Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C.

Mycobacterium tuberculosis virulence is highly metal-dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu2+ ≥ Zn2+ > Ni2+ > Co2+ ) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for kcat rather than KM . Removal of the artificial N-terminal 6xHis-tag did not change the metal-dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn2+ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal-dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+ /Mn2+ heterobinucleation.

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+ , Mn2+ , Mg2+ , Fe2+ , and Zn2+ . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+ /Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+ /Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.

Allosteric regulation of GRASP protein-dependent Golgi membrane tethering by mitotic phosphorylation.

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.

ADAR proteins: structure and catalytic mechanism.

Since the discovery of the adenosine deaminase (ADA) acting on RNA (ADAR) family of proteins in 1988 (Bass and Weintraub, Cell 55:1089-1098, 1988) (Wagner et al. Proc Natl Acad Sci U S A 86:2647-2651, 1989), we have learned much about their structure and catalytic mechanism. However, much about these enzymes is still unknown, particularly regarding the selective recognition and processing of specific adenosines within substrate RNAs. While a crystal structure of the catalytic domain of human ADAR2 has been solved, we still lack structural data for an ADAR catalytic domain bound to RNA, and we lack any structural data for other ADARs. However, by analyzing the structural data that is available along with similarities to other deaminases, mutagenesis and other biochemical experiments, we have been able to advance the understanding of how these fascinating enzymes function.

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis.

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies.

Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing.

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

A transition state analogue for an RNA-editing reaction.

Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6-N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2's two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR.RNA complexes.

Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding.

Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs.