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The endo-β-1,4-xylanase glycosyl hydrolase (GH11) decomposes the backbone of xylan into xylooligosaccharides or xylose. These enzymes are important for industrial applications in the production of biofuel, feed, food, and value-added materials. β-D-xylopyranose (XYP, also known as β-D-xylose) is the fundamental unit of the substrate xylan, and understanding its recognition is fundamental for the initial steps of GH11's molecular mechanism. However, little is known about the recognition of a single XYP molecule by GH11. In this study, the crystal structures of GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) complexed with an XYP molecule were determined at a resolution of 1.7–1.9 Å. The XYP molecule binds to subsite −2 of the substrate-binding cleft. The XYP molecule is mainly stabilized by a π–π interaction with the conserved Trp36 residue. The O2 and O3 atoms of XYP are stabilized by hydrogen bond interactions with the hydroxyl groups of Tyr96 and Tyr192. The conformation of the thumb domain of TsaGH11 does not play a critical role in XYP binding, and XYP binding induces a shift in the thumb domain of TsaGH11 toward the XYP molecule. A structural comparison of TsaGH11 with other GH11 xylanases revealed that the XYP molecule forms π–π stacking with the center between the phenyl and indoline ring of Trp36, whereas the XYP molecule unit from xylobiose or xylotetraose forms π–π stacking with the indoline of Trp36, which indicates that the binding modes of the substrate and XYP differ. These structural results provide a greater understanding of the recognition of XYP by the GH11 family. [ABSTRACT FROM AUTHOR]

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. [ABSTRACT FROM AUTHOR]

The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed. [ABSTRACT FROM AUTHOR]

Radiation damage is an inherent challenge in macromolecular crystallography (MX). This diminishes the diffraction quality and also compromises the accuracy of the crystal structure. Investigating the impact of radiation damage on the crystal quality and structure can offer valuable insights into the structural interpretation and data collection strategy. Selenomethionine (SeMet, Mse) is an amino acid that exists in nature and contains a high-Z atom, i.e., selenium (Se), which is sensitive to radiation damage; however, little is known regarding the radiation damage of this amino acid. To better understand the radiation damage that affects SeMet, we investigated the radiation damage to a SeMet-substituted substrate-binding protein from Rhodothermus marinus. As the X-ray dose increased, the quality of the data statistics deteriorated. In particular, an increase in the X-ray dose increased the negative Fo-Fc electron density map near the Se atom of the Mse residue, while no negative Fo-Fc electron density map was observed in the other atoms (O, C, and N). Radiation damage increased the absolute B-factor value of the Se atom in the Mse residue, which was higher than that of the other atoms. This indicates that Se is more sensitive to radiation damage than other atoms. These results will contribute to advancing our knowledge of the radiation damage that can occur in MX. [ABSTRACT FROM AUTHOR]

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-β-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg−1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL−1 and 34,015.3 s−1, respectively, resulting in kcat/Km value of 2658.7 mL mg−1 s−1, higher by 102–103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a β-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation. [ABSTRACT FROM AUTHOR]

Endo-1,4-β-xylanases belonging to the glycoside hydrolase (GH) 11 family hydrolyze the β-1,4-glycosidic linkages in the xylan backbone to convert polymeric xylan into xylooligosaccharides. GH11 xylanases play an essential role in sugar metabolism and are one of the most widely used enzymes in various industries, such as pulp and paper, food and feed, biorefinery, textile, and pharmaceutical industries. pH is a crucial factor influencing the biochemical properties of GH11 xylanase and its application in bioprocessing. For the optimal pH shifting of GH11 xylanase in industrial applications, various protein engineering studies using directed evolution, rational engineering, and in silico approaches have been adopted. Here, we review the functions, structures, and engineering methods developed for the optimal pH shifting of GH11 xylanases. The various GH11 engineering techniques and key residues involved in pH shifting are discussed based on their crystal and modeled structure. This review provides an overview of recent advancements in the characterization and engineering of GH11 xylanases, providing a guide for future research in this field. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) enables the determination of the structure of macromolecules or small molecules with minimal radiation damage. In particular, biomolecule structures determined using the SX technique have the advantage of providing room-temperature crystal structures with high biological relevance. The SX technique requires numerous crystals to be collected to complete three-dimensional structural information. To minimize crystal sample consumption, we introduced SX data collection with fixed-target (FT) pink-beam serial synchrotron crystallography (SSX) at the 1C beamline of Pohang Light Source II. A new sample holder consisting of a magnetic frame with a nylon mesh was developed for easy sample handling. The FT-pink-SSX diffraction data were collected by continuously scanning X-rays using a stepping motor. The room-temperature structures of glucose isomerase and lysozyme were successfully determined at a resolution of 1.7 and 2.2 Å, respectively. The use of pink-beam FT-SSX in experimental applications and data acquisition for large beam sizes is discussed. Our results provide useful information for future pink-beam SSX and SX data collection using large X-ray beams. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family. [ABSTRACT FROM AUTHOR]

Human lysozyme is a natural non-specific immune protein that participates in the immune response of infants against bacterial and viral infections. Lysozyme is a well-known hydrolase that cleaves peptidoglycan in bacterial cell walls. Several crystal structures of human lysozyme have been reported, but little is known regarding how it recognizes sugar molecules. In this study, the crystal structures of human lysozyme in its native and two N-acetyl-α-d-glucosamine (α-D-NAG)-bound forms were determined at 1.3 Å and 1.55/1.60 Å resolution, respectively. Human lysozyme formed a typical c-type lysozyme fold and the α-D-NAG molecule was bound to the middle of subsites C and D. The N-acetyl and glucosamine groups of α-D-NAG were stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges. Conformational changes of Arg80, Tyr81, Val128, and Arg131 of human lysozyme were observed due to the interactions of α-D-NAG with the active-site cleft. The binding configuration of α-D-NAG in human lysozyme was distinct compared with that of other sugar-bound lysozymes. Findings from this structural analysis provide a better understanding of the sugar recognition of human lysozyme during the immune response to microbial pathogens. [ABSTRACT FROM AUTHOR]

Sensitive and accurate detection of specific metal ions is important for sensor development and can advance analytical science and support environmental and human medical examinations. Fluorescent proteins (FPs) can be quenched by specific metal ions and spectroscopically show a unique fluorescence-quenching sensitivity, suggesting their potential application as FP-based metal biosensors. Since the characteristics of the fluorescence quenching are difficult to predict, spectroscopic analysis of new FPs is important for the development of FP-based biosensors. Here we reported the spectroscopic and structural analysis of metal-induced fluorescence quenching of the photoconvertible fluorescent protein DendFP. The spectroscopic analysis showed that Fe2+, Fe3+, and Cu2+ significantly reduced the fluorescence emission of DendFP. The metal titration experiments showed that the dissociation constants (Kd) of Fe2+, Fe3+, and Cu2+ for DendFP were 24.59, 41.66, and 137.18 μM, respectively. The tetrameric interface of DendFP, which the metal ions cannot bind to, was analyzed. Structural comparison of the metal-binding sites of DendFP with those of iq-mEmerald and Dronpa suggested that quenchable DendFP has a unique metal-binding site on the β-barrel that does not utilize the histidine pair for metal binding. [ABSTRACT FROM AUTHOR]

Serial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments. [ABSTRACT FROM AUTHOR]

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X‐ray beam position in a stable manner. Using this system, the room‐temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Å and 1.75 Å, respectively, were determined. This straightforward syringe pump‐based sample delivery system can be utilized in SX. [ABSTRACT FROM AUTHOR]

Many fluorescent proteins ( FPs) show fluorescence quenching by specific metal ions, which can be applied towards metal biosensor development. In this study, we investigated the significant fluorescence quenching of Dronpa by Co2+ and Cu2+ ions. Crystal structures of Co2+-, Ni2+- and Cu2+ -bound Dronpa revealed previously unseen, unique, metal-binding sites for fluorescence quenching. These metal ions commonly interact with surface-exposed histidine residues (His194-His210 and His210-His212), and interact indirectly with chromophores. Structural analysis of the Co2+- and Cu2+- binding sites of Dronpa provides insight into FP-based metal biosensor engineering. [ABSTRACT FROM AUTHOR]

Site-specific Xer recombination plays a pivotal role in reshuffling genetic information. Here, we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C-shaped clamp with an angle of ~ 48° and a distance of 57 Å between the core-binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage mode by XerA's C-term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase. [ABSTRACT FROM AUTHOR]

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3′-to-5′ ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca2+ was located at strategic positions in the oligomerization interface. We further showed that removal of Ca2+ ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca2+ ions. [ABSTRACT FROM AUTHOR]

Cryocrystallography is a widely used method for determining the crystal structure of macromolecules. This technique uses a cryoenvironment, which significantly reduces the radiation damage to the crystals and has the advantage of requiring only one crystal for structural determination. In standard cryocrystallography, a single crystal is used for collecting diffraction data, which include single-crystal diffraction patterns. However, the X-ray data recorded often may contain diffraction patterns from several crystals. The indexing of multicrystal diffraction patterns in cryocrystallography requires more precise data processing techniques and is therefore time consuming. Here, an approach for processing multicrystal diffraction data using a serial crystallography program is introduced that allows for the integration of multicrystal diffraction patterns from a single image. Multicrystal diffraction data were collected from lysozyme crystals and processed using the serial crystallography program CrystFEL. From 360 images containing multicrystal diffraction patterns, 1138 and 691 crystal lattices could be obtained using the XGANDALF and MOSFLM indexing algorithms, respectively. Using this indexed multi-lattice information, the crystal structure of the lysozyme could be determined successfully at a resolution of 1.9 Å. Therefore, the proposed approach, which is based on serial crystallography, is suitable for processing multicrystal diffraction data in cryocrystallography. [ABSTRACT FROM AUTHOR]

Abstract: Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2Å; the ADP–Mg2+, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP–Mg2+-complex at 2.0Å. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP–Mg2+-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP–Mg2+ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK. [Copyright &y& Elsevier]

Serial crystallography (SX) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment. [ABSTRACT FROM AUTHOR]

Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu2+. During Cu2+-induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors. [ABSTRACT FROM AUTHOR]

Various peptides or non-structural amino acids are recognized by their specific target proteins, and perform a biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein–peptide complexes. To obtain structural information on the substrate recognition of the peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as the main-chain of the peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at a 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of the main-chain. One of the triglycine-binding conformations was identical to the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application in X-ray crystallography in order to identify the substrate recognition sites in the peptide binding enzymes. [ABSTRACT FROM AUTHOR]

X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX. [ABSTRACT FROM AUTHOR]