Sensing stressful conditions and adjusting the cellular metabolism to adapt to the environment are essential activities for bacteria to survive in variable situations. Here, we describe a stress-related protein, YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine-5 0 -monophosphate to a protein tyrosine/histidine residue, an unusual modification defined as UMPylation. Mn 2+ serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn 2+ binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. A detailed mechanism is proposed based on the crystal structures of Apo-YdiU and YdiU-AMPNPP-Mn 2+ and on molecular dynamics simulation models of YdiU-UTP-Mn 2+ and YdiU-UTP-peptide. In vivo data demonstrate that YdiU effectively protects Salmonella from stress-induced ATP depletion through UM-Pylation.

Five different murine monoclonal antibodies (MAbs) to Mycobacterium tuberculosis were examined for degree of cross-reactivity with other mycobacterial species by enzyme-linked immunosorbent assay and immunoblotting. One MAb reacted solely with M. tuberculosis and M. bovis BCG. Two of the MAbs reacted with all mycobacterial species examined, whereas two MAbs demonstrated a limited reactivity pattern. The epitopes are located on molecules susceptible to protease treatment, and two of these molecules possess concanavalin A-binding moieties. Two of the antigens defined by these five MAbs are present in tuberculin purified protein derivative.

ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu were observed in the catalytic center. Our observations suggest that Mg ions together with Glu and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.

To enhance the efficiency of firefly luciferase/luciferin bioluminescence imaging, a series of N-cycloalkylaminoluciferins (cyaLucs) were developed by introducing lipophilic N-cycloalkylated substitutions. The experimental results demonstrate that these cyaLucs are effective substrates for native firefly luciferase (Fluc) and can produce elevated bioluminescent signals in vitro, in cellulo, and in vivo. It should be noted that, in animal studies, N-cyclobutylaminoluciferin (cybLuc) at 10 μM (0.1 mL), which is 0.01% of the standard dose of d-luciferin (dLuc) used in mouse imaging, can radiate 20-fold more bioluminescent light than d-luciferin (dLuc) or aminoluciferin (aLuc) at the same concentration. Longer in vivo emission imaging using cybLuc suggests that it can be used for long-time observation. Regarding the mechanism of cybLuc, our cocrystal structure data from firefly luciferase with oxidized cybLuc suggested that oxidized cybLuc fits into the same pocket as oxyluciferin. Most interestingly, our results demonstrate that the sensitivity of cybLuc in brain tumor imaging contributes to its extended application in deep tissues.

Salmonella reduces flagella biogenesis to avoid detection within host cells by a largely unknown mechanism. We identified an EAL-like protein STM1697 as required and sufficient for this process. STM1697 surges to a high level after Salmonella enters host cells and restrains the expression of flagellar genes by regulating the function of flagellar switch protein FlhD4C2, the transcription activator of all other flagellar genes. Unlike other anti-FlhD4C2 factors, STM1697 does not prevent FlhD4C2 from binding to target DNA. A 2.0 Å resolution STM1697–FlhD structure reveals that STM1697 binds the same region of FlhD as STM1344, but with weaker affinity. Further experiments show that STM1697 regulates flagella biogenesis by restricting FlhD4C2 from recruiting RNA polymerase and the regulatory effect of STM1697 on flagellar biogenesis and virulence are all achieved by interaction with FlhD. Finally, we describe a novel mechanism mediated by STM1697 in which Salmonella can inhibit the production of flagella antigen and escape from the host immune system.

SummaryThe vast majority of oceanic dimethylsulfoniopropionate (DMSP) is thought to be catabolized by bacteria via the DMSP demethylation pathway. This pathway contains four enzymes termed DmdA, DmdB, DmdC and DmdD/AcuH, which together catabolize DMSP to acetylaldehyde and methanethiol as carbon and sulfur sources respectively. While molecular mechanisms for DmdA and DmdD have been proposed, little is known of the catalytic mechanisms of DmdB and DmdC, which are central to this pathway. Here, we undertake physiological, structural and biochemical analyses to elucidate the catalytic mechanisms of DmdB and DmdC. DmdB, a 3‐methylmercaptopropionate (MMPA)‐coenzyme A (CoA) ligase, undergoes two sequential conformational changes to catalyze the ligation of MMPA and CoA. DmdC, a MMPA‐CoA dehydrogenase, catalyzes the dehydrogenation of MMPA‐CoA to generate MTA‐CoA with Glu435 as the catalytic base. Sequence alignment suggests that the proposed catalytic mechanisms of DmdB and DmdC are likely widely adopted by bacteria using the DMSP demethylation pathway. Analysis of the substrate affinities of involved enzymes indicates that Roseobacters kinetically regulate the DMSP demethylation pathway to ensure DMSP functioning and catabolism in their cells. Altogether, this study sheds novel lights on the catalytic and regulative mechanisms of bacterial DMSP demethylation, leading to a better understanding of bacterial DMSP catabolism.

No abstract

53BP1 performs essential functions in DNA double-strand break (DSB) repair and it was recently reported that Tudor interacting repair regulator (TIRR) negatively regulates 53BP1 during DSB repair. Here, we present the crystal structure of the 53BP1 tandem Tudor domain (TTD) in complex with TIRR. Our results show that three loops from TIRR interact with 53BP1 TTD and mask the methylated lysine-binding pocket in TTD. Thus, TIRR competes with histone H4K20 methylation for 53BP1 binding. We map key interaction residues in 53BP1 TTD and TIRR, whose mutation abolishes complex formation. Moreover, TIRR suppresses the relocation of 53BP1 to DNA lesions and 53BP1-dependent DNA damage repair. Finally, despite the high-sequence homology between TIRR and NUDT16, NUDT16 does not directly interact with 53BP1 due to the absence of key residues required for binding. Taken together, our study provides insights into the molecular mechanism underlying TIRR-mediated suppression of 53BP1-dependent DNA damage repair.