Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.

In vitro production of N-degron fused proteins and its application.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy.

LC3/ATG8 has long been appreciated to play a central role in autophagy, by which a variety of cytoplasmic materials are delivered to lysosomes and eventually degraded. However, information on the molecular functions of LC3 in RNA biology is very limited. Here, we show that LC3B is an RNA-binding protein that directly binds to mRNAs with a preference for a consensus AAUAAA motif corresponding to a polyadenylation sequence. Autophagic activation promotes an association between LC3B and target mRNAs and triggers rapid degradation of target mRNAs in a CCR4-NOT-dependent manner before autolysosome formation. Furthermore, our transcriptome-wide analysis reveals that PRMT1 mRNA, which encodes a negative regulator of autophagy, is one of the major substrates. Rapid degradation of PRMT1 mRNA by LC3B facilitates autophagy. Collectively, we demonstrate that LC3B acts as an RNA-binding protein and an mRNA decay factor necessary for efficient autophagy.

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex.

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N1- and N3-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.

Structural and Biochemical Study of the Mono-ADP-Ribosyltransferase Domain of SdeA, a Ubiquitylating/Deubiquitylating Enzyme from Legionella pneumophila.

Conventional ubiquitylation occurs through an ATP-dependent three-enzyme cascade (E1, E2, and E3) that mediates the covalent conjugation of the C-terminus of ubiquitin to a lysine on the substrate. SdeA, which belongs to the SidE effector family of Legionella pneumophila, can transfer ubiquitin to endoplasmic reticulum-associated Rab-family GTPases in a manner independent of E1 and E2 enzymes. The novel ubiquitin-modifying enzyme SdeA utilizes NAD+ as a cofactor to attach ubiquitin to a serine residue of the substrate. Here, to elucidate the coupled enzymatic reaction of NAD+ hydrolysis and ADP-ribosylation of ubiquitin in SdeA, we characterized the mono-ADP-ribosyltransferase domain of SdeA and show that it consists of two sub-domains termed mART-N and mART-C. The crystal structure of the mART-C domain of SdeA was also determined in free form and in complex with NAD+ at high resolution. Furthermore, the spatial orientations of the N-terminal deubiquitylase, phosphodiesterase, mono-ADP-ribosyltransferase, and C-terminal coiled-coil domains within the 180-kDa full-length SdeA were determined. These results provide insight into the unusual ubiquitylation mechanism of SdeA and expand our knowledge on the structure-function of mono-ADP-ribosyltransferases.

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils.

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction.