Browsing by Author "Guan, Hong-Hsiang"

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Crystal structure of a secondary vitamin D(3) binding site of milk beta-lactoglobulin
(2008-05-15)
Yang, Ming-Chi
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Guan, Hong-Hsiang
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Liu, Ming-Yih
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Lin, Yih-Hung
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Yang, Jinn-Moon
beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 angstrom resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 angstrom). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an a-helix and a beta-strand I with 17.91 angstrom in length, while the span of vitamin D(3) is about 12.51 angstrom. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure Of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).
Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2
(2006-09-01)
Kuo, Chueh-Yuan
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Wu, Yue-Jin
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Hsieh, Yin-Cheng
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Guan, Hong-Hsiang
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Tsai, Huei-Ju
Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 angstrom resolution, the crystal belongs to space group P2(1), with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 angstrom, beta = 99.31 degrees. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.
Rational crystallization of beta -lactoglobulin and vitamin D-3 complex reveal a secondary binding site
(2008-01-01)
Chen, Chun-Jung
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Guan, Hong-Hsiang
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Yang, Ming-Chi
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Moo, Simon J. T.
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生物科技學系
Rational Design for Crystallization of beta-Lactoglobulin and Vitamin D(3) Complex: Revealing a Secondary Binding Site
(2008-12-01)
Yang, Ming Chi
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Guan, Hong-Hsiang
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Yang, Jirin-Moon
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Ko, Cheng-Neng
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Liu, Ming-Yih
beta-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D(3) binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D3 to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D3 in the complex by altering the pH and initial ratios of vitamin D(3)/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D(3)/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D(3) in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.
Structural insights into the histidine-containing phosphotranster protein and receiver domain of sensor histidine kinase suggest a complex model in the two-component regulatory system in Pseudomonas aeruginosa
(2020-09-01)
Chen, Shao-Kang
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Guan, Hong-Hsiang
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Wu, Pei-Hsun
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Lin, Li-Ting
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Wu, Meng-Chun
In Pseudomonas aeruginosa, an important opportunistic pathogen that causes numerous acute and chronic infections, the hybrid two-component system (TCS) regulates the swarming ability and biofilm formation with a multistep phosphorelay, and consists of hybrid-sensor histidine kinase (HK), histidine-containing phosphotransfer protein (Hpt) and response regulator (RR). In this work, two crystal structures of HptB and the receiver domain of HK PA1611 (PA1611REC) of P. aeruginosa have been determined in order to elucidate their interactions for the transfer of the phosphoryl group. The structure of HptB folds into an elongated four-helix bundle - helices alpha 2, alpha 3, alpha 4 and alpha 5, covered by the short N-terminal helix alpha 1. The imidazole side chain of the conserved activesite histidine residue His57, located near the middle of helix alpha 3, protrudes from the bundle and is exposed to solvent. The structure of PA1611REC possesses a conventional (beta/alpha)(5) topology with five-stranded parallel beta-sheets folded in the central region, surrounded by five alpha-helices. The divalent Mg2+ ion is located in the negatively charged active-site cleft and interacts with Asp522, Asp565 and Arg567. The HptB-PA1611REC complex is further modeled to analyze the binding surface and interactions between the two proteins. The model shows a shape complementarity between the convex surface of PA1611REC and the kidney-shaped HptB with fewer residues and a different network involved in interactions compared with other TCS complexes, such as SLN1-R1/YPD1 from Saccharomyces cerevisiae and AHK5(RD)/AHP1 from Arabidopsis thaliana. These structural results provide a better understanding of the TCS in P. aeruginosa and could potentially lead to the discovery of a new treatment for infection.
Structure of a Secondary Vitamin D-3 Binding Site of Milk beta-Lactoglobulin for Vitamin D Transport.
(2009-01-01)
Chen, Chun-Jung
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Yang, Ming-Chi
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Guan, Hong-Hsiang
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Yang, Jinn-Moon
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Chen, Wen-Liang