Molecular basis of dual anti-CRISPR and auto-regulatory functions of AcrIF24.

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.

The structure of MucD from Pseudomonas syringae revealed N-terminal loop-mediated trimerization of HtrA-like serine protease.

Protein quality control mechanisms are essential for maintaining cellular integrity, and the HtrA family of serine proteases plays a crucial role in handling folding stress in prokaryotic periplasm. Escherichia coli harbors three HtrA members, namely, DegS, DegP, and DegQ, which share a common domain structure. MucD, a putative HtrA family member that resembles DegP, is involved in alginate biosynthesis regulation and the stress response. Pseudomonas syringae causes plant diseases and opportunistic infections in humans. This study presents the high-resolution structure of MucD from Pseudomonas syringae (psMucD), revealing its composition as a typical HtrA family serine protease with protease and PDZ domains. Its findings suggest that psMucD containing one PDZ domain is a trimer in solution, and psMucD trimerization is mediated by its N-terminal loop. Sequence and structural analyses revealed similarities and differences with other HtrA family members. Additionally, this study provides a model of psMucD's catalytic process, comparing it with other members of the HtrA family of serine proteases.

Molecular basis for SMC rod formation and its dissolution upon DNA binding.

SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive "hinge" dimerization domain with an ATP-regulated "head" dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB's association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc's dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.

Crystal structure of PYCH_01220 from Pyrococcus yayanosii potentially involved in binding nucleic acid.

We report the crystal structure of PYCH_01220, a hypothetical protein in Pyrococcus yayanosii CH1. This protein is composed of two domains, named Domain A and Domain B. While Domain B is not significantly homologous to known protein structures, Domain A is structurally analogous to the C-terminal ribonuclease domain of Escherichia coli colicin D. Domain A has a positively charged surface patch rendered by 13 basic residues, eight arginine or lysine residues of which are evolutionarily conserved. Electrophoretic mobility shift assays showed that PYCH_01220 binds to DNA, and charge-inversion mutations on this patch negatively affect the DNA binding, suggesting that the function of PYCH_01220 might involve nucleic acid-binding via the positively charged patch.

Upgrade of BL-5C as a highly automated macromolecular crystallography beamline at Pohang Light Source II.

BL-5C is an in-vacuum undulator beamline dedicated to macromolecular crystallography (MX) at the 3 GeV Pohang Light Source II in Korea. The beamline delivers X-ray beams with a focal spot size of 200 µm × 40 µm (FWHM, H × V) over the energy range 6.5-16.5 keV. The measured flux is 7 × 1011 photons s-1 at 12.659 keV through an aperture size of 50 µm. The experimental station is newly equipped with the photon-counting detector EIGER 9M, the multi-axis micro-diffractometer MD2, and a robotic sample changer with a high-capacity dewar. These instruments enable the operation of this beamline as an automated MX beamline specialized in X-ray fragment screening. This beamline can collect more than 400 data sets a day without human intervention, and a difference map can be automatically calculated by using the data processing pipeline for ligand or fragment identification.

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.

A 1.3 Å high-resolution crystal structure of an anti-CRISPR protein, AcrI E2.

As a result of bacterial infection with viruses, bacteria have developed CRISPR-Cas as an adaptive immune system, which allows them to destroy the viral genetic material introduced via infection. However, viruses have also evolved to develop multiple anti-CRISPR proteins, which are capable of inactivating the CRISPR-Cas adaptive immune system to combat bacteria. In this study, we aimed to elucidate the molecular mechanisms associated with anti-CRISPR proteins by determining a high-resolution crystal structure (1.3 Å) of Type I-E anti-CRISPR protein called AcrIE2. Our structural analysis revealed that AcrIE2 was composed of unique folds comprising five antiparallel ß-sheets (ß1∼ß5) surrounding one α-helix (α1) in the order, ß2ß1α1ß5ß4ß3. Structural comparison of AcrIE2 with a structural homolog called AcrIF9 showed that AcrIE2 contained a long and flexible ß4-ß5 connecting loop and a distinct surface feature. These results indicated that the inhibitory mechanism of AcrIE2 might be different from that of AcrIF9. This unique structure of AcrIE2 indicates its special mode of CRISPR-Cas inhibitory activity. Therefore, this study helps us understand the diversity in the inhibitory mechanisms of Acr family.

SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling.

Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.

CIDE domains form functionally important higher-order assemblies for DNA fragmentation.

Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a family with diverse functions ranging from cell death to lipid homeostasis. Here we show that the CIDE domains of Drosophila and human apoptotic nucleases Drep2, Drep4, and DFF40 all form head-to-tail helical filaments. Opposing positively and negatively charged interfaces mediate the helical structures, and mutations on these surfaces abolish nuclease activation for apoptotic DNA fragmentation. Conserved filamentous structures are observed in CIDE family members involved in lipid homeostasis, and mutations on the charged interfaces compromise lipid droplet fusion, suggesting that CIDE domains represent a scaffold for higher-order assembly in DNA fragmentation and other biological processes such as lipid homeostasis.

Structural Basis for Broad Substrate Selectivity of Alcohol Dehydrogenase YjgB from Escherichia coli.

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π-π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3.

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.

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 Structures of Penicillin-Binding Protein D2 from Listeria monocytogenes and Structural Basis for Antibiotic Specificity.

ß-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of ß-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the ß-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of ß-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important ß-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of ß-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against ß-lactam-resistant PBPs.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

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 dual nucleotide exchange and GDI displacement activity of SidM/DrrA.

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber.

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine-lysine catalytic dyad. We report the 2.0-Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight- and weak-binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP-driven protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.

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.

Crystal structure of the Gtr1p(GTP)-Gtr2p(GDP) protein complex reveals large structural rearrangements triggered by GTP-to-GDP conversion.

The heterodimeric Rag GTPases consisting of RagA (or RagB) and RagC (or RagD) are the key regulator activating the target of rapamycin complex 1 (TORC1) in response to the level of amino acids. The heterodimer between GTP-loaded RagA/B and GDP-loaded RagC/D is the most active form that binds Raptor and leads to the activation of TORC1. Here, we present the crystal structure of Gtr1p(GTP)-Gtr2p(GDP), the active yeast Rag GTPase heterodimer. The structure reveals that GTP-to-GDP conversion on Gtr2p results in a large conformational transition of this subunit, including a large scale rearrangement of a long segment whose corresponding region in RagA is involved in binding to Raptor. In addition, the two GTPase domains of the heterodimer are brought to contact with each other, but without causing any conformational change of the Gtr1p subunit. These features explain how the nucleotide-bound statuses of the two GTPases subunits switch the Raptor binding affinity on and off.

Crystal structure and nucleic acid-binding activity of the CRISPR-associated protein Csx1 of Pyrococcus furiosus.

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C-terminal domain of PF1127 has a unique ß-hairpin structure that protrudes out of an α-helix and contains several positively charged residues. We demonstrate that PF1127 binds double-stranded DNA and RNA and that this activity requires an intact ß-hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the ß-hairpin structure and exhibits no DNA-binding properties under the same experimental conditions. Notably, the C-terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N-terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid-binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities.