Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices

Abstract

The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique alpha-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.

Figure 1. The starch utilization system (Sus) of Bacteroides thetaiotaomicron

Cartoon representation of the Sus operon and its protein products (Cho and Salyers, 2001; D’Elia and Salyers, 1996a; Shipman et al., 2000; Shipman et al., 1999). The stoichiometry of the Sus complex is not known.

Figure 2. Growth of Bacteroides thetaiotaomicron and derivative strains on starch-like oligosaccharides and polysaccarides

Shown are the log-phase growth rates of wild-type, ΔsusD, and complemented ΔsusD (ΔsusD::P_susB-susD) strains on glucose (G1), maltooligosaccharides of varying length (G2-G7), amylopectin (AP), and pullulan (Pull) and dextran (Dex). The ΔsusD strain is unable to grow on substrates >5 glucose units and exhibits significantly slower rates on G4 and G5 compared to wild-type on the same substrates (P<0.01, denoted by an asterisk). Complementation with a single copy of susD (expressed at ∼10% of wild-type levels; see Fig. S1) restores either full or partial growth characteristics depending on substrate size.

Figure 3. Atomic structure of SusD

(A) Stereo ribbon diagram of apo-SusD, color-ramped from dark blue to red as the chain extends from the amino to the carboxyl end of the protein. An ordered Ca²⁺ ion is represented by a magenta-colored sphere while polyethylene glycol and ethylene glycol are shown as ball-and-stick figures. As a reference, a molecule of maltoheptaose from the structure of the SusD-maltoheptose complex is shown as a transparent ball-and-stick. (B) Stereo figure of SusD (yellow) highlighting residues 31-172 of PilF (blue) which contain the TPR units.

Figure 4. SusD complexed with maltoheptaose

A) Shown here is the electron density of bound maltoheptaose from the corresponding omit map contoured at 3σ. B) In this panel, important hydrophobic-stacking and hydrogen-bonding interactions between the maltoheptaose and SusD are detailed. C) This panel shows a stereo diagram of SusD in the presence (blue) and absence (mauve) of bound maltoheptaose to highlight the conformational changes that occur upon oligosaccaride binding.

Figure 5. SusD complexed with β-cyclodextrin and maltotriose

Panels (A) and (C) show the electron densities of β-cyclodextrin and maltotriose, respectively, from omit maps contoured at 3σ. Panels B and D highlight important hydrophobic-stacking and hydrogen-bonding interactions for bound β-cyclodextrin and maltotriose, respectively.

Figure 6. SusD complexed with α-cyclodextrin

A) Ribbon and surface rendering of α-cyclodextrin complexed with two copies of SusD. B) Omit map contoured at 3σfor bound α-cyclodextrin. C) Important ring-stacking and hydrogen-bonding interactions (distances in Å) are shown for the α-cyclodextrin/SusD complex.

Figure 7. Isothermal titration calorimetry of the binding of various oligosaccharides to SusD

As described in the Methods, the heat of binding was converted to % of the maximum binding of the ligands to SusD and fitted to a single class of binding sites equation.