Background Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. Methods We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. Results BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. Conclusions BstA is a bacillithiol transferase from Staphylococcus aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. General Significance BstA is an active bacillithiol transferase from Staphylococcus aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from Bacillus subtilis YfiT.

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation, however the way in which ASB9 interacts with CK is not yet known. We present complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight and no dissociation could be observed. Deletion of the N-terminal 34 amino acids forming ASB9(35-252) resulted in weakening of the binding so that a binding affinity of 2.6 nM could be measured. Amide hydrogen/deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.

Summary Cullin-RING E3 Ligases (CRLs) are elongated and bowed protein complexes that transfer ubiquitin over 60 Å to proteins targeted for proteasome degradation. One such CRL contains the ankyrin repeat and SOCS box protein 9 (ASB9), which binds to and partially inhibits creatine kinase (CK). While current models for the ASB9-CK complex contain some known interface residues, the overall structure and precise interface of the ASB9-CK complex remains unknown. Through an integrative modeling approach, we report a third generation model that reveals precisely the interface interactions and also fits the shape of the ASB9-CK complex as determined by SAXS. We constructed an atomic model for the entire CK-targeting CRL to uncover dominant modes of motion that could permit ubiquitin transfer. Remarkably, only the correctly docked CK-containing E3 ligase and not incorrectly docked structures permitted close approach of ubiquitin to the CK substrate.

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transient intra-rather than intermolecular interactions. Furthermore, even disulfide crosslinking of z cyt N-termini, in a configuration reminiscent of T cell receptor clustering, fails to lead to an association of protomers. SEC-MALS confirms the monomeric state of z cyt but reveals a curious concentration-dependent shift of the elution volume of z cyt that may previously have been interpreted as dimerization. Our data show that z cyt does not form a highly disordered protein complex but leave open the question as to whether completely disordered dimers or other oligomers exist in nature. Interactions of intrinsically disordered proteins (IDP) with their binding partners often involve coupled binding and folding. A long-standing question is the extent to which folding of the IDP is mediated by selection of a folded conformer from the disordered state ensemble rather than folding induced by interaction with the binding partner. Answering this question requires detailed information about the disordered state ensemble, in particular the extent to which the IDP possesses residual structure. Yet obtaining this type of information at near-atomic resolution remains challenging. To address this need, we have developed an approach based on millisecond quench-flow amide H/D exchange and mass spectrometry to measure residual structure. In the present work, we examine residual structure in the disordered CBP-binding domain of ACTR as a model system for validation. Following millisecond H/D exchange and acid quench, digestion with pepsin produced a set of 67 highly-overlapping fragments covering the entire 77-resdidue sequence. Residue-by-residue analysis of empirically-determined H/D exchange halflife obtained from each ACTR fragment provided exchange kinetics at nearresidue resolution. In ACTR, we found that the regions that are known adopt an a-helical fold upon binding to CBP became more protected from H/D exchange than the structured loop regions. We also found that most of the N-terminal region, which does not appear in the solved structure, was the least protected. There was also evidence of slight protection in a short stretch of the N-terminal region. Our results are consistent both with a recent analysis of residual structure obtained from NMR secondary shift measurements and with the AGADIR helicity prediction algorithm. Our results demonstrate the utility of millisecond H/D exchange for mapping secondary structural propensity in disordered state ensembles with near-residue resolution.