Da-Cheng Wang

Crystal Structures of a Populus tomentosa 4-Coumarate:CoA Ligase Shed Light on Its Enzymatic Mechanisms

The crystal structure of Chinese white poplar 4-coumaric acid:coenzyme A ligase, the enzyme that catalyzes the synthesis of an important precursor of lignin, was determined in the apo, adenosine monophosphate-, and adenosine 5′-(3-(4-hydroxyphenyl)propyl)phosphate-complexed forms. Enzymatic mechanisms were proposed for the protein. Residues responsible for substrate specificity were identified. 4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 5′-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 81° relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.

Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv

Two antifungal peptides, named EAFP1 and EAFP2, have been purified from the bark of Eucommia ulmoides Oliv. Each of the sequences consists of 41 residues with a N‐terminal blockage by pyroglutamic acid determined by automated Edman degradation in combination with the tandem mass spectroscopy and the C‐terminal ladder sequencing analysis. The primary structurs all contain 10 cysteines, which are cross‐linked to form five disulfide bridges with a pairing pattern (C1–C5, C2–C9, C3–C6, C4–C7, C8–C10). This is the first finding of a plant antifungal peptide with a five‐disulfide motif. EAFP1 and EAFP2 show characteristics of hevein domain and exhibit chitin‐binding properties similar to the previously identified hevein‐like peptides. They exhibit relatively broad spectra of antifungal activities against eight pathogenic fungi from cotton, wheat, potato, tomato and tobacco. The inhibition activity of EAFP1 and EAFP2 can be effective on both chitin‐containing and chitin‐free fungi. The values of IC50 range from 35 to 155 μg/ml for EAFP1 and 18 to 109 μg/ml for EAFP2. Their antifungal effects are strongly antagonized by calcium ions.

Importance of the Conserved Aromatic Residues in the Scorpion α-Like Toxin BmK M1 THE HYDROPHOBIC SURFACE REGION REVISITED

About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called “conserved hydrophobic surface.” At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded β-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the β2-sheet and in the loop between the β2- and β3-sheets, respectively) are most likely involved in the pharmacological function of the toxin.

Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita

Lectin AAL (Agrocybe aegerita lectin) from the edible mushroom A. aegerita is an antitumor protein that exerts its tumor-suppressing function via apoptosis-inducing activity in cancer cells. The crystal structures of ligand-free AAL and its complex with lactose have been determined. The AAL structure shows a dimeric organization, and each protomer adopts a prototype galectin fold. To identify the structural determinants for antitumor effects arising from the apoptosis-inducing activity of AAL, 11 mutants were prepared and subjected to comprehensive investigations covering oligomerization detection, carbohydrate binding test, apoptosis-inducing activity assay, and X-ray crystallographic analysis. The results show that dimerization of AAL is a prerequisite for its tumor cell apoptosis-inducing activity, and both galactose and glucose are basic moieties of functional carbohydrate ligands for lectin bioactivity. Furthermore, we have identified a hydrophobic pocket that is essential for the proteins apoptosis-inducing activity but independent of its carbohydrate binding and dimer formation. This hydrophobic pocket comprises a hydrophobic cluster including residues Leu33, Leu35, Phe93, and Ile144, and is involved in AALs function mechanism as an integrated structural motif. Single mutants such as F93G or I144G do not disrupt carbohydrate binding and homodimerization capabilities, but abolish the bioactivity of the protein. These findings reveal the structural basis for the antitumor property of AAL, which may lead to de novo designs of antitumor drugs based on AAL as a prototype model.

Structural and Mechanistic Insights into NDM-1 Catalyzed Hydrolysis of Cephalosporins.

Cephalosporins constitute a large class of β-lactam antibiotics clinically used as antimicrobial drugs. New Dehli metallo-β-lactamase (NDM-1) poses a global threat to human health as it confers on bacterial pathogen resistance to almost all β-lactams, including penicillins, cephalosporins, and carbapenems. Here we report the first crystal structures of NDM-1 in complex with cefuroxime and cephalexin, as well as NMR spectra monitoring cefuroxime and cefixime hydrolysis catalyzed by NDM-1. Surprisingly, cephalosporoate intermediates were captured in both crystal structures determined at 1.3 and 2.0 Å. These results provide detailed information concerning the mechanism and pathways of cephalosporin hydrolysis. We also present the crystal structure and enzyme assays of a D124N mutant, which reveals that D124 most likely plays a more structural than catalytic role.

Structural basis for distinct binding properties of the human galectins to thomsen-friedenreich antigen

The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif (51AHGDA55) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins.

Molecular basis of the mammalian potency of the scorpion α-like toxin, BmK M1

In‐depth structure‐function studies of voltage‐gated Na+ channels and peptide toxins are continuously increasing our understanding of their interaction. In this study, an effective yeast expression system was used to study the role of 14 N‐ and C‐terminal residues from the α‐like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. With the use of site‐directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage‐gated Na+ channels (Nav1.2, Nav1.5, and para), and a circular dichroism analysis. Our results reveal large mutant‐dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids, we were able to redirect the α‐like characteristics of BmK M1 (active on both mammals and insects) to either much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion α‐like toxin, BmK M1 on voltage‐gated Na+ channels.

Structural insights into the Pseudomonas aeruginosa type VI virulence effector Tse1 bacteriolysis and self-protection mechanisms

Background: Pseudomonas aeruginosa employs Tse1 to kill rival cells and Tsi1 to inactivate Tse1 for self-protection. Results: Tse1 features a conserved catalytic site for murein hydrolysis, and Tsi1 specifically occupies the substrate-binding sites of Tse1. Conclusion: Tse1 acts as a murein peptidase, and Tsi1 blocks its substrate binding. Significance: This work builds a novel understanding of niche competition among bacteria. Recently, it was identified that Pseudomonas aeruginosa competes with rival cells to gain a growth advantage using a novel mechanism that includes two interrelated processes as follows: employing type VI secretion system (T6SS) virulence effectors to lyse other bacteria, and at the same time producing specialized immunity proteins to inactivate their cognate effectors for self-protection against mutual toxicity. To explore the structural basis of these processes in the context of functional performance, the crystal structures of the T6SS virulence effector Tse1 and its complex with the corresponding immunity protein Tsi1 were determined, which, in association with mutagenesis and Biacore analyses, provided a molecular platform to resolve the relevant structural questions. The results indicated that Tse1 features a papain-like structure and conserved catalytic site with distinct substrate-binding sites to hydrolyze its murein peptide substrate. The immunity protein Tsi1 interacts with Tse1 via a unique interactive recognition mode to shield Tse1 from its physiological substrate. These findings reveal both the structural mechanisms for bacteriolysis and the self-protection against the T6SS effector Tse1. These mechanisms are significant not only by contributing to a novel understanding of niche competition among bacteria but also in providing a structural basis for antibacterial agent design and the development of new strategies to fight P. aeruginosa.

Crystal structure of DNA gyrase B′ domain sheds lights on the mechanism for T-segment navigation

DNA gyrase is an indispensible marvelous molecular machine in manipulating the DNA topology for the prokaryotes. In the ‘two-gate’ mechanism of DNA topoisomerase, T-segment navigation from N- to DNA-gate is a critical step, but the structural basis supporting this scheme is unclear. The crystal structure of DNA gyrase B′ subfragment from Mycobacterium tuberculosis reveals an intrinsic homodimer. The two subunits, each consisting of a Tail and a Toprim domain, are tightly packed one another to form a ‘crab-like’ organization never observed previously from yeast topo II. Structural comparisons show two orientational alterations of the Tail domain, which may be dominated by a 43-residue peptide at the B′ module C-terminus. A highly conserved pentapeptide mediates large-scale intrasubunit conformational change as a hinge point. Mutational studies highlight the significant roles of a negatively charge cluster on a groove at dimer interface. On the basis of structural analysis and mutation experiments, a sluice-like model for T-segment transport is proposed.

Structural Mechanism Governing the Quaternary Organization of Monocot Mannose-binding Lectin Revealed by the Novel Monomeric Structure of an Orchid Lectin

Two isoforms of an antifungal protein, gastrodianin, were isolated from two subspecies of the orchid Gastrodia elata, belonging to the protein superfamily of monocot mannose-specific lectins. In the context that all available structures in this superfamily are oligomers so far, the crystal structures of the orchid lectins, both at 2.0 Å, revealed a novel monomeric structure. It resulted from the rearrangement of the C-terminal peptide inclusive of the 12th β-strand, which changes from the “C-terminal exchange” into a “C-terminal self-assembly” mode. Thus, the overall tertiary scaffold is stabilized with an intramolecular β-sheet instead of the hybrid observed on subunit/subunit interface in all known homologous dimeric or tetrameric lectins. In contrast to the constrained extended conformation with a cis peptide bond between residues 98 and 99 commonly occurring in oligomers, a β-hairpin forms from position 97 to 101 with a normal trans peptide bond at the corresponding site in gastrodianin, which determines the topology of the C-terminal peptide and thereby its unique fold pattern. Sequence and structure comparison shows that residue replacement and insertion at the position where the β-hairpin occurs in association with cis-trans inter-conversion of the specific peptide bond (97–98) are possibly responsible for such a radical structure switch between monomers and oligomers. Moreover, this seems to be a common melody controlling the quaternary states among bulb lectins through studies on sequence alignment. The observations revealed a structural mechanism by which the quaternary organization of monocot mannose binding lectins could be governed. The mutation experiment performed on maltose-binding protein-gastrodianin fusion protein followed by a few biochemical detections provides direct evidence to support this conclusion. Potential carbohydrate recognition sites and biological implications of the orchid lectin based on its monomeric state are also discussed in this paper.