Redesign of Substrate Specificity and Identification of the Aminoglycoside Binding Residues of Eis from Mycobacterium tuberculosis

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis. Eis from M. tuberculosis (Eis_<i>Mtb</i>) and M. smegmatis (Eis_<i>Msm</i>) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_<i>Mtb</i> is divided into two narrow channels, whereas Eis_<i>Msm</i> contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_<i>Mtb</i>, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_<i>Msm</i>, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_<i>Mtb</i> and for the functional differences from Eis_<i>Msm</i>, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_<i>Mtb</i> by mutating them into their Eis_<i>Msm</i> counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_<i>Mtb</i>. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_<i>Mtb</i> nomenclature) dictate the differences between the acetylation efficiencies of Eis_<i>Mtb</i> and Eis_<i>Msm</i> for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_<i>Mtb</i> into Ala plays a role in APR acetylation

Synthesis and Evaluation of Hetero- and Homodimers of Ribosome-Targeting Antibiotics: Antimicrobial Activity, in Vitro Inhibition of Translation, and Drug Resistance

In this study, we describe the synthesis of a full set of homo- and heterodimers of three intact structures of different ribosome-targeting antibiotics: tobramycin, clindamycin, and chloramphenicol. Several aspects of the biological activity of the dimeric structures were evaluated including antimicrobial activity, inhibition of in vitro bacterial protein translation, and the effect of dimerization on the action of several bacterial resistance mechanisms that deactivate tobramycin and chloramphenicol. This study demonstrates that covalently linking two identical or different ribosome-targeting antibiotics may lead to (i) a broader spectrum of antimicrobial activity, (ii) improved inhibition of bacterial translation properties compared to that of the parent antibiotics, and (iii) reduction in the efficacy of some drug-modifying enzymes that confer high levels of resistance to the parent antibiotics from which the dimers were derived

Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications

Cysteine <i>S</i>-nitrosation and <i>S</i>-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous <i>S</i>-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of <i>S</i>-nitrosation. In parallel, <i>S</i>-nitrosothiol-linked probes enable enrichment and detection of endogenous <i>S</i>-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases <i>S</i>-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both <i>S</i>-nitrosation and <i>S</i>-sulfination. Quantitative proteomic analysis demonstrates that both <i>S</i>-nitrosation and <i>S</i>-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Post-translational <i>S</i>-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute <i>S</i>-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of <i>S</i>-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development