Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in α-aminoadipate aminotransferase from Thermus thermophilus

To clarify the mechanism for substrate recognition of α-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5′-phosphopyridoxyl)-l-glutamate (PPE) was determined at 1.67 Å resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5′-phosphopyridoxyl)-l-α-aminoadipate (PPA) recognition; however, to bind the γ-carboxyl group of Glu at a fixed position, the Cα atom of the Glu moiety moves 0.80 Å toward the γ-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the Cα atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of γ (δ)-carboxyl group of Glu (AAA) and (ii) rearrangement of α2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.     

P. aeruginosa PilT Structures with and without Nucleotide Reveal a Dynamic Type IV Pilus Retraction Motor

Type IV pili are bacterial extracellular filaments that can be retracted to create force and motility. Retraction is accomplished by the motor protein PilT. Crystal structures of Pseudomonas aeruginosa PilT with and without bound β,γ-methyleneadenosine-5′-triphosphate have been solved at 2.6 Å and 3.1 Å resolution, respectively, revealing an interlocking hexamer formed by the action of a crystallographic 2-fold symmetry operator on three subunits in the asymmetric unit and held together by extensive ionic interactions. The roles of two invariant carboxylates, Asp Box motif Glu163 and Walker B motif Glu204, have been assigned to Mg²⁺ binding and catalysis, respectively. The nucleotide ligands in each of the subunits in the asymmetric unit of the β,γ-methyleneadenosine-5′-triphosphate-bound PilT are not equally well ordered. Similarly, the three subunits in the asymmetric unit of both structures exhibit differing relative conformations of the two domains. The 12° and 20° domain rotations indicate motions that occur during the ATP-coupled mechanism of the disassembly of pili into membrane-localized pilin monomers. Integrating these observations, we propose a three-state “Ready, Active, Release” model for the action of PilT.     

Structure of the class IV adenylyl cyclase reveals a novel fold

The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 A resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.     

Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-Å resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic α-helix (A′) at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Crystal Structure of Bacillus cereusd-Alanyl Carrier Protein Ligase (DltA) in Complex with ATP

d-Alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA (d-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-Å-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the β-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.     

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)₈ domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the − 1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the − 1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.     

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.     

Crystal structure and biochemical studies of Brucella melitensis 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.     

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs

Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-γ-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1′ site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1′ sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-γ-glutamate, aspartyl-glutamate, and γ-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 Å resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.     

Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD⁺ utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5′,3′-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5′-AMP, 3′-AMP, and 2′-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5′-nucleotides and 3′-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5′ substrates in an anti conformation and 3′ substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.     

Analysis of Flavivirus NS5 Methyltransferase Cap Binding

The flavivirus 2′-O-nucleoside N-terminal RNA methyltransferase (MTase) enzyme is responsible for methylating the viral RNA cap structure. To increase our understanding of the mechanism of viral RNA cap binding we performed a detailed structural and biochemical characterization of the guanosine cap-binding pocket of the dengue (DEN) and yellow fever (YF) virus MTase enzymes. We solved an improved 2.1 Å resolution crystal structure of DEN2 Mtase, new 1.5 Å resolution crystal structures of the YF virus MTase domain in apo form, and a new 1.45 Å structure in complex with guanosine triphosphate and RNA cap analog. Our structures clarify the previously reported DEN MTase structure, suggest novel protein-cap interactions, and provide a detailed view of guanine specificity. Furthermore, the structures of the DEN and YF proteins are essentially identical, indicating a large degree of structural conservation amongst the flavivirus MTases. Guanosine triphosphate analog competition assays and mutagenesis analysis, performed to analyze the biochemical characteristics of cap binding, determined that the major interaction points are (i) guanine ring via π−π stacking with Phe24, N1 hydrogen interaction with the Leu19 backbone carbonyl via a water bridge, and C2 amine interaction with Leu16 and Leu19 backbone carbonyls; (ii) ribose 2′ hydroxyl interaction with Lys13 and Asn17; and (iii) α-phosphate interactions with Lys28 and Ser215. Based on our mutational and analog studies, the guanine ring and α-phosphate interactions provide most of the energy for cap binding, while the combination of the water bridge between the guanine N1 and Leu19 carbonyl and the hydrogen bonds between the C2 amine and Leu16/Leu19 carbonyl groups provide for specific guanine recognition. A detailed model of how the flavivirus MTase protein binds RNA cap structures is presented.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Helix Dynamics in LacY: Helices II and IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H⁺ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼ 4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.     

Distal-to-proximal NO conversion in hemoproteins: the role of the proximal pocket

Hemoproteins play central roles in the formation and utilization of nitric oxide (NO) in cellular signaling, as well as in protection against nitrosative stress. Key to heme-nitrosyl function and reactivity is the Fe coordination number (5 or 6). For (five-coordinate) 5c-NO complexes, the potential for NO to bind on either heme face exists, as in the microbial cytochrome c′ from Alcaligenes xylosoxidans (AxCYTcp), which forms a stable proximal 5c-NO complex via a distal six-coordinate NO intermediate and a putative dinitrosyl species. Strong parallels between the NO-binding kinetics of AxCYTcp, the eukaryotic NO sensor soluble guanylate cyclase, and the ferrocytochrome c/cardiolipin complex have led to the suggestion that a distal-to-proximal NO switch could contribute to the selective ligand responses in gas-sensing hemoproteins. The proximal NO-binding site in AxCYTcp is close to a conserved basic (Arg124) residue that is postulated to modulate NO reactivity. We have replaced Arg124 by five different amino acids and have determined high-resolution (1.07-1.40 Å) crystallographic structures with and without NO. These, together with kinetic and resonance Raman data, provide new insights into the mechanism of distal-to-proximal heme-NO conversion, including the determinants of Fe-His bond scission. The Arg124Ala variant allowed us to determine the structure of an analog of the previously unobserved key 5c-NO distal intermediate species. The very high resolution structures combined with the extensive spectroscopic and kinetic data have allowed us to provide a fresh insight into heme reactivity towards NO, a reaction that is of wide importance in biology.     

Crystal structures of SULT1A2 and SULT1A1∗3: Insights into the substrate inhibition and the role of Tyr149 in SULT1A2

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1∗3, bound with 3′-phosphoadenosine 5′-phosphate (PAP), at 2.4 and 2.3 Å resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1 Å further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1∗3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K_m and two times lower V_max with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Functional characterization of the Caenorhabditis elegans DNA repair enzyme APN-1

Caenorhabditis elegans possesses two distinct DNA repair enzymes EXO-3 and APN-1 that have been identified by cross-specie complementation analysis of the Saccharomyces cerevisiae apn1Δ apn2Δ tpp1Δ triple mutant deficient in the ability to repair apurinic/apyrimidinc (AP) sites and DNA strand breaks with blocked 3′-ends. While purified EXO-3 directly incises AP sites and removes 3′-blocking groups, such characterization has not been previously reported for APN-1. We recently documented that C. elegans knockdown for apn-1 is unable to maintain integrity of the genome. Despite the presence of EXO-3, the apn-1 knockdown animals are also defective in the division of the P1 blastomere, an observation consistent with the accumulation of unrepaired DNA lesions suggesting a unique role for APN-1 DNA repair functions. Herein, we show that C. elegans APN-1 is stably expressed as GST-fusion protein in S. cerevisiae only when it carries a nuclear localization signal, and with this requirement rescued the DNA repair defects of the S. cerevisiae apn1Δ apn2Δ tpp1Δ triple mutant. We purified the APN-1 from the yeast expression system and established that it displays AP endonuclease and 3′-diesterase activities. In addition, we showed that APN-1 also possesses a 3′- to 5′-exonuclease and the nucleotide incision repair activity. This latter activity is capable of directly incising DNA at the 5′-side of various oxidatively damaged bases, as previously observed for Escherichia coli endonuclease IV and S. cerevisiae Apn1, underscoring the importance of this family of enzymes in removing these types of lesions. Glycine substitution of the conserved amino acid residue Glu261 of APN-1, corresponding to Glu145 involved in coordinating Zn²⁺ ions in the active site pocket of E. coli endonuclease IV, resulted in an inactive variant that lose the ability to rescue the DNA repair defects of S. cerevisiae apn1Δ apn2Δ tpp1Δ mutant. Interestingly, the Glu261Gly variant did not sustain purification and yielded a truncated polypeptide. These data suggest that the Glu261 residue of APN-1 may have a broader role in maintaining the structure of the protein.     

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 Å resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3′ overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 Å, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.     

A structural study of the hydrated and the dimethylsulfoxide, N,N′-dimethylpropyleneurea, and N,N-dimethylthioformamide solvated iron(II) and iron(III) ions in solution and solid state

The structures of the solvated iron(II) and iron(III) ions have been studied in solution and solid state by extended X-ray absorption fine structure (EXAFS) in three oxygen donor solvents, water, dimethylsulfoxide (Me₂SO), N,N′-dimethylpropyleneurea (DMPU), and one sulfur donor solvent, N,N-dimethylthioformamide (DMTF); these solvents have different coordination and solvation properties. In addition, the structure of hexakis(dimethylsulfoxide)iron(III) perchlorate has been determined crystallographically to support the determination of the corresponding solvate in solution. The hydrated, the dimethylsulfoxide and N,N-dimethylthioformamide solvated iron(II) ions show regular octahedral coordination in both solution and solid state with mean Fe-O, Fe-O, and Fe-S bond distances of 2.10, 2.10, and 2.52 Å, respectively, whereas the N,N′-dimethylpropyleneurea iron(II) solvate is five-coordinated, d(Fe-O) = 2.06 Å. The compounds vary in color from light green (hydrate) to dark orange or red (DMPU). The hydrated iron(III) ion in aqueous solution and the dimethylsulfoxide solvated iron(III) ions in solution and solid state show the expected octahedral coordination, the Fe-O bond distances are 2.00 Å for both, whereas the N,N′-dimethylpropyleneurea iron(III) solvate is found to be five-coordinated with a mean Fe-O bond distance of 1.99 Å. The N,N-dimethylthioformamide solvated iron(III) ion in the solid perchlorate salt is tetrahedrally four-coordinated, the mean Fe-S bond distance is 2.20 Å. Iron(III) is reduced with time to iron(II) in N,N-dimethylthioformamide solution. The compounds vary in color from pale yellow (hydrate) to blackish red (DMPU).