Direct phasing algorithm for protein crystals with high solvent content using low-resolution diffraction data.

Over the past decade, iterative projection algorithms, an effective approach to recovering phases from a single intensity measurement, have found application in protein crystallography to directly surmount the `phase problem'. However, previous studies have always assumed that some prior knowledge constraints (i.e. a low-resolution envelope about the protein structure in the crystal cell or histogram matching requiring a similar density distribution to the target crystal) must be known for successful phase retrieval, thus hindering its widespread application. In this study, a novel phase-retrieval workflow is proposed that eliminates the need for a reference density distribution by utilizing low-resolution diffraction data in phasing algorithms. The approach involves randomly assigning one out of 12 possible phases at 30° intervals (or two for centric reflections) to produce an initial envelope, which is then refined through density modification after each run of phase retrieval. To evaluate the success of the phase-retrieval procedure, information entropy is introduced as a new metric. This approach was validated using ten protein structures with high solvent content, demonstrating its effectiveness and robustness.

Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.

Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3Cpro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3Cpro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3Cpro. Our lipid-binding assays showed that SVA 3Cpro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3Cpro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3Cpro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3Cpro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3Cpro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3Cpro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme's proteolytic activity during infection.

Structural basis for the transformation of the traditional medicine berberine by bacterial nitroreductase.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB-BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Šfrom the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure-function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

Morinda officinalis oligosaccharides increase serotonin in the brain and ameliorate depression via promoting 5-hydroxytryptophan production in the gut microbiota.

Morinda officinalis oligosaccharides (MOO) are an oral drug approved in China for the treatment of depression in China. However, MOO is hardly absorbed so that their anti-depressant mechanism has not been elucidated. Here, we show that oral MOO acted on tryptophan â†’ 5-hydroxytryptophan (5-HTP) â†’ serotonin (5-HT) metabolic pathway in the gut microbiota. MOO could increase tryptophan hydroxylase levels in the gut microbiota which accelerated 5-HTP production from tryptophan; meanwhile, MOO inhibited 5-hydroxytryptophan decarboxylase activity, thus reduced 5-HT generation, and accumulated 5-HTP. The raised 5-HTP from the gut microbiota was absorbed to the blood, and then passed across the blood-brain barrier to improve 5-HT levels in the brain. Additionally, pentasaccharide, as one of the main components in MOO, exerted the significant anti-depressant effect through a mechanism identical to that of MOO. This study reveals for the first time that MOO can alleviate depression via increasing 5-HTP in the gut microbiota.

Gut dysbacteriosis attenuates resistance to Mycobacterium bovis infection by decreasing cyclooxygenase 2 to inhibit endoplasmic reticulum stress.

The role of gut microbiota has been described as an important influencer of the immune system. Gut-lung axis is critical in the prevention of mycobacterium infection, but the specific mechanism, by which dysbiosis affects tuberculosis, has not been reported. In this study, we attempted to provide more information on how the gut-lung axis contributes to Mycobacterium bovis (M. bovis) infection. Mice are pre-treated with broad-spectrum antibiotics cocktail (Abx) to induce gut dysbiosis. Interestingly, dysbiosis of microbes showed a significant increase in the bacterial burden in the lungs and inhibited the level of COX-2. After faecal transplantation, cyclooxygenase 2 (COX-2) expression was restored and the inflammatory lesion in the lungs was reduced. Further research found that the deficiency of COX-2 inhibited endoplasmic reticulum stress (ER stress). This mechanism was completed by COX-2 interaction with BIP. Moreover, we found a positive feedback mechanism by which blocking ER stress could reduce COX-2 levels by the NF-κB pathway. Taken together, we reveal for the first time gut dysbacteriosis exacerbates M. bovis disease by limiting the COX-2/ER stress pathway. The finding strengthens the foundation of gut microbiota-targeted therapy for tuberculosis treatment.

Structural basis of a multi-functional deaminase in chlorovirus PBCV-1.

2-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The large double-stranded DNA-containing chlorovirus PBCV-1 encodes a dCD family enzyme PBCV1dCD that was reported to be able to deaminize both dCMP and dCTP, which makes PBCV1dCD unique in the dCD family proteins. In this study, we report the crystal structure of PBCV1dCD in complex with dCTP/dCMP and dTTP/dTMP, respectively. We further proved the ability of PBCV1dCD in the deamination of dCDP, which makes PBCV1dCD a multi-functional deaminase. The structural basis for the versatility of PBCV1dCD is analyzed and discussed, with the finding of a unique Trp121 residue key to the deamination and substrate binding ability. Our findings may broaden the understanding of dCD family proteins and provide novel insights into the multi-functional enzyme.

Mamba: a systematic software solution for beamline experiments at HEPS.

To cater for the diverse experiment requirements at the High Energy Photon Source (HEPS) with often limited human resources, Bluesky was chosen as the basis for our software framework, Mamba. In our attempt to address Bluesky's lack of integrated graphical user interfaces (GUIs), command injection with feedback was chosen as the main way for the GUIs to cooperate with the command line interface; a remote-procedure-call service is also provided, which covers functionalities unsuitable for command injection, as well as pushing of status updates. In order to fully support high-frequency applications like fly scans, Bluesky's support for asynchronous control is being improved; furthermore, to support high-throughput experiments, Mamba Data Worker is being developed to cover the complexity in asynchronous online data processing for these experiments. To systematically simplify the specification of metadata, scan parameters and data-processing graphs for each type of experiment, an experiment parameter generator will be developed; experiment-specific modules to automate preparation steps will also be made. The integration of off-the-shelf code in Mamba for domain-specific needs is under investigation, and Mamba GUI Studio is being developed to simplify the implementation and integration of GUIs.

Insights into the Neutralization and DNA Binding of Toxin-Antitoxin System ParESO-CopASO by Structure-Function Studies.

ParESO-CopASO is a new type II toxin-antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParESO severely inhibits cell growth, while CopASO functions as an antitoxin to neutralize ParESO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParESO-CopASO complex that adopted an open V-shaped heterotetramer with the organization of ParESO-(CopASO)2-ParESO. The structure showed that upon ParESO binding, the intrinsically disordered C-terminal domain of CopASO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParESO. Thermodynamics analysis showed the DNA-binding affinity of CopASO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParESO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParESO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParESO-CopASO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.

A reference-based multi-lattice indexing method integrating prior information correction and iterative refinement in protein crystallography.

A new multi-lattice indexing method based on the principle of whole-pattern matching given cell dimensions and space-group symmetry is presented for macromolecular crystallography. The proposed method, termed the multi-crystal data processing suite (MCDPS), features a local correction for prior information accompanied by iterative refinement of experimental parameters, both of which are numerically and experimentally demonstrated to be critical for accurately identifying multiple crystal lattices. Further analysis of data reduction and structure determination with conventional single-crystal programs reveals that the processed multi-lattice data sets are comparable in quality to typical single-crystal ones in terms of crystallographic metrics. Importantly, it is confirmed that careful exclusion of overlapping reflections prior to scaling is necessary to guarantee an accurate data reduction result. The potential for multi-lattice indexing in solving the general macroscopic twinning problem is also explored.

Structure and Biochemical Characteristic of the Methyltransferase (MTase) Domain of RNA Capping Enzyme from African Swine Fever Virus.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease and it is urgently needed to develop effective anti-ASFV vaccines and drugs. The process of mRNA 5'-end capping is a common characteristic in eukaryotes and many viruses, and the cap structure is required for mRNA stability and efficient translation. The ASFV protein pNP868R was found to have guanylyltransferase (GTase) activity involved in mRNA capping. Here we report the crystal structure of pNP868R methyltransferase (MTase) domain (referred as pNP868RMT) in complex with S-adenosyl-L-methionine (AdoMet). The structure shows the characteristic core fold of the class I MTase family and the AdoMet is bound in a negative-deep groove. Remarkably, the N-terminal extension of pNP868RMT is ordered and keeps away from the AdoMet-binding site, distinct from the close conformation over the active site of poxvirus RNA capping D1 subunit or the largely disordered conformation in most cellular RNA capping MTases. Structure-based mutagenesis studies based on the pNP868RMT-cap analog complex model revealed essential residues involved in substrate recognition and binding. Functional studies suggest the N-terminal extension may play an essential role in substrate recognition instead of AdoMet-binding. A positively charged path stretching from the N-terminal extension to the region around the active site was suggested to provide a favorable electrostatic environment for the binding and approaching of substrate RNA into the active site. Our structure and biochemical studies provide novel insights into the methyltransfer process of mRNA cap catalyzed by pNP868R.IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic viral disease in pigs that is caused by African swine fever virus (ASFV). There are no effective drugs or vaccines for protection against ASFV infection till now. The protein pNP868R was predicted to be responsible for process of mRNA 5'-end capping in ASFV, which is essential for mRNA stability and efficient translation. Here, we solved the high-resolution crystal structure of the methyltransferase (MTase) domain of pNP868R. The MTase domain structure shows a canonical class I MTase family fold and the AdoMet binds into a negative pocket. Structure-based mutagenesis studies revealed critical and conserved residues involved in AdoMet-binding and substrate RNA-binding. Notably, both the conformation and the role in MTase activities of the N-terminal extension are distinct from those of previously characterized poxvirus MTase domain. Our structure-function studies provide the basis for potential anti-ASFV inhibitor design targeting the critical enzyme.

A micro-focusing and high-flux-throughput beamline design using a bending magnet for microscopic XAFS at the High Energy Photon Source.

An optical design study of a bending-magnet beamline, based on multi-bend achromat storage ring lattices, at the High Energy Photon Source, to be built in Beijing, China, is described. The main purpose of the beamline design is to produce a micro-scale beam from a bending-magnet source with little flux loss through apertures. To maximize the flux of the focal spot, the synchrotron source will be 1:1 imaged to a virtual source by a toroidal mirror; a mirror pair will be used to collimate the virtual source into quasi-parallel light which will be refocused by a Kirkpatrick-Baez mirror pair. In the case presented here, a beamline for tender X-rays ranging from 2.1 keV to 7.8 keV, with a spot size of approximately 7 µm (H) × 6 µm (V) and flux up to 2 × 1012 photons s-1, can be achieved for the purpose of X-ray absorption fine-structure (XAFS)-related experiments, such as scanning micro-XAFS and full-field nano-XAFS.

Conformational changes of antitoxin HigA from Escherichia coli str. K-12 upon binding of its cognate toxin HigB reveal a new regulation mechanism in toxin-antitoxin systems.

HigA functions as the antitoxin in HigB-HigA toxin-antitoxin system. It neutralizes HigB-mediated toxicity by forming a stable toxin-antitoxin complex. Here the crystal structure of isolated HigA from Escherichia coli str. K-12 has been determined to 2.0 Šresolution. The structural differences between HigA and HigA in HigBA complex imply that HigA undergoes drastic conformational changes upon the binding of HigB. The conformational changes are achieved by rigid motions of N-terminal and C-terminal domains of HigA around its central linker domain, which is different from other known forms of regulation patterns in other organisms. As a transcriptional regulator, HigA bind to its operator DNA through the C-terminal HTH motif, in which key residues were identified in this study.

Structural characterization of the Imm52 family protein TsiT in Pseudomonas aeruginosa.

The bacterial type VI secretion system (T6SS) utilizes many toxic effectors to gain advantage over interbacterial competition and eukaryotic host infection. Meanwhile, the cognate immunity proteins of these effectors are employed to protect themselves from the virulence. TseT and TsiT form an effector-immunity (E-I) protein pair secreted by T6SS of Pseudomonas aeruginosa. TseT is toxic for other bacteria, whereas TsiT can suppress the virulence of TseT. Here, we report the crystal structure of TsiT at 1.6 Å resolution. TsiT is a typical α + ß class protein and belongs to a novel Imm52 protein family of the polymorphic toxin system. Apart from TsiT, only one structure of the Imm52 family proteins is present in the Protein Data Bank (PDB), but that structure is not characterized and shares low sequence identity with TsiT. We characterized the basic features of TsiT structure and identified conserved residues of the Imm52 family proteins according to homology comparison. Our work provided structural information of a new protein family and should aid future functional studies.

The crystal structure of KSHV ORF57 reveals dimeric active sites important for protein stability and function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 Å and monomer at 3.0 Å. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167-222 of ORF57-CTD protrudes a long "arm" and holds the globular domains of the neighboring monomer, while the C-terminal residues 445-454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the "arm" region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the "arm" region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases.

Structure-function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN-MNT toxin-antitoxin system.

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Shewanella oneidensis Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite RX4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the RX4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family.

Crystal structure of the putative cytoplasmic protein STM0279 (Hcp2) from Salmonella typhimurium.

STM0279 is a putative cytoplasmic protein from Salmonella typhimurium and was recently renamed haemolysin co-regulated protein 2 (Hcp2), with the neighbouring STM0276 being Hcp1. Both of them are encoded by the type VI secretion system (T6SS) of the Salmonella pathogenicity island 6 (SPI-6) locus and have high sequence identity. The Hcp proteins may function as a vital component of the T6SS nanotube and as a transporter and chaperone of diverse effectors from the bacterial T6SS. In this study, the crystal structure and the oligomeric state in solution of Hcp2 from S. typhimurium (StHcp2) were investigated. The crystal structure refined to 3.0 Šresolution showed that the protein is composed of a ß-barrel domain with extended loops and can form hexameric rings as observed in known Hcp homologues. Mutation of the extended loop was found to partly destabilize the hexameric conformation into monomers or cause the production of inclusion bodies, suggesting it has an important role in hexameric ring formation.

Structural and SAXS analysis of Tle5-Tli5 complex reveals a novel inhibition mechanism of H2-T6SS in Pseudomonas aeruginosa.

Widely spread in Gram-negative bacteria, the type VI secretion system (T6SS) secretes many effector-immunity protein pairs to help the bacteria compete against other prokaryotic rivals, and infect their eukaryotic hosts. Tle5 and Tle5B are two phospholipase effector protein secreted by T6SS of Pseudomonas aeruginosa. They can facilitate the bacterial internalization process into human epithelial cells by interacting with Akt protein of the PI3K-Akt signal pathway. Tli5 and PA5086-5088 are cognate immunity proteins of Tle5 and Tle5B, respectively. They can interact with their cognate effector proteins to suppress their virulence. Here, we report the crystal structure of Tli5 at 2.8Å resolution and successfully fit it into the Small angle X-ray scattering (SAXS) model of the complete Tle5-Tli5 complex. We identified two important motifs in Tli5 through sequence and structural analysis. One is a conserved loop-ß-hairpin motif that exists in the Tle5 immunity homologs, the other is a long and sharp α-α motif that directly interacts with Tle5 according to SAXS data. We also distinguished the structural features of Tle5 and Tle5B family immunity proteins. Together, our work provided insights into a novel inhibition mechanism that may enhance our understanding of phospholipase D family proteins.

Structural analysis of Pseudomonas aeruginosa H3-T6SS immunity proteins.

The Pseudomonas aeruginosa PldB protein is a transkingdom effector secreted by the Type VI Secretion System (T6SS). PA5088, PA5087, and PA5086 are three immunity proteins that can suppress the virulence of PldB. We report the crystal structures of PA5088 and PA5087 at 2.0 and 2.1 Å resolution, respectively. PA5088 and PA5087 both consist of several Sel1-like Repeats (SLRs) and form super-ring folds. Our structural analysis of these proteins revealed key differences among PA5088, PA5087, and their homologs. Our docking experiments have shed light on the putative interaction mechanism of their function as phospholipase D inhibitors.

Structural insights into the inhibition mechanism of bacterial toxin LsoA by bacteriophage antitoxin Dmd.

Bacteria have obtained a variety of resistance mechanisms including toxin-antitoxin (TA) systems against bacteriophages (phages), whereas phages have also evolved to overcome bacterial anti-phage mechanisms. Dmd from T4 phage can suppress the toxicities of homologous toxins LsoA and RnlA from Escherichia coli, representing the first example of a phage antitoxin against multiple bacterial toxins in known TA systems. Here, the crystal structure of LsoA-Dmd complex showed Dmd is inserted into the deep groove between the N-terminal repeated domain (NRD) and the Dmd-binding domain (DBD) of LsoA. The NRD shifts significantly from a 'closed' to an 'open' conformation upon Dmd binding. Site-directed mutagenesis of Dmd revealed the conserved residues (W31 and N40) are necessary for LsoA binding and the toxicity suppression as determined by pull-down and cell toxicity assays. Further mutagenesis identified the conserved Dmd-binding residues (R243, E246 and R305) of LsoA are vital for its toxicity, and suggested Dmd and LsoB may possess different inhibitory mechanisms against LsoA toxicity. Our structure-function studies demonstrate Dmd can recognize LsoA and inhibit its toxicity by occupying the active site possibly via substrate mimicry. These findings have provided unique insights into the defense and counter-defense mechanisms between bacteria and phages in their co-evolution.

Structural basis for the interaction of BamB with the POTRA3-4 domains of BamA.

In Escherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3-4 domains of BamA is reported at 2.1 Šresolution. Based on this structure, the POTRA3 domain is associated with BamB via hydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.