Chemical logic of MraY inhibition by antibacterial nucleoside natural products

Abstract

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot 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.

Fig. 1

X-ray crystal structures of MraY_AA bound to nucleoside inhibitors. a Top: the MraY_AA-carbacaprazamycin complex structure as viewed from the membrane, with one protomer shown in surface representation and one in cartoon. Carbacaprazamycin is shown in magenta. For simplicity, one protomer of MraY_AA with bound carbacaprazamycin is shown from membrane and cytoplasmic views. a Bottom: chemical structure of carbacaprazamycin with its substructures labeled. (b, top) Membrane and cytoplasmic views of an MraY_AA protomer bound to capuramycin (yellow). Loop E in is distorted and is represented by a dashed line. b Bottom: chemical structure of capuramycin. c Top: membrane and cytoplasmic views of an MraY_AA protomer bound to 3′-hydroxymureidomycin A (green). c Bottom: chemical structure of 3′-hydroxymureidomycin A. Each MraY inhibitor binds distinctly to a site on the cytoplasmic face of MraY formed by TMs 5, 8, and 9b and Loops C, D, and E (labeled throughout)

Fig. 2

Carbacaprazamycin binds to the hydrophobic groove in MraY_AA. a The binding sites recognized by carbacaprazamycin (magenta) on the cytoplasmic side of MraY_AA include the uridine (red), uridine-adjacent (lime green), TM9b/Loop E (purple), and hydrophobic (cyan) pockets. b A zoomed-in view of the carbacaprazamycin binding site in the same orientation as shown in a. Residues forming interactions with carbacaprazamycin are labeled and color-coded according to the binding pocket to which they belong. Hydrogen bonds are represented by dashed lines. c A view of the carbacaprazamycin binding site rotated 90° relative to the orientation shown in a to highlight the aliphatic tail binding site (cyan dashes lines). TMs (numbers) and Loops (letters) are labeled throughout

Fig. 3

Capuramycin forms a unique interaction with the caprolactam site on MraY_AA. a The binding pockets occupied by capuramycin (yellow) on the cytoplasmic face of MraY_AA include the uridine (red), uridine-adjacent (lime green), and caprolactam (pink) sites. b A zoomed-in view of the capuramycin binding site in the same orientation as shown in a. Residues forming interactions with capuramycin are labeled and color-coded according to the binding pocket to which they belong. Hydrogen bonds are represented by dashed lines. c A view of the capuramycin binding site rotated 60° relative to the orientation in a to highlight the caprolactam binding site. The surface of MraY_AA is shown in transparent gray with residues forming the shallow caprolactam binding pocket (pink dashes lines) labeled. TMs (numbers) and Loops (letters) are labeled throughout. The side chains of residues K70 and K121 are disordered in the MraY_AA-capuramycin complex structure

Fig. 4

A detailed view of the interactions between MraY_AA and 3′-hydroxymureidomycin A. a The binding pockets recognized by 3′-hydroxymureidomycin A (bright green) on the cytoplasmic side of MraY_AA include the uridine (red), uridine-adjacent (lime green), TM9b/Loop E (purple), and Mg²⁺ (gold) sites. b A zoomed-in view of the 3′-hydroxymureidomycin A binding site in the same orientation as shown in a. Residues forming interactions with 3′-hydroxymureidomycin A are labeled and color-coded according the binding pocket to which they belong. Hydrogen bonds are represented by dashed lines. c A view of the 3′-hydroxymureidomycin A binding site rotated 45° relative to the orientation shown in a. TMs (numbers) and Loops (letters) are labeled throughout. The side chain of residue K70 is disordered in the MraY_AA-3′-hydroxymureidomycin A complex structure

Fig. 5

Structurally diverse moieties occupy the uridine-adjacent binding pocket in MraY_AA. a Carbacaprazamycin (magenta), b muraymycin D2 (orange), c capuramycin (yellow), d 3′-hydroxymureidomycin A (green), e tunicamycin (slate) binding interactions at the uridine-adjacent pocket. Residues labeled in black and blue form side chain and backbone interactions with the inhibitor, respectively. Hydrogen bonds are represented by black dashed lines. Residue numbering is shown for MraY_AA except in e, which shows residue numbering for MraY_CB with the corresponding residues in MraY_AA provided in parentheses

Fig. 6

Summary of the hot spots of MraY inhibition. a Structural overlay of MraY_AA bound to carbacaprazamycin (magenta), capuramycin (yellow), 3′-hydroxymureidomycin A (green), and muraymycin D2 (orange), and MraY_CB bound to tunicamycin (slate) viewed from the cytoplasm. b Structure of MraY_AA in surface representation with inhibitor binding site hot spots (HSs) color-coded and labeled as follows: uridine (red), uridine-adjacent (HS1; lime green), TM9b/LoopE (HS2; purple), caprolactam (HS3; pink), hydrophobic (HS4; cyan), Mg²⁺ (HS5; gold), and tunicamycin (HS6; brown). c A barcode representing the interactions each nucleoside inhibitor makes with HS1–6. The residues shown underneath each HS label are found at that site in MraY. Amino acid residue numbering is shown for MraY_AA and color-coding is consistent with b. Each row represents a different compound: carbacaprazamycin (CAR), capuramycin (CAP), 3′-hydroxymureidomycin A (MUR), muraymycin D2 (MD2), and tunicamycin (TUN). A dark gray square represents an interaction between the corresponding inhibitor and residue. A white square indicates that no contact is made. Squares colored light gray represent that either the amino acid residue side chain or the inhibitor substructure is not resolved in the crystal structure, but likely makes the relevant binding interaction. The side chains of residues K70 and K121 are disordered in the MraY_AA-capuramycin complex structure. The side chain of residue K70 is disordered in the MraY_AA-3′-hydroxymureidomycin A complex structure