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Abstract: Inositol monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47 Å resolution crystal structure (PDB entry 6zk0), are presented. In the complex with human IMPase, ebselen in a ring-opened conformation is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and in the crystal structure two adjacent dimers share four ebselen molecules, creating a tetramer with approximate 222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor or may block the binding of partner proteins.

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Abstract: The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Å resolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.

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Abstract: Ribonucleotide reductase (RNR) is a central enzyme for DNA building block synthesis. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1-R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, electron microscopy, x-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1-R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral, but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1-R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

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Abstract: Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin’s Gly76. Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+. Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.

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Abstract: Antibody therapeutics are one of the most important classes of drugs. Antibody structures have become an integral part of predicting the behavior of potential therapeutics, either directly or as the basis of modeling. Structures of Fab:antigen complexes have even greater value. While the crystallization and structure determination of Fabs is easy relative to many other protein classes, especially membrane proteins, broad screening and optimization of crystalline hits is still necessary. Through a comprehensive review of rabbit Fab crystal contacts and their incompatibility with human Fabs, we identified a small secondary structural element from the rabbit light chain constant domain potentially responsible for hindering the crystallization of human Fabs. Upon replacing the human kappa constant domain FG loop (HQGLSSP) with the two residue shorter rabbit loop (QGTTS), we dramatically improved the crystallization of human Fabs and Fab:antigen complexes. Our design, which we call “Crystal Kappa”, enables rapid crystallization of human fabs and fab complexes in a broad range of conditions, with less material in smaller screens or from dilute solutions.