We addressed this challenge by obtaining different crystal forms of MraYAA in the presence of camelid single-chain antibodies called nanobodies. present crystal structures of MraY in complex with representative users of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable warm spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design. species with promising activity against pathogenic bacteria: the liposidomycins/caprazamycins, capuramycins, mureidomycins, muraymycins, and tunicamycins. Each MraY inhibitor contains a uridine moiety, but they normally differ in their core chemical structures. Nucleoside natural product inhibitors exhibit differing antibacterial activity, structure-activity-relationship (SAR) profiles6,7, mechanisms of action8,9, and inhibitor kinetics8C10 Tunicamycin inhibits both MraY and its eukaryotic paralog GlcNAc-1-P-transferase (GPT), leading to cytotoxicity11, but users of the other classes of nucleoside inhibitors are selective for bacterial MraY9,12. The mechanistic and structural basis for the unique pharmacological properties observed among MraY-targeted nucleoside inhibitors is usually poorly comprehended. Recent structures of tunicamycin bound to MraY13 and GPT14, 15 show that this tunicamycin binding pocket is usually deep and occluded in GPT, while in MraY it is shallow and largely exposed to the cytosol. The MraY inhibitor binding site around the cytoplasmic face of MraY is AT7519 usually unlike the large, deep, and enclosed binding pouches typically found in enzyme active sites16. This observation raises an intriguing and important question: what strategy does nature employ to target the shallow cytosolic surface of MraY using nucleoside inhibitors with very different core chemical structures? One possibility is that the structural plasticity of MraY helps to accommodate structurally diverse inhibitors, as suggested by comparison of apoenzyme and muraymycin D2-bound MraY17,18. Alternatively, it is possible that this shallow surface of MraY contains many cryptic druggable sites, which can be exploited in different Rabbit Polyclonal to GPRIN3 combinations by each nucleoside inhibitor. To address this question, we solved structures of MraY from (MraYAA) individually bound to carbacaprazamycin (a member of the caprazamycin class), capuramycin, and 3-hydroxymureidomycin A (a ribose?derivative of mureidomycin A). These three classes of nucleoside inhibitors are unique in their chemical structures, mechanisms of inhibition, and antibacterial activity. For example, liposidomycin is usually competitive for C55-P, the lipid carrier substrate of MraY8, while capuramycin is usually noncompetitive AT7519 for C55-P and exhibits mixed type inhibition with respect to UM5A9. The liposidomycins/caprazamycins demonstrate potent antibacterial activity against Gram-positive bacteria, mycobacteria, and various drug-resistant bacterial strains, including MRSA and VRE19. Mureidomycin and AT7519 its analogs appear to have a narrower spectrum of activity, primarily against species20,21, while the capuramycins are particularly effective against mycobacteria22,23; capuramycin analog SQ641 kills faster than existing antitubercular drugs24. Our structures cover the chemical space sampled by MraY natural product inhibitors, revealing that they occupy both overlapping and unique sites around the cytoplasmic surface of MraY. This region of MraY is usually highly conserved among Gram-positive and Gram-negative bacteria, with 34 invariant amino acid residues comprising the active site17,18. Therefore, our crystal structures collectively serve as a generalizable MraY structural model by which nucleoside inhibitor AT7519 SAR data can be analyzed and understood in order to achieve a comprehensive picture of MraY inhibition. Results Crystal structures of MraY bound to nucleoside inhibitors We previously recognized a biochemically stable ortholog of MraY from thermophile (MraYAA), with which we obtained crystal structures of MraY in its apoenzyme form17 as well as bound to muraymycin D218. MraYAA is a good model with which to study MraY activity and inhibition because it recognizes the same substrates and catalyzes the same enzymatic reaction as do pathogenic Gram-positive and Gram-negative bacteria17. MraYAA enzymatic activity is usually potently inhibited by carbacaprazamycin, capuramycin, and 3-hydroxymureidomycin A with IC50 values.