Figure 4. The C-terminal mini-domain of PBP4 directly interacts with GpsB.

(A) Domain representation of the four/five S. aureus (COL)/methicillin-resistant S. aureus (MRSA) (USA300) penicillin-binding proteins (PBPs). Each protein is shown from the N-terminus (left) to the C-terminus (right). All four transpeptidase PBPs - PBP1, PBP2, PBP3, and PBP2a - lack a GpsB recognition motif on their N-terminal, cytosolic mini-domain. In contrast, the C-terminal mini-domain of PBP4, the sole S. aureus class C PBP, contains this motif (NRLFRKRK, red). The dissociation constants were determined with SPR (n=2). (B) Crystal structure of Sa GpsB R24A in complex with PBP4 C-terminal peptide fragment at 2.40 Å resolution. The middle panel includes the electron density map of the Sa PBP4 heptapeptide, 2F_o-F_c=1.0σ. The right panel shows a superimposition of the Bs PBP1 mini-domain from the Bs GpsB+PBP1 complex (PDB ID 6GP7, purple) highlighting similar binding features.

Figure 4—figure supplement 1. The crystal packing interface of Sa GpsB dimers form interactions that mimic those between GpsB-PBP pairs.

(A) Sa GpsB dimers assemble in a head-to-head, antiparallel arrangement, where the membrane-binding loop interacts with the PBP-binding groove of the other. (B) Enhanced viewpoint of the crystal packing interactions. 2F_o-F_c map, shown in gray, is contoured at 1.0σ. R28, R24, and K18 adopt similar interactions with the negatively charged PBP-binding groove, as the arginine fingers of (C, D) of Bs PBP1a with Bs GpsB (6GP7). PBP, penicillin-binding protein.

Figure 4—figure supplement 2. Surface plasmon resonance (SPR) titration of Sa FtsZ C-terminal peptides against Sa GpsB^ΔMAD_FL.

K_D values are reported in Supplementary file 2.

Figure 4—figure supplement 3. AlphaFold2-Multimer prediction of GpsB:FtsZ^385-390 complex (cyan) superimposed onto the GpsB:PBP4 peptide crystal structure (green:orange).

FtsZ residues are labeled. PBP, penicillin-binding protein.