ABSTRACT
Recent technological breakthroughs in machine-learning-based AlphaFold2 (AF2) are pushing the prediction accuracy of protein structures to an unprecedented level that is on par with experimental structural quality. Despite its outstanding structural modeling capability, further experimental validations and performance assessments of AF2 predictions are still required, thus necessitating the development of integrative structural biology in synergy with both computational and experimental methods. Focusing on the B318L protein that plays an essential role in the African swine fever virus (ASFV) for viral replication, we experimentally demonstrate the high quality of the AF2 predicted model and its practical utility in crystal structural determination. Structural alignment implies that the AF2 model shares nearly the same atomic arrangement as the B318L crystal structure except for some flexible and disordered regions. More importantly, side-chain-based analysis at the individual residue level reveals that AF2's performance is likely dependent on the specific amino acid type and that hydrophobic residues tend to be more accurately predicted by AF2 than hydrophilic residues. Quantitative per-residue RMSD comparisons and further molecular replacement trials suggest that AF2 has a large potential to outperform other computational modeling methods in terms of structural determination. Additionally, it is numerically confirmed that the AF2 model is accurate enough so that it may well potentially withstand experimental data quality to a large extent for structural determination. Finally, an overall structural analysis and molecular docking simulation of the B318L protein are performed. Taken together, our study not only provides new insights into AF2's performance in predicting side-chain conformations but also sheds light upon the significance of AF2 in promoting crystal structural determination, especially when the experimental data quality of the protein crystal is poor.
Subject(s)
African Swine Fever Virus , Amino Acids , Swine , Animals , Molecular Docking Simulation , Furylfuramide , Proteins/chemistry , Protein ConformationABSTRACT
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.
Subject(s)
DCMP Deaminase , Deoxycytidine Monophosphate , Crystallography, X-Ray , DCMP Deaminase/chemistry , DCMP Deaminase/metabolism , Substrate SpecificityABSTRACT
Electron tomography, a powerful imaging tool for studying 3D structures of macromolecular assemblies, always suffers from imperfect reconstruction with limited resolution due to the intrinsic low signal-to-noise ratio (SNR) and inaccessibility to certain tilt angles induced by radiation damage or mechanical limitation. In order to compensate for such insufficient data with low SNR and further improve imaging resolution, prior knowledge constraints about the objects in both real space and reciprocal space are thus exploited during tomographic reconstruction. However, direct Fast Fourier transform (FFT) between real space and reciprocal space remains extraordinarily challenging owing to their inconsistent grid sampling modes, e.g. regular and uniform grid sampling in real space whereas radial or polar grid sampling in reciprocal space. In order to solve such problem, a technique of non-uniform fast Fourier transform (NFFT) has been developed to transform efficiently between non-uniformly sampled grids in real and reciprocal space with sufficient accuracy. In this work, a Non-Uniform fast Fourier transform based Dual-space constraint Iterative reconstruction Method (NUDIM) applicable to biological electron tomography is proposed with a combination of basic concepts from equally sloped tomography (EST) and NFFT based reconstruction. In NUDIM, the use of NFFT can circumvent such grid sampling inconsistency and thus alleviate the stringent equally-sloped sampling requirement in EST reconstruction, while the dual-space constraint iterative procedure can dramatically enhance reconstruction quality. In comparison with conventional reconstruction methods, NUDIM is numerically and experimentally demonstrated to produce superior reconstruction quality with higher contrast, less noise and reduced missing wedge artifacts. More importantly, it is also capable of retrieving part of missing information from a limited number of projections.
Subject(s)
Electron Microscope Tomography , Image Processing, Computer-Assisted , Algorithms , Electron Microscope Tomography/methods , Fourier Analysis , Image Processing, Computer-Assisted/methods , Tomography, X-Ray Computed/methodsABSTRACT
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 2.0â¯Å resolution. 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.
Subject(s)
Escherichia coli K12/metabolism , Escherichia coli Proteins/metabolism , Crystallography, X-Ray , Escherichia coli Infections/microbiology , Escherichia coli K12/chemistry , Escherichia coli Proteins/chemistry , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein MultimerizationABSTRACT
The recently described T6SS (type VI secretion system) acts as a needle that punctures the membrane of the target cells to deliver effector proteins. Type VI amidase effectors can be classified into four divergent families (Tae1-Tae4). These effectors are secreted into the periplasmic space of neighbouring cells via the T6SS and subsequently rupture peptidoglycan. However, the donor cells are protected from damage because of the presence of their cognate immunity proteins [Tai1 (type VI amidase immunity 1)-Tai4]. In the present paper, we describe the structure of Tae3 in complex with Tai3. The Tae3-Tai3 complex exists as a stable heterohexamer, which is composed of two Tae3 molecules and two Tai3 homodimers (Tae3-Tai34-Tae3). Tae3 shares a common NlpC/P60 fold, which consists of N-terminal and C-terminal subdomains. Structural analysis indicates that two unique loops around the catalytic cleft adopt a closed conformation, resulting in a narrow and extended groove involved in the binding of the substrate. The inhibition of Tae3 is attributed to the insertion of the Ω-loop (loop of α3-α4) of Tai3 into the catalytic groove. Furthermore, a cell viability assay confirmed that a conserved motif (Gln-Asp-Xaa) in Tai3 members may play a key role in the inhibition process. Taken together, the present study has revealed a novel inhibition mechanism and provides insights into the role played by T6SS in interspecific competition.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Secretion Systems/immunology , Ralstonia pickettii/immunology , Bacterial Proteins/physiology , Crystallography, X-Ray , Protein Multimerization/immunology , Protein Structure, SecondaryABSTRACT
DrRRA, a vital and recently discovered gene product of Deinococcus radiodurans, is a member of the OmpR/PhoB family of response regulators that couple with the cognate histidine kinase (HK) to form a typical two component system (TCS). It is known that the DrRRA is responsible for the transcriptional levels of numerous genes mostly relating to the stress response and DNA repair. In this paper, the crystal structures of the effector domain and full-length protein of DrRRA with resolutions of 1.6 and 2.3Å, respectively, are determined. These crystal structures depicted that DrRRA has the structural features of the OmpR/PhoB subfamily and were also confirmed by SAXS investigation of the protein in solution. Our data suggest that the receiver domain blocks the binding of DNA to the DNA recognition helix of effector domain; while the interdomain interface would be unwrapped, via the phosphorylation of receiver domain and/or the inducement of DNA, in order to provide DNA binding.
Subject(s)
Bacterial Proteins/chemistry , Deinococcus/metabolism , Trans-Activators/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , Deinococcus/genetics , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Scattering, Small Angle , Trans-Activators/genetics , Trans-Activators/metabolism , X-Ray DiffractionABSTRACT
PldA, a phospholipase D (PLD) effector, catalyzes hydrolysis of the phosphodiester bonds of glycerophospholipids-the main component of cell membranes-and assists the invasion of the opportunistic pathogen Pseudomonas aeruginosa. As a cognate immunity protein, PA3488 can inhibit the activity of PldA to avoid self-toxicity. However, the precise inhibitory mechanism remains elusive. We determine the crystal structures of full-length and truncated PldA and the cryogenic electron microscopy structure of the PldA-PA3488 complex. Structural analysis reveals that there are different intermediates of PldA between the "open" and "closed" states of the catalytic pocket, accompanied by significant conformational changes in the "lid" region and the peripheral helical domain. Through structure-based mutational analysis, we identify the key residues responsible for the enzymatic activity of PldA. Together, these data provide an insight into the molecular mechanisms of PldA invasion and its neutralization by PA3488, aiding future design of PLD-targeted inhibitors and drugs.
Subject(s)
Phospholipase D , Pseudomonas aeruginosa , Bacterial Proteins/metabolism , Glycerophospholipids , Phospholipase D/genetics , Phospholipase D/metabolism , Pseudomonas aeruginosa/metabolismABSTRACT
The bacterial type VI secretion system (T6SS) secretes many toxic effectors to gain advantage in inter-bacterial competition and for eukaryotic host infection. The cognate immunity proteins of these effectors protect bacteria from the virulence of their own effectors. The T6SS injects its inner-needle Hcp tube, the sharpening tip complex -consisting of VgrG and proline-alanine-alanine-arginine repeats (PAAR) proteins- and toxic effectors into neighboring cells. Its functions are largely determined by the activities of its delivered effectors. Five PAAR proteins were found in the Pseudomonas aeruginosa PAO1 genome with three of them shown to facilitate the delivery of various effectors. Here, we report a putative virus-type replication-repair nuclease domain-containing effector TseV encoded by the least investigated P. aeruginosa PAAR2 cluster. The crystal structure of its putative cognate effector TsiV is presented at 1.6 Å resolution. Through structure and sequence comparisons, we propose TseV-TsiV to be a putative novel effector-immunity (E-I) pair and we discuss the roles of other PAAR2 cluster encoded proteins.
Subject(s)
Bacterial Infections/genetics , Bacterial Proteins/genetics , Pseudomonas aeruginosa/genetics , Type VI Secretion Systems/genetics , Amino Acid Sequence/genetics , Bacterial Infections/microbiology , Genome, Bacterial/genetics , Host-Pathogen Interactions/genetics , HumansABSTRACT
The bacterial type VI secretion system (T6SS) is a powerful arsenal that fires many toxic effectors into neighboring cells to gain advantage over inter-bacterial competition and eukaryotic host infection. Meanwhile, the cognate immunity proteins of these effectors are employed to protect themselves from the virulence. TseT-TsiT is a newly discovered effector-immunity (E-I) protein pair secreted by T6SS of Pseudomonas aeruginosa. Our group had reported the crystal structure of TsiT before. Here, we report the crystal structure of P. aeruginosa TseT-TsiT complex at 3.1 Å resolution. The interface of TseT-TsiT is characterized in this work. Through structure and small angle X-ray scattering (SAXS) studies, we discover that the long C-terminal helix of TseT may be flexible. Combining the homolog comparison results, we propose that TseT may form an oligomer in favor of its putative nuclease activity. Although TsiT doesn't directly block the putative active-site of TseT, it may hinder the TseT's oligomerization process to neutralize its virulence.
Subject(s)
Bacterial Proteins/ultrastructure , Pseudomonas aeruginosa/ultrastructure , Type VI Secretion Systems/ultrastructure , Virulence/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Crystallography, X-Ray , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Pseudomonas aeruginosa/pathogenicity , Scattering, Small Angle , Type VI Secretion Systems/chemistry , Type VI Secretion Systems/genetics , X-Ray DiffractionABSTRACT
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.
Subject(s)
Algorithms , Proteins , Crystallography , Macromolecular SubstancesABSTRACT
Orotate phosphoribosyltransferase (OPRTase) catalyzes the OMP-forming step in de novo pyrimidine-nucleotide biosynthesis. Here, the crystal structure of OPRTase from the caries pathogen Streptococcus mutans is reported at 2.4 A resolution. S. mutans OPRTase forms a symmetric dimer and each monomer binds two sulfates at the active sites. The structural symmetry of the sulfate-binding sites and the missing loops in this structure are consistent with a symmetric catalysis mechanism.
Subject(s)
Orotate Phosphoribosyltransferase/chemistry , Streptococcus mutans/enzymology , Binding Sites , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
The bacterial type VI secretion system (T6SS) secretes many toxic effectors to gain advantage in interbacterial competition and for eukaryotic host infection. The cognate immunity proteins of these effectors protect bacteria from their own effectors. PldB is a T6SS trans-kingdom effector in Pseudomonas aeruginosa that can infect both prokaryotic and eukaryotic cells. Three proteins, PA5086, PA5087 and PA5088, are employed to suppress the toxicity of PldB-family proteins. The structures of PA5087 and PA5088 have previously been reported, but the identification of further distinctions between these immunity proteins is needed. Here, the crystal structure of PA5086 is reported at 1.90â Å resolution. A structural comparison of the three PldB immunity proteins showed vast divergences in their electrostatic potential surfaces. This interesting phenomenon provides an explanation of the stockpiling mechanism of T6SS immunity proteins.
Subject(s)
Bacterial Proteins/chemistry , Pseudomonas aeruginosa/chemistry , Type VI Secretion Systems/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Protein Conformation, alpha-Helical/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Static ElectricityABSTRACT
The genome of the thermophilic bacteriophage GVE2 encodes a putative tailspike protein (GVE2 TSP). Here we report the crystal structure of the truncated GVE2 TSP at 2.0-Å resolution lacking 204 amino acid residues at its N-terminus (ΔnGVE2 TSP), possessing a "vase" outline similar to other TSP's structures. However, ΔnGVE2 TSP displays structural characteristics distinct from other TSPs. Despite lacking 204 amino acid residues, the head domain forms an asymmetric trimer compared to symmetric in other TSPs, suggesting that its long N-terminus may be unique to the long-tailed bacteriophages. Furthermore, the α-helix of the neck is 5-7 amino acids longer than that of other TSPs. The most striking feature is that its binding domain consists of a ß-helix with 10 turns, whereas other TSPs have 13 turns, even including the phage Sf6 TSP, which is the closest homologue of GVE2 TSP. The C-terminal structure is also quite different with those of other TSPs. Furthermore, we observed that ΔnGVE2 TSP can slow down growth of its host, demonstrating that this TSP is essential for the phage GVE2 to infect its host. Overall, the structural characteristics suggest that GVE2 TSP may be more primitive than other phage TSPs.
Subject(s)
Aquatic Organisms , Bacteriophages/physiology , Models, Molecular , Protein Conformation , Viral Tail Proteins/chemistry , Viral Tail Proteins/metabolism , Amino Acid Sequence , Bacteriophages/classification , Bacteriophages/genetics , Cloning, Molecular , Enzyme Activation , Gene Expression , Glycoside Hydrolases , Phylogeny , Protein Domains , Recombinant Proteins , Structure-Activity Relationship , Viral Tail Proteins/geneticsABSTRACT
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.
Subject(s)
Pseudomonas aeruginosa/metabolism , Type VI Secretion Systems/chemistry , Crystallography, X-Ray , Models, Molecular , Multigene Family , Protein Conformation , Pseudomonas aeruginosa/chemistryABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Pseudomonas aeruginosa/metabolism , Type VI Secretion Systems/chemistry , Type VI Secretion Systems/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Models, Molecular , Phospholipases/metabolism , Pseudomonas aeruginosa/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , Static Electricity , Type VI Secretion Systems/genetics , X-Ray DiffractionABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Secretion Systems/chemistry , Phospholipase D/chemistry , Pseudomonas aeruginosa/genetics , Bacterial Proteins/genetics , Bacterial Proteins/therapeutic use , Bacterial Secretion Systems/genetics , Crystallography, X-Ray , Phospholipase D/genetics , Protein Conformation/drug effects , Pseudomonas Infections/drug therapy , Pseudomonas Infections/genetics , Pseudomonas aeruginosa/pathogenicityABSTRACT
The bacterial type VI secretion system (T6SS), a dynamic organelle, participates in microbial competition by transporting toxic effector molecules to neighbouring cells to kill competitors. TsiV3, a recently defined T6SS immunity protein in Vibrio cholerae, possesses self-protection against killing by T6SS predatory cells by directly binding to and inhibiting their effector protein VgrG-3. Structural information about TsiV3 could help to illuminate its specific mechanism. In this study, TsiV3 from V. cholerae was cloned, expressed and crystallized and single-crystal X-ray diffraction data sets were collected to a resolution of 2.55â Å. Specifically, the crystal belonged to space group P212121, with unit-cell parameters a = 73.3, b = 78.12, c = 106.18â Å. Matthews coefficient calculations indicated that the crystal may contain six TsiV3 molecules in one asymmetric unit, with a VM value of 2.25â Å(3)â Da(-1) and a solvent content of 45.42%.
Subject(s)
Bacterial Proteins/chemistry , Vibrio cholerae , Bacterial Proteins/isolation & purification , Bacterial Secretion Systems , Chromatography, Affinity , Crystallization , Crystallography, X-RayABSTRACT
In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3-Csm2 sub-complex is presented at 1.8Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU-protein complex coupled with DNA.