Zengqiang Gao

Structure of the type VI effector-immunity complex (Tae4-Tai4) provides novel insights into the inhibition mechanism of the effector by its immunity protein

Background: The bacteria effector Tae4 is injected into the recipient cells to kill them and the immunity protein Tai4 is produced to inactivate Tae4. Results: Tae4 displays a papain-like fold, and Tai4 dimer is responsible for inhibiting Tae4 activity. Conclusion: The inactivation of Tae4 is required by collaboration of both subunits of Tai4 dimer. Significance: Our results add new insights into the effector-immunity interaction module. The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The Gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a DL-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.

High-resolution structure of a new crystal form of BamA POTRA4-5 from Escherichia coli.

In Escherichia coli, the BAM complex is employed to mediate correct folding of the outer membrane (OM) proteins into β-barrels and their insertion into the OM. BamA, which is an essential component of the complex, consists of a C-terminal transmembrane region and five N-terminal polypeptide transport-associated (POTRA) domains. Although deletion studies have shown that each of the POTRA domains plays an important role in the process of BAM complex formation, only POTRA5 is essential for cell viability. Here, the crystal structure of POTRA4-5 has been determined to 1.50 Å resolution with an R factor of 14.7% and an Rfree of 18.9%.

The crystal structure of the MPN domain from the COP9 signalosome subunit CSN6

CSN6 and CSN6 bind by x ray scattering ( View interaction )

Crystal structure of type VI effector Tse1 from Pseudomonas aeruginosa

The type VI secretion systems (T6SS) have emerging roles in interspecies competition. In order to have an advantage in defense against other organisms, this system in Pseudomonas aeruginosa delivers a peptidoglycan amidase (Tse1) to the periplasmic space of a competitor. An immune protein (Tsi1) is also produced by the bacterium to protect itself from damage caused by Tse1. Tsi1 directly interacts with Tse1. We report that the crystal structure of Tse1 displays a common CHAP protein fold. Strikingly, our structures showed that the third residue in the catalytic triad may be novel as this residue type has not been observed previously.

Structural and SAXS analysis of the budding yeast SHU-complex proteins

PSY3 and CSM2 bind by X‐ray crystallography ( View interaction ) PSY3 , CSM2 , Shu 1 and Shu 2 physically interact by x ray scattering (View interaction)

Structural and Functional Characterization of Escherichia coli Toxin-Antitoxin Complex DinJ-YafQ

Background: The type II TA dinJ-yafQ module autoregulates bacterial growth in response to environmental stimuli. Results: The crystal structures of the DinJ-YafQ complex and free YafQ unveiled their structural details. Conclusion: The DinJ-YafQ complex as a transcription repressor interacts with a defined region in its operator via the RHH domain of DinJ. Significance: This study defines YafQ as a ribosome-dependent ribonuclease in vivo. Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)2-(YafQ)2 complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)2-(YafQ)2 revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.

Structure-function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance.

Escherichia coli RnlA–RnlB is a newly identified toxin–antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA‐Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 Å. RnlA is composed of three independent domains: NTD (N‐terminal domain), NRD (N repeated domain) and DBD (Dmd‐binding domain), which is an organization not previously observed among known toxin structures. Small‐angle X‐ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C‐terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure–function relationship.

Structural Basis for Interaction between Mycobacterium smegmatis Ms6564, a TetR Family Master Regulator, and Its Target DNA

Background: The structural basis for interaction between a master regulator and DNA remains unclear. Results: We solved the crystal structures of a broad regulator Ms6564 and its protein-operator complex. Conclusion: Ms6564 binds DNA with strong affinity but makes flexible contacts with DNA. Significance: Ms6564 might slide more easily along the genomic DNA and extensively regulate the expression of diverse genes. Master regulators, which broadly affect expression of diverse genes, play critical roles in bacterial growth and environmental adaptation. However, the underlying mechanism by which such regulators interact with their cognate DNA remains to be elucidated. In this study, we solved the crystal structure of a broad regulator Ms6564 in Mycobacterium smegmatis and its protein-operator complex at resolutions of 1.9 and 2.5 Å, respectively. Similar to other typical TetR family regulators, two dimeric Ms6564 molecules were found to bind to opposite sides of target DNA. However, the recognition helix of Ms6564 inserted only slightly into the DNA major groove. Unexpectedly, 11 disordered water molecules bridged the interface of TetR family regulator DNA. Although the DNA was deformed upon Ms6564 binding, it still retained the conformation of B-form DNA. Within the DNA-binding domain of Ms6564, only two amino acids residues directly interacted with the bases of cognate DNA. Lys-47 was found to be essential for the specific DNA binding ability of Ms6564. These data indicate that Ms6564 can bind DNA with strong affinity but makes flexible contacts with DNA. Our study suggests that Ms6564 might slide more easily along the genomic DNA and extensively regulate the expression of diverse genes in M. smegmatis.

Crystal and solution structures of methyltransferase RsmH provide basis for methylation of C1402 in 16S rRNA

RsmH is a specific AdoMet-dependent methyltransferase (MTase) responsible for N(4)-methylation of C1402 in 16S rRNA and conserved in almost all species of bacteria. The methylcytidine interacts with the P-site codon of the mRNA and increases ribosomal decoding fidelity. In this study, high resolution crystal structure (2.25Å) of Escherichia coli RsmH in complex with AdoMet and cytidine (the putative rRNA binding site) was determined. The structural analysis demonstrated that the complex consists of two distinct but structurally related domains: the typical MTase domain and the putative substrate recognition and binding domain. A deep pocket was found in the conserved AdoMet binding domain. It was also found that the cytidine bound far from AdoMet with the distance of 25.9Å. It indicates that the complex is not in a catalytically active state, and structural rearrangement of RsmH or the nucleotides neighboring C1402 may be necessary to trigger catalysis. Although there is only one molecule in the asymmetric unit of the crystals, RsmH can form a compact dimer across a crystallographic twofold axis. Further analysis of RsmH by small-angle X-ray scattering (SAXS) also revealed the dimer in solution, but with a more flexible conformation than that in crystal, likely resulting from the absence of the substrate. It implies that an active status of RsmH in vivo is achieved by a formation of the dimeric architecture. In general, crystal and solution structural analysis provides new information on the mechanism of the methylation of the fine-tuning ribosomal decoding center by the RsmH.

Insights into the Catalytic Mechanism of 16S rRNA Methyltransferase RsmE (m(3)U1498) from Crystal and Solution Structures

RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine-specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S-adenosyl-l-methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.