BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

Sequence preference and structural heterogeneity of BZ junctions.

BZ junctions, which connect B-DNA to Z-DNA, are necessary for local transformation of B-DNA to Z-DNA in the genome. However, the limited information on the junction-forming sequences and junction structures has led to a lack of understanding of the structural diversity and sequence preferences of BZ junctions. We determined three crystal structures of BZ junctions with diverse sequences followed by spectroscopic validation of DNA conformation. The structural features of the BZ junctions were well conserved regardless of sequences via the continuous base stacking through B-to-Z DNA with A-T base extrusion. However, the sequence-dependent structural heterogeneity of the junctions was also observed in base step parameters that are correlated with steric constraints imposed during Z-DNA formation. Further, circular dichroism and fluorescence-based analysis of BZ junctions revealed that a base extrusion was only found at the A-T base pair present next to a stable dinucleotide Z-DNA unit. Our findings suggest that Z-DNA formation in the genome is influenced by the sequence preference for BZ junctions.

Crystal structure of Arabidopsis thaliana RabA1a.

RabGTPase is a member of the Ras superfamily of small GTPases, which share a GTP-binding pocket containing highly conserved motifs that promote GTP hydrolysis. In Arabidopsis, the RabA group, which corresponds to the Rab11 group in animals, functions in the recycling of endosomes that control docking and fusion during vesicle transport. However, their molecular mechanisms remain unknown. In this study, we determined the crystal structures of the GDP-bound inactive form and both GppNHp- and GTP-bound active forms of RabA1a, at resolutions of 2.8, 2.6, and 2.6 Å, respectively. A bound sulfate ion in the active site of the GDP-bound structure stabilized Switch II by bridging the interaction between a magnesium ion and Arg74. Comparisons of the two states of RabA1a with Rab11 proteins revealed clear differences in the Switch I and II loops. These results suggested that conformational change of the Switch regions of RabA1a, derived by GTP or GDP binding, could maintain subcellular membrane traffic through the specific interaction of effector molecules.

Crystal Structure of NisI in a Lipid-Free Form, the Nisin Immunity Protein, from Lactococcus lactis.

Nisin is a lantibiotic, a member of a family of polypeptides containing lanthionine with antimicrobial activity. Nisin-producing microorganisms require immunity proteins for self-protection from nisin itself. Lactococcus lactis, a microorganism that synthesizes nisin, has an integral NisFEG ABC transporter and an NisI lipoprotein that function in nisin immunity. Here, we present the crystal structure of the full length of NisI22-C, a lipid-free form of NisI, determined at 1.9-Å resolution. As with the nuclear magnetic resonance (NMR) structures of the N- and C-terminal domains of NisI, NisI22-C is composed of N- and C-terminal domains, both of which display a fold similar to that found in SpaI, a lipoprotein with immunity against subtilin in Bacillus subtilis The full-length structure of NisI22-c reveals a large, deep cleft by the interdomain association, one side of which is occupied by the residues important for immunity. Opposite the cleft, a shallow groove is found where nisin-interacting residues are distributed in the periphery composed of the C-terminal negative patch. Based on a sulfate ion found in the large and deep cleft, a model of NisI in complex with a farnesyl diphosphate backbone of lipid II is proposed, suggesting a mechanism for increasing the chances of encountering nisin.

Structural basis of the cystein protease inhibitor Clonorchis sinensis Stefin-1.

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

Structural Basis for Carbohydrate Recognition and Anti-inflammatory Modulation by Gastrointestinal Nematode Parasite Toxascaris leonina Galectin.

Toxascaris leonina galectin (Tl-gal) is a galectin-9 homologue protein isolated from an adult worm of the canine gastrointestinal nematode parasite, and Tl-gal-vaccinated challenge can inhibit inflammation in inflammatory bowel disease-induced mice. We determined the first X-ray structures of full-length Tl-gal complexes with carbohydrates (lactose, N-acetyllactosamine, lacto-N-tetraose, sialyllactose, and glucose). Bonds were formed on concave surfaces of both carbohydrate recognition domains (CRDs) in Tl-gal. All binding sites were found in the HXXXR and WGXEER motifs. Charged Arg61/Arg196 and Glu80/Glu215 on the conserved motif of Tl-gal N-terminal CRD and C-terminal CRD are critical amino acids for recognizing carbohydrate binding, and the residues can affect protein folding and structure. The polar amino acids His, Asn, and Trp are also important residues for the interaction with carbohydrates through hydrogen bonding. Hemagglutination activities of Tl-gal were inhibited by interactions with carbohydrates and mutations. We found that the mutation of Tl-gal (E80A/E215A) at the carbohydrate binding region induced protein aggregation and could be caused in many diseases. The short linker region between the N-terminal and C-terminal CRDs of Tl-gal was very stable against proteolysis and maintained its biological activity. This structural information is expected to elucidate the carbohydrate recognition mechanism of Tl-gal and improve our understanding of anti-inflammatory mediators and modulators of immune response.

Structural and functional study of ChuY from Escherichia coli strain CFT073.

The uropathogenic Escherichia coli strain CFT073 contains multiple iron and heme transport systems, which facilitate infection of the host urinary tract. To elucidate the molecular and cellular function of ChuY, a hypothetical gene in the heme degradation/utilization pathway, we solved the crystal structure of ChuY at 2.4 Å resolution. ChuY has high structural homology with human biliverdin and flavin reductase. We confirmed that ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. A chuY deletion-insertion strain showed reduced survival potential compared to wild-type and complemented strains in mammalian cells. Current results suggest ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of E. coli CFT073.

Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ).

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the ß-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the ß-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.

Structure of the hypothetical protein Ton1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left-handed helical turn in normal α-solenoid protein.

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa.

Structural insights into the histidine trimethylation activity of EgtD from Mycobacterium smegmatis.

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.

Structural and kinetic bases for the metal preference of the M18 aminopeptidase from Pseudomonas aeruginosa.

The peptidases in clan MH are known as cocatalytic zinc peptidases that have two zinc ions in the active site, but their metal preference has not been rigorously investigated. In this study, the molecular basis for metal preference is provided from the structural and biochemical analyses. Kinetic studies of Pseudomonas aeruginosa aspartyl aminopeptidase (PaAP) which belongs to peptidase family M18 in clan MH revealed that its peptidase activity is dependent on Co(2+) rather than Zn(2+): the kcat (s(-1)) values of PaAP were 0.006, 5.10 and 0.43 in no-metal, Co(2+), and Zn(2+)conditions, respectively. Consistently, addition of low concentrations of Co(2+) to PaAP previously saturated with Zn(2+) greatly enhanced the enzymatic activity, suggesting that Co(2+)may be the physiologically relevant cocatalytic metal ion of PaAP. The crystal structures of PaAP complexes with Co(2+) or Zn(2+) commonly showed two metal ions in the active site coordinated with three conserved residues and a bicarbonate ion in a tetragonal geometry. However, Co(2+)- and Zn(2+)-bound structures showed no noticeable alterations relevant to differential effects of metal species, except the relative orientation of Glu-265, a general base in the active site. The characterization of mutant PaAP revealed that the first metal binding site is primarily responsible for metal preference. Similar to PaAP, Streptococcus pneumonia glutamyl aminopeptidase (SpGP), belonging to aminopeptidase family M42 in clan MH, also showed requirement for Co(2+) for maximum activity. These results proposed that clan MH peptidases might be a cocatalytic cobalt peptidase rather than a zinc-dependent peptidase.

Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a potential redox-regulated function.

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.

Crystal structures of bifunctional penicillin-binding protein 4 from Listeria monocytogenes.

Penicillin-binding proteins (PBPs), which catalyze the biosynthesis of the peptidoglycan chain of the bacterial cell wall, are the major molecular target of bacterial antibiotics. Here, we present the crystal structures of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase (TP) PBP4 from Listeria monocytogenes in the apo-form and covalently linked to two ß-lactam antibiotics, ampicillin and carbenicillin. The orientation of the TP domain with respect to the GT domain is distinct from that observed in the previously reported structures of bifunctional PBPs, suggesting interdomain flexibility. In this structure, the active site of the GT domain is occluded by the close apposition of the linker domain, which supports the hypothesis that interdomain flexibility is related to the regulation of GT activity. The acylated structures reveal the mode of action of ß-lactam antibiotics toward the class A PBP4 from the human pathogen L. monocytogenes. Ampicillin and carbenicillin can access the active site and be acylated without requiring a structural rearrangement. In addition, the active site of the TP domain in the apo-form is occupied by the tartrate molecule via extensive hydrogen bond interactions with the catalytically important residues; thus, derivatives of the tartrate molecule may be useful in the search for new antibiotics to inhibit PBPs.

Structural basis for the toxic activity of MafB2 from maf genomic island 2 (MGI-2) in N. meningitidis B16B6.

The Maf polymorphic toxin system is involved in conflict between strains found in pathogenic Neisseria species such as Neisseria meningitidis and Neisseria gonorrhoeae. The genes encoding the Maf polymorphic toxin system are found in specific genomic islands called maf genomic islands (MGIs). In the MGIs, the MafB and MafI encode toxin and immunity proteins, respectively. Although the C-terminal region of MafB (MafB-CT) is specific for toxic activity, the underlying enzymatic activity that renders MafB-CT toxic is unknown in many MafB proteins due to lack of homology with domain of known function. Here we present the crystal structure of the MafB2-CTMGI-2B16B6/MafI2MGI-2B16B6 complex from N. meningitidis B16B6. MafB2-CTMGI-2B16B6 displays an RNase A fold similar to mouse RNase 1, although the sequence identity is only ~ 14.0%. MafB2-CTMGI-2B16B6 forms a 1:1 complex with MafI2MGI-2B16B6 with a Kd value of ~ 40 nM. The complementary charge interaction of MafI2MGI-2B16B6 with the substrate binding surface of MafB2-CTMGI-2B16B6 suggests that MafI2MGI-2B16B6 inhibits MafB2-CTMGI-2B16B6 by blocking access of RNA to the catalytic site. An in vitro enzymatic assay showed that MafB2-CTMGI-2B16B6 has ribonuclease activity. Mutagenesis and cell toxicity assays demonstrated that His335, His402 and His409 are important for the toxic activity of MafB2-CTMGI-2B16B6, suggesting that these residues are critical for its ribonuclease activity. These data provide structural and biochemical evidence that the origin of the toxic activity of MafB2MGI-2B16B6 is the enzymatic activity degrading ribonucleotides.

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40.

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed ß-propeller catalytic domain. The structure of the enzyme-ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.

Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases.

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B-Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B-Z junction as revealed by X-ray crystallography at 2.6 A resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA.

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZbeta(DAI)) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZbeta(DAI)/Z-DNA complex reveals that hZbeta(DAI) has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first beta-strand rather than residues in the beta-loop contributes to DNA binding, and part of the (alpha3) recognition helix adopts a 3(10) helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.

Elucidation of the electron transfer environment in the MMOR FAD-binding domain from Methylosinus sporium 5.

By facilitating electron transfer to the hydroxylase diiron center, MMOR-a reductase-serves as an essential component of the catalytic cycle of soluble methane monooxygenase. Here, the X-ray structure analysis of the FAD-binding domain of MMOR identified crucial residues and its influence on the catalytic cycle.

A H-REV107 Peptide Inhibits Tumor Growth and Interacts Directly with Oncogenic KRAS Mutants.

Kirsten-RAS (KRAS) has been the target of drugs because it is the most mutated gene in human cancers. Because of the low affinity of drugs for KRAS mutations, it was difficult to target these tumor genes directly. We found a direct interaction between KRAS G12V and tumor suppressor novel H-REV107 peptide with high binding affinity. We report the first crystal structure of an oncogenic mutant, KRAS G12V-H-REV107. This peptide was shown to interact with KRAS G12V in the guanosine diphosphate (GDP)-bound inactive state and to form a stable complex, blocking the activation function of KRAS. We showed that the peptide acted as an inhibitor of mutant KRAS targets by [α-32P] guanosine triphosphate (GTP) binding assay. The H-REV107 peptide inhibited pancreatic cancer and colon cancer cell lines in cell proliferation assay. Specially, the H-REV107 peptide can suppress pancreatic tumor growth by reduction of tumor volume and weight in xenotransplantation mouse models. Overall, the results presented herein will facilitate development of novel drugs for inhibition of KRAS mutations in cancer patients.