Hong-Hsiang Guan

Crystal structure of a secondary vitamin D3 binding site of milk β-lactoglobulin

β‐lactoglobulin (β‐LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly β structure. The structural function of the only α‐helix with three turns at the C‐terminus is unknown. Vitamin D3 binds to the central calyx formed by the β‐strands. Whether there are two vitamin D binding‐sites in each β‐LG molecule has been a subject of controversy. Here, we report a second vitamin D3 binding site identified by synchrotron X‐ray diffraction (at 2.4 Å resolution). In the central calyx binding mode, the aliphatic tail of vitamin D3 clearly inserts into the binding cavity, where the 3‐OH group of vitamin D3 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 Å). The second binding site, however, is near the surface at the C‐terminus (residues 136–149) containing part of an α‐helix and a β‐strand I with 17.91 Å in length, while the span of vitamin D3 is about 12.51 Å. A remarkable feature of the second exosite is that it combines an amphipathic α‐helix providing nonpolar residues (Phe‐136, Ala‐139, and Leu‐140) and a β‐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 D3 binding. This finding provides a new insight into the interaction between vitamin D3 and β‐LG, in which the exosite may provide another route for the transport of vitamin D3 in vitamin D3 fortified dairy products. Atomic coordinates for the crystal structure of β‐LG‐vitamin D3 complex described in this work have been deposited in the PDB (access code 2GJ5). Proteins 2008.

Crystal structures of Aspergillus japonicus fructosyltransferase complex with donor/acceptor substrates reveal complete subsites in the active site for catalysis.

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade β-propeller fold linked to a C-terminal β-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (−1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the −1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

Structure of Bacillus amyloliquefaciens alpha-amylase at high resolution: implications for thermal stability.

The crystal structure of Bacillus amyloliquefaciens alpha-amylase (BAA) at 1.4 A resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type alpha-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis alpha-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying alpha-amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.

Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman-Birk inhibitor from Oryza sativa

Bowman-Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 A, the unit cell belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 A. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.

Direct phase selection of initial phases from single-wavelength anomalous dispersion (SAD) for the improvement of electron density and ab initio structure determination

A novel direct phase-selection method to select optimized phases from the ambiguous phases of a subset of reflections to replace the corresponding initial SAD phases has been developed. With the improved phases, the completeness of built residues of protein molecules is enhanced for efficient structure determination.

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1–100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

Ab initio phasing by molecular averaging in real space with new criteria: application to structure determination of a betanodavirus

A two-step process of phase determination in the X-ray structural analysis of the coat protein of a betanodavirus is described. A new indicator, the free fraction, for molecular averaging in real space is introduced to effectively evaluate the phasing power in order to enhance the success of determining new structures.

Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa

Rice bifunctional alpha-amylase/subtilisin inhibitor (RASI) can inhibit both alpha-amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 angstroms resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 angstroms. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.

Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2

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 A resolution, the crystal belongs to space group P2(1), with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 A, beta = 99.31 degrees . Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.