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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., (Copyright © 2019 Liepman, Vijayalakshmi, Peisach, Hulsebus, Olsen and Saper.)

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.

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 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.

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.

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.

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.

D-Amino acid transaminase is a bacterial enzyme that uses pyridoxal phosphate (PLP) as a cofactor to catalyze the conversion of D-amino acids into their corresponding alpha-keto acids. This enzyme has already been established as a target for novel antibacterial agents through suicide inactivation by a number of compounds. To improve their potency and specificity, the detailed enzyme mechanism, especially the role of its PLP cofactor, is under investigation. Many PLP-dependent transaminases have a negatively charged amino acid residue forming a salt-bridge with the pyridine nitrogen of its cofactor that promotes its protonation to stabilize the formation of a ketimine intermediate, which is subsequently hydrolyzed in the normal transaminase reaction pathway. However, alanine racemase has a positively charged arginine held rigidly in place by an extensive hydrogen bond network that may destabilize the ketimine intermediate, and make it too short-lived for a transaminase type of hydrolysis to occur. To test this hypothesis, we changed Glu-177 into a titratable, positively charged lysine (E177K). The crystal structure of this mutant shows that the positive charge of the newly introduced lysine side chain points away from the nitrogen of the cofactor, which may be due to electrostatic repulsions not being overcome by a hydrogen bond network such as found in alanine racemase. This mutation makes the active site more accessible, as exemplified by both biochemical and crystallographic data: CD measurements indicated a change in the microenvironment of the protein, some SH groups become more easily titratable, and at pH 9.0 the PMP peak appeared around 315 nm rather than at 330 nm. The ability of this mutant to convert L-alanine into D-alanine increased about 10-fold compared to wild-type and to about the same extent as found with other active site mutants. On the other hand, the specific activity of the E177K mutant decreased more than 1000-fold compared to wild-type. Furthermore, titration with L-alanine resulted in the appearance of an enzyme-substrate quinonoid intermediate absorbing around 500 nm, which is not observed with usual substrates or with the wild-type enzyme in the presence of L-alanine. The results overall indicate the importance of charged amino acid side chains relative to the coenzyme to maintain high catalytic efficiency.

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.

The three-dimensional structures of two forms of the D-amino acid aminotransferase (D-aAT) from Bacillus sp. YM-1 have been determined crystallographically: the pyridoxal phosphate (PLP) form and a complex with the reduced analogue of the external aldimine, N-(5'-phosphopyridoxyl)-d-alanine (PPDA). Together with the previously reported pyridoxamine phosphate form of the enzyme [Sugio et al. (1995) Biochemistry 34, 9661], these structures allow us to describe the pathway of the enzymatic reaction in structural terms. A major determinant of the enzyme's stereospecificity for D-amino acids is a group of three residues (Tyr30, Arg98, and His100, with the latter two contributed by the neighboring subunit) forming four hydrogen bonds to the substrate alpha-carboxyl group. The replacement by hydrophobic groups of the homologous residues of the branched chain L-amino acid aminotransferase (which has a similar fold) could explain its opposite stereospecificity. As in L-aspartate aminotransferase (L-AspAT), the cofactor in D-aAT tilts (around its phosphate group and N1 as pivots) away from the catalytic lysine 145 and the protein face in the course of the reaction. Unlike L-AspAT, D-aAT shows no other significant conformational changes during the reaction.

Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E approximately P.Mg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+ increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+ on the free enzyme, high concentrations of lumenal Ca2+ would convert all of the enzyme to the same maximal concentration of Ca2.E approximately P.Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+ on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.