Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

International audienceA simple and cost effective method to independently and stereo-specifically incorporate [1H,13C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [1H,13C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [1H,13C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-13C,2,3-2H2,15N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[13CH3]γ2 labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for 2H-glycine to prevent scrambling

Structure of the ubiquitin-binding zinc finger domain of human DNA Y-polymerase η

The ubiquitin-binding zinc finger (UBZ) domain of human DNA Y-family polymerase (pol) η is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol η UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C(2)H(2) zinc-finger structure characterized by a ββα fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the α-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single α-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol η UBZ domain represents a novel member of the C(2)H(2) zinc finger family that interacts with ubiquitin to regulate translesion synthesis

A Celecoxib Derivative Eradicates Antibiotic-Resistant Staphylococcus aureus and Biofilms by Targeting YidC2 Translocase

The treatment of Staphylococcus aureus infections is impeded by the prevalence of MRSA and the formation of persisters and biofilms. Previously, we identified two celecoxib derivatives, Cpd36 and Cpd46, to eradicate MRSA and other staphylococci. Through whole-genome resequencing, we obtained several lines of evidence that these compounds might act by targeting the membrane protein translocase YidC2. Our data showed that ectopic expression of YidC2 in S. aureus decreased the bacterial susceptibility to Cpd36 and Cpd46, and that the YidC2-mediated tolerance to environmental stresses was suppressed by both compounds. Moreover, the membrane translocation of ATP synthase subunit c, a substrate of YidC2, was blocked by Cpd46, leading to a reduction in bacterial ATP production. Furthermore, we found that the thermal stability of bacterial YidC2 was enhanced, and introducing point mutations into the substrate-interacting cavity of YidC2 had a dramatic effect on Cpd36 binding via surface plasmon resonance assays. Finally, we demonstrated that these YidC2 inhibitors could effectively eradicate MRSA persisters and biofilms. Our findings highlight the potential of impeding YidC2-mediated translocation of membrane proteins as a new strategy for the treatment of bacterial infections

Solution Structure of a Novel Tryptophan-Rich Peptide with Bidirectional Antimicrobial Activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an α-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes

Structural basis of adaptor-mediated protein degradation by the tail-specific PDZ-protease Prc

MepS is a peptidoglycan (PG) cross-link specific hydrolase needed for cell wall expansion and its cellular levels must be tightly regulated. Here the authors present the structure of the MepS degrading protease Prc bound to its adaptor NlpI and propose a model how the NlpI-Prc complex mediates MepS degradation

Interaction between <it>Serotonin Transporter</it> and <it>Serotonin Receptor 1 B</it> genes polymorphisms may be associated with antisocial alcoholism

<p>Abstract</p> <p>Background</p> <p>Several studies have hypothesized that genes regulating the components of the serotonin system, including <it>serotonin transporter</it> (<it>5-HTTLPR</it>) and <it>serotonin 1 B receptor (5-HT1B),</it> may be associated with alcoholism, but their results are contradictory because of alcoholism’s heterogeneity. Therefore, we examined whether the <it>5-HTTLPR</it> gene and <it>5-HT1B</it> gene <it>G861C</it> polymorphism are susceptibility factors for a specific subtype of alcoholism, antisocial alcoholism in Han Chinese in Taiwan.</p> <p>Methods</p> <p>We recruited 273 Han Chinese male inmates with antisocial personality disorder (ASPD) [antisocial alcoholism (AS-ALC) group (<it>n</it> = 120) and antisocial non-alcoholism (AS-N-ALC) group (<it>n</it> = 153)] and 191 healthy male controls from the community. Genotyping was done using PCR-RFLP.</p> <p>Results</p> <p>There were no significant differences in the genotypic frequency of the <it>5-HT1B G861C</it> polymorphism between the 3 groups. Although AS-ALC group members more frequently carried the <it>5-HTTLPR S/S, S/L</it>_<it>G</it><it>,</it> and <it>L</it>_<it>G</it><it>/L</it>_<it>G</it> genotypes than controls, the difference became non-significant after controlling for the covarying effects of age. However, the <it>5-HTTLPR S/S, S/L</it>_<it>G</it><it>,</it> and <it>L</it>_<it>G/</it><it>L</it>_<it>G</it> genotypes may have interacted with the <it>5-HT1B G861C C/C</it> polymorphism and increased the risk of becoming antisocial alcoholism.</p> <p>Conclusion</p> <p>Our study suggests that neither the <it>5-HTTLPR</it> gene nor the <it>5-HT1B G861C</it> polymorphism alone is a risk factor for antisocial alcoholism in Taiwan’s Han Chinese population, but that the interaction between both genes may increase susceptibility to antisocial alcoholism.</p

Structural basis of antizyme-mediated regulation of polyamine homeostasis

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az(1)) and antizyme inhibitor (AzIN). Az(1) suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az(1) binding. The structural basis of the Az(1)-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az(1) complexed with either ODC or AzIN. Structural analysis revealed that Az(1) sterically blocks ODC homodimerization. Moreover, Az(1) binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az(1) for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az(1)-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az(1) structure suggests how AzIN may effectively compete with ODC for Az(1) to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation