Yong-Xing He

Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 A resolution revealed a novel two-strand antiparallel beta-sheet opposite the GSH binding groove. This extra beta-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes themultiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr⁹⁴ in OpuCC compared with Asp⁹⁶ in OpuBC.

Crystal structure of LZ‐8 from the medicinal fungus Ganoderma lucidium

Ling Zhi (Ganoderma lucidium) and other medicinal fungi have a long history of use as traditional herbs in China. The mechanism of their effects for a wide range of ailments remains unclear. To elucidate the functional compounds in these herbs, a new family of fungal immunomodulatory proteins (Fips) has been identified, including four Fips isolated from Ganoderma lucidium, Flammulina veltipes, Volvariella volvacea, and Ganoderma tsugae, termed LZ-8, Fip-gts, Fip-fve, and Fip-vvo, respectively.1–4 The native LZ-8 has a series of biological activities which include immunomodulatory responses,3 alleviation of transplant rejection,4 and antitumor activity. In terms of antitumor activity, LZ-8 plays a role as a mitogen in mouse spleen cells,5 human peripheral lymphocytes,6 and peripheral mononuclear cells.4 Fip-gts from G. tsugae, which shares an identical primary sequence to LZ-8, was found to perform its antitumor activity by triggering calcium signaling in repression of telomerase activity and inhibiting the transcription of telomerase in human lung adenocarcinoma cell line A549, via exporting the telomerase reverse transcriptase out of the nuclei.7,8 Despite being highly conserved in primary sequence, members of Fips family are quite different from each other in biological activity.9 For instance, LZ-8 has a strong antitumor activity against HL60 cells, whereas Fip-fve has no such an activity (unpublished data), even though they share a sequence similarity of 81%. To elucidate the structural basis of this functional divergence, we solved the crystal structure of LZ-8 at 2.10 Å resolution (PDB code 3F3H), representing the second structure in this family after Fip-fve.10 Comparative structural analyses revealed significant local conformation changes at two loop regions of the fibronectin type III (FNIII) domain which might be potential active sites. This finding provided us clues for engineering these loops to further improve the medicine activity of LZ-8 and its homologs.

Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103

BackgroundThe carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: α-, β-, γ-, δ-, ε-, ζ- CAs, with β-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of β-CA Nce103/YNL036W.ResultsWe determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 Å resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His112 and Cys115 in addition to a water molecule. Residues Asp59, Arg61, Gly111, Leu102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. Activity assays of full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.ConclusionThe quaternary structure of Nce103 resembles the typical plant type β-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket.

Crystal structure of the mucin-binding domain of Spr1345 from Streptococcus pneumoniae

The surface protein Spr1345 from Streptococcus pneumoniae R6 is a 22-kDa mucin-binding protein (MucBP) involved in adherence and colonization of the human lung and respiratory tract. It is composed of a mucin-binding domain (MucBD) and a proline-rich domain (PRD) followed by an LPxTG motif, which is recognized and cleaved by sortase, resulting in a mature form of 171 residues (MF171) that is anchored to the cell wall. We found that the MucBD alone possesses comparable in vitro mucin-binding affinity to the mature form, and can be specifically enriched at the surface of human lung carcinoma A549 cells. Using single-wavelength anomalous dispersion (SAD) phasing method with the iodine signals, we solved the crystal structure of the MucBD at 2.0Å resolution, the first structure of MucBDs from pathogenic bacteria. The overall structure adopts an immunoglobulin-like fold with an elongated rod-like shape, composed of six anti-parallel β-strands and a long loop. Structural comparison suggested that the conserved C-terminal moiety may participate in the recognition of mucins. These findings provided structural insights into host-pathogen interaction mediated by mucins, which might be useful for designing novel vaccines and antibiotic drugs against human diseases caused by pneumococci.

Crystal structure of Saccharomyces cerevisiae glutamine synthetase Gln1 suggests a nanotube‐like supramolecular assembly

Glutamine synthetase (GS, EC 6.3.1.2) is an enzyme that catalyzes the condensation of glutamate and ammonium to form glutamine, with concomitant hydrolysis of ATP.1 There are three different classes of GS enzymes, referred to as GSI, GSII, and GSIII. GSI enzymes are specific to prokaryotes and form oligomers of 12 identical subunits.2 The activity of GSI enzyme is regulated by the adenylation of a tyrosine residue.3 GSII enzymes are found in eukaryotes and some bacteria (Rhizobiaceae, Frankiaceae, and Streptomycetaceae families, which also have GSI). They form decamers of identical subunits.4 In mammals, GSII enzymes eliminate free ammonia and convert the excitotoxic glutamate into glutamine, which is not neurotoxic.5 In plants, there are two or more isoenzymes of GSII, which are targets of some herbicides because of their roles in ammonia assimilation. GSIII enzymes were first found in Bacteroides fragilis and identified afterward in a few more anaerobic bacteria and cyanobacteria.6–8 They are hexamers of identical subunits which are much larger (about 700 residues) than that of GSI (450–470 residues) or GSII (350–420 residues) enzymes.9 Over the course of two decades, GSI enzymes have been biophysically and structurally characterized.1– 3,10,11 However, little was known about the catalytic mechanism and quaternary structure of the GSII enzymes until the recent publication of GSII structures from maize, human, and canine.4,12 Structural analyses indicated that GSII and GSI enzymes share a similar catalytic mechanism, implying a common ancestor of these two classes of GS. Moreover, the crystal structures revealed that GSII has the decamer structure consisting of two stacking pentameric rings, instead of the formerly claimed octamer structure inferred from the electronic microscopy studies or sedimentation experiments.1,13 Because of the relatively fewer crystallographic studies on GSII enzymes, it is necessary to elucidate GSII structures of other species to complete our understandings on GSII enzymes from the structural and biochemical perspective. In this article, we report the crystal structure of Gln1, a GSII enzyme from Saccharomyces cerevisiae that shares high sequence homology with maize GS and human GSII ( 55% sequence identity), at the resolution of 2.95 Å.

Structures of yeast glutathione-S-transferase Gtt2 reveal a new catalytic type of GST family.

Glutathione‐S‐transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand‐bound forms, at 2.23 Å, 2.20 Å and 2.10 Å, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues—tyrosine, serine and cysteine—distinguishes it from all other cytosolic GSTs of known structure. Site‐directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino‐terminus of helix‐α1 because of stereo‐hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.

Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels.

Brachydactyly type A1 (BDA1), the first recorded Mendelian autosomal dominant disorder in humans, is characterized by a shortening or absence of the middle phalanges. Heterozygous missense mutations in the Indian Hedgehog (IHH) gene have been identified as a cause of BDA1; however, the biochemical consequences of these mutations are unclear. In this paper, we analyzed three BDA1 mutations (E95K, D100E, and E131K) in the N-terminal fragment of Indian Hedgehog (IhhN). Structural analysis showed that the E95K mutation changes a negatively charged area to a positively charged area in a calcium-binding groove, and that the D100E mutation changes the local tertiary structure. Furthermore, we showed that the E95K and D100E mutations led to a temperature-sensitive and calcium-dependent instability of IhhN, which might contribute to an enhanced intracellular degradation of the mutant proteins via the lysosome. Notably, all three mutations affected Hh binding to the receptor Patched1 (PTC1), reducing its capacity to induce cellular differentiation. We propose that these are common features of the mutations that cause BDA1, affecting the Hh tertiary structure, intracellular fate, binding to the receptor/partners, and binding to extracellular components. The combination of these features alters signaling capacity and range, but the impact is likely to be variable and mutation-dependent. The potential variation in the signaling range is characterized by an enhanced interaction with heparan sulfate for IHH with the E95K mutation, but not the E131K mutation. Taken together, our results suggest that these IHH mutations affect Hh signaling at multiple levels, causing abnormal bone development and abnormal digit formation.

Crystal structure and computational analyses provide insights into the catalytic mechanism of 2, 4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr121, Tyr229, and Asn132, which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.