Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis

Abstract

Extended Data Fig. 2 RagAB binds a wide range of oligopeptides EDFig2.tiff a-c, LC-MS/MS analysis of peptides bound to RagAB W83 KRAB, showing length distribution (a), total charge (b) and pI (c). d-f, Analysis of peptides bound to RagAB W83 wild-type, showing length distribution (d), total charge (e) and pI (f). For charge calculations, the pH was assumed to be 7.0 and contributions of any His residues were ignored. g, Amino acid frequency of RagAB-bound peptides (KRAB and wild-type combined; black) vs. the amino acid composition in the P. gingivalis proteome (gray), showing a substantial enrichment of Ala, Glu, Lys, Thr and Val. By contrast, aromatics (Phe, Trp) and bulky hydrophobics (Leu) appear to be underrepresented. The peptides bound to RagAB from W83 KRAB vary in length from 7 to 29 residues, with a broad maximum of around 13 residues that fits well with the peptide density observed in the structures. Assuming equal abundance of each detected peptide, there is a slight preference for neutral to slightly acidic peptides, and the pI distribution has a bimodal shape, with maxima for acidic and slightly basic peptides. Analysis of the smaller RagAB-bound peptide set from wild-type W83 (d-f) yields a slightly wider size range from 5-36 residues, but overall there are no dramatic differences in the collective length, net charge and pI of the RagAB-bound peptide populations from W83 KRAB and wild-type strains. Extended Data Fig. 3 Molecular dynamics simulations of RagAB show lid opening EDFig3.tiff a, C α-rmsd values of RagB lids in apo-RagAB (red) and peptide-bound RagAB (green) with reference to the starting crystal structure in the closed conformation. The C α-rmsd values of the RagB lids in RagA 2 B 2 are shown in blue with reference to the OO EM state. Each point indicates an average of 50 ns simulation trajectory. b, Comparison of the RagA 2 B 2 open conformation from EM (magenta) Data description i.e.

TonB‐dependent transporters in the Bacteroidetes: Unique domain structures and potential functions

“…The SusD protein encoded within the starch utilization system locus of B. theta was the first in this protein family to be functionally characterized and validated as binding starch (Koropatkin et al, 2008;Shipman et al, 2000). Early work on the B. theta Sus provided direct evidence of an interaction between the SusC and SusD proteins, which has been validated in the recent crystal structures from homologous systems such as the levan BT1763/2 and the peptide-targeting BT2264/3 complexes (Glenwright et al, 2017;Gray et al, 2021;Madej et al, 2020;Shipman et al, 2000).…”

Towards defining the outer membrane proteome of Porphyromonas gingivalis

“…Multiple alignments showed that 10 sequences exhibited a conserved region of ~32 aa in the plug domain (Figure 2c) while the remaining five did not. The function of the Cpdr domain is not known but is located on the periplasmic side of the barrel in the RagA structure (Madej et al., 2020). Structural homology searches with Phyre‐2 for most P. gingivalis TDRs matched best to the ferripyoverdine receptor (FpvA) and the ferrioxamine b transporter (FoxA) of Pseudomonas aeruginosa .…”

“…Of the 15 predicted TDRs, only two have a definitive function. RagA (PG0185) was shown to complex with the RagB lipoprotein and was determined to be a peptide transporter based on structural studies, binding studies and growth experiments (Glew et al., 2014; Madej et al., 2020; Nagano et al., 2007). RagB serves as the initial peptide binder and also functions as a hinged lid on the extracellular side of RagA (Figure 2d).…”

The surface lipoproteins of gram-negative bacteria: Protectors and foragers in harsh environments

“…Recent structures of SLP bipartite systems in Bacteroidetes involved in oligopeptide and glycan uptake (72,73) demonstrates that the field is expanding to include the study of SLPs that are involved in the uptake of nutrients that are not restricted by host specific proteins. Bacteroidetes possess enzymes that degrade complex, inaccessible sugars and proteins to produce simpler molecules more amenable to uptake for growth and perhaps signaling (72,73). Xray and cryo-electron microscopy structures coupled with functional studies support a model in which the Bacteroidetes starch utilization system (Sus) and receptor antigen gene (Rag) SLPs function as a lid that operates according to a pedal bin mechanism.…”