proteins, where its structural function is to assist in maintaining the proper refractive index in the lens. In addition to its structural role, it has been shown to function in a chaperone-like manner. The chaperone-like function of alpha-crystallin will help prevent the formation of large light-scattering aggregates and possibly cataract. In the lens, alpha-crystallin is a polydisperse molecule consisting of a 3:1 ratio of alpha A to alpha B subunits. In this study, we expressed recombinant alpha A- and alpha B-crystallin in E. coli and compared the polydispersity, structure and aggregation state between each other and native bovine lens alpha-crystallin. Using gel permeation chromatography to assay for polydispersity, we found native alpha-crystallin to be significantly more polydisperse than either recombinant alpha A- or alpha B-crystallin, with alpha B-crystallin having the most homogeneous structure of the three. Reconstructed images of alpha B-crystallin obtained with cryo-electron microscopy support the concept that alpha B-crystallin is an extremely dynamic molecule and demonstrated that it has a hollow interior. Interestingly, we present evidence that native alpha-crystallin is significantly more thermally stable than either alpha A- or alpha B-crystallin alone. In fact, our experiments suggest that a 3:1 ratio of alpha A to alpha B subunit composition in an alpha-crystallin molecule is optimal in terms of thermal stability. This fascinating result explains the stoichiometric ratios of alpha A- and alpha B-crystallin subunits in the mammalian lens.