Dong-Cai Liang

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-Å resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 Å, c = 189.4 Å, α = β = 90°, γ = 120°. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 Å. The r.m.s. deviations of bond length and angles are 0.015 Å and 2.9°, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1α84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2β of another monomer. In R-PE and C-PC, chromophore 1α84 only forms 1 hydrogen bond with 2β77 residue in subunit 2β. This result may support and explain great spectrum difference exists between APC trimer and monomer.

Crystal Structure of Earthworm Fibrinolytic Enzyme Component A: Revealing the Structural Determinants of its Dual Fibrinolytic Activity

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEas S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.

From Structure to Function: Insights into the Catalytic Substrate Specificity and Thermostability Displayed by Bacillus subtilis Mannanase BCman

BCman, a beta-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-beta-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the beta4 and beta7 strands, respectively. The 1.45-A crystal structure reveals that BCman is a typical (beta/alpha)(8) folding type. One large difference from the saddle-shaped active center of other endo-beta-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and -1) within the active site of BCman. These two sites are involved in the enzymes mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzymes stability.

Crystal Structure of R-Phycocyanin and Possible Energy Transfer Pathways in the Phycobilisome

The crystal structure of R-phycocyanin from Polysiphonia urceolata (R-PC-PU) at 2.4 A is reported. The R-PC-PU crystal belongs to space group P4(3)2(1)2 with cell parameters a = 135.1 A, c = 210.0 A, and alpha = beta = gamma = 90 degrees. The structure was determined by molecular replacement. The crystallographic R-factor of the refined model is 0.189 (R(free) = 0.239). Comparison of the microenvironment of chromophore beta 155 in R-PC-PU and in C-PC from Fremyolla diphosiphon (C-PC-FD) reveals that their spectral differences may be caused by their different alpha 28 residues. In the R-PC-PU crystal structure, two (alpha beta)(3) trimers assemble face to face to form a hexamer, and two such hexamers assemble in two novel side-to-side arrangements. Possible models for the energy transfer from phycoerythrin to phycocyanin and from phycocyanin to allophycocyanin are proposed based on several phycobiliprotein crystal structures.

Structure of C-phycocyanin from Spirulina platensis at 2.2 Å resolution: a novel monoclinic crystal form for phycobiliproteins in phycobilisomes

The crystal structure of C-phycocyanin from the cyanobacterium S. platensis has been determined at 2.2 A resolution. The crystals belong to the monoclinic crystal form, which has not been previously reported for phycobiliprotein structures. The structure was solved using the molecular-replacement method with a final R value of 18.9% (R(free) = 23.7%) after model building and refinement. In the crystals used for the study, the C-phycocyanin hexamers formed by face-to-face association of two trimers are arranged in layers rather than in columns. Three different kinds of packing between adjacent hexamers in the layer were compared. The tight packing of two adjacent hexamers formed by four trimers in the asymmetric unit brings beta155 PCB chromophores close together, so it is possible that lateral energy transfer takes place through the beta155-beta155 route.

Structure and function of chromophores in R‐phycoerythrin at 1.9 Å resolution

The crystal structure of R‐Phycoerythrin (R‐PE) from Polysiphonia urceolata has been refined to a resolution of 1.9 Å, based on the atomic coordinates of R‐PE determined at 2.8 Å resolution, through the use of difference Fourier method and steorochemistry parameters restrained refinement with model adjustment according to the electron density map. Crystallographic R‐factor of the refined model is 0.195 (Rfree = 0.282) from 8–1.9 Å. High resolution structure of R‐PE showed precise interactions between the chromophores and protein residues, which explained the spectrum characteristic and function of chromophores. Four chiral atoms of phycourobilin (PUB) were identified as C(4)‐S, C(16)‐S, C(21)‐S, and C(20)‐R. In addition to the coupling distances of 19 Å to 45 Å between the chromophores which were observed and involved in the energy transfer pathway, high resolution structure of R‐PE suggested other pathways of energy transfer, such as the ultrashort distance between α140a and β155. It has been proposed that aromatic residues in linker proteins not only influence the conformation of chromophore, but may also bridge chromophores to improve the energy transfer efficiency. Proteins 1999;34:224–231.

Structural Basis for Thermostability of beta-Glycosidase from the Thermophilic Eubacterium Thermus nonproteolyticus HG102.

The three-dimensional structure of a thermostable beta-glycosidase (Gly(Tn)) from the thermophilic eubacterium Thermus nonproteolyticus HG102 was determined at a resolution of 2.4 A. The core of the structure adopts the (betaalpha)(8) barrel fold. The sequence alignments and the positions of the two Glu residues in the active center indicate that Gly(Tn) belongs to the glycosyl hydrolases of retaining family 1. We have analyzed the structural features of Gly(Tn) related to the thermostability and compared its structure with those of other mesophilic glycosidases from plants, eubacteria, and hyperthermophilic enzymes from archaea. Several possible features contributing to the thermostability of Gly(Tn) were elucidated.

Structure of potato calmodulin PCM6: the first report of the three-dimensional structure of a plant calmodulin

The crystal structure of a potato calmodulin (PCM6) was solved by molecular replacement and refined to a crystallographic R factor of 22.8% (R(free) = 25.0%) using X-ray diffraction data in the resolution range 8.0-2.0 A. This is the first report of the three-dimensional structure of a plant Ca(2+)-calmodulin. PCM6 crystallizes in a crystal form that belongs to space group P2(1)2(1)2(1), which is different to that of most other calmodulin crystals. The main structural difference between PCM6 and the other calmodulins is in the central helix region and appears to be caused by crystal packing. The surface properties of PCM6 molecules were compared with those of animal calmodulins, which provided an explanation for the unique crystal-packing state of PCM6.

Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion‐dependent enzyme: Insight into the catalytic mechanism

Glycerophosphodiester phosphodiesterase (GDPD; EC 3.1.4.46) catalyzes the hydrolysis of a glycerophosphodiester to an alcohol and glycerol 3‐phosphate in glycerol metabolism. It has an important role in the synthesis of a variety of products that participate in many biochemical pathways. We report the crystal structure of the Thermoanaerobacter tengcongensis GDPD (ttGDPD) at 1.91 Å resolution, with a calcium ion and glycerol as a substrate mimic coordinated at this calcium ion (PDB entry 2pz0). The ttGDPD dimer with an intermolecular disulfide bridge and two hydrogen bonds is considered as the potential functional unit. We used site‐directed mutagenesis to characterize ttGDPD as a metal ion‐dependent enzyme, identified a cluster of residues involved in substrate binding and the catalytic reaction, and we propose a possible general acid‐base catalytic mechanism for ttGDPD. Superposing the active site with the homologous structure GDPD from Agrobacterium tumefaciens (PDB entry 1zcc), which binds a sulfate ion in the active site, the sulfate ion can represent the phosphate moiety of the substrate, simulating the binding mode of the true substrate of GDPD. Proteins 2008.

Crystallization and preliminary X-ray analysis of earthworm fibrinolytic enzyme component A from Eisenia fetida.

Earthworm fibrinolytic enzyme component A, a protein which functions both as a direct fibrinolytic enzyme and a plasminogen activator, was purified from the earthworm Eisenia fetida. Diffraction-quality single crystals of the protein were grown by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.6, b = 127.5, c = 129.2 A and three molecules per asymmetric unit. The data set reached a resolution of 1.95 A.