Crystal Structure Of Photorespiratory Alanine:Glyoxylate Aminotransferase 1 (AGT1) From Arabidopsis thaliana.

Photorespiration is an energetically costly metabolic pathway for the recycling of phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to phosphoglycerate. Arabidopsis alanine:glyoxylate aminotransferase 1 (AGT1) is a peroxisomal aminotransferase with a central role in photorespiration. This enzyme catalyzes various aminotransferase reactions, including serine:glyoxylate, alanine:glyoxylate, and asparagine:glyoxylate transaminations. To better understand structural features that govern the specificity of this enzyme, its crystal structures in the native form (2.2-Å resolution) and in the presence of l-serine (2.1-Å resolution) were solved. The structures confirm that this enzyme is dimeric, in agreement with studies of the active enzyme in solution. In the crystal, another dimer related by noncrystallographic symmetry makes close interactions to form a tetramer mediated in part by an extra carboxyl-terminal helix conserved in plant homologs of AGT1. Pyridoxal 5'-phosphate (PLP) is bound at the active site but is not held in place by covalent interactions. Residues Tyr35' and Arg36', entering the active site from the other subunits in the dimer, mediate interactions between AGT and l-serine when used as a substrate. In comparison, AGT1 from humans and AGT1 from Anabaena lack these two residues and instead position a tyrosine ring into the binding site, which accounts for their preference for l-alanine instead of l-serine. The structure also rationalizes the phenotype of the sat mutant, Pro251 to Leu, which likely affects the dimer interface near the catalytic site. This structural model of AGT1 provides valuable new information about this protein that may enable improvements to the efficiency of photorespiration.

Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.

Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNA-binding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43- and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.

The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response.

The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1alpha. The monomer of the luminal domain comprises a unique fold of a triangular assembly of beta-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1alpha molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.

Crystal structure of the C-terminal domain of the two-component system transmitter protein nitrogen regulator II (NRII; NtrB), regulator of nitrogen assimilation in Escherichia coli.

The kinase/phosphatase nitrogen regulator II (NRII, NtrB) is a member of the transmitter protein family of conserved two-component signal transduction systems. The kinase activity of NRII brings about the phosphorylation of the transcription factor nitrogen regulator I (NRI, NtrC), causing the activation of Ntr gene transcription. The phosphatase activity of NRII results in the inactivation of NRI-P. The activities of NRII are regulated by the signal transduction protein encoded by glnB, PII protein, which upon binding to NRII inhibits the kinase and activates the phosphatase activity. The C-terminal ATP-binding domain of NRII is required for both the kinase and phosphatase activities and contains the PII binding site. Here, we present the crystal structure of the C-terminal domain of a mutant form of NRII, NRII-Y302N, at 1.6 A resolution and compare this structure to the analogous domains of other two-component system transmitter proteins. While the C-terminal domain of NRII shares the general tertiary structure seen in CheA, PhoQ, and EnvZ transmitter proteins, it contains a distinct beta-hairpin projection that is absent in these related proteins. This projection is near the site of a well-characterized mutation that reduces the binding of PII and near other less-characterized mutations that affect the phosphatase activity of NRII. Sequence alignment suggests that the beta-hairpin projection is present in NRII proteins from various organisms, and absent in other transmitter proteins from Escherichia coliK-12. This unique structural element in the NRII C-terminal domain may play a role in binding PII or in intramolecular signal transduction.

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold.

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.