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The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch

Nature Structural & Molecular Biology volume 31, pages 255–265 (2024)Cite this article

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Abstract

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

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Fig. 1: Ruminococcus bromii Sas6 is a starch-binding protein that contains two CBMs.
Fig. 2: Sas6 is a compact protein with two BIg domains that orient RbCBM26 and RbCBM74.
Fig. 3: RbCBM74 has an extended groove that accommodates starch double helices.
Fig. 4: Conservation of binding residues among select CBM74 family members.
Fig. 5: W373A, F326A and H289A mediate starch binding by RbCBM74.
Fig. 6: RbCBM74 and RbCBM26 bind separate molecules of G10 in solution.

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Data availability

The X-ray structures and diffraction data reported in this paper have been deposited in the Protein Data Bank under accession codes 7UWU, 7UWVand 7UWW. The SAXS data are deposited in the small angle X-ray scattering database (SASDB) under accession code SASDPE2 (ref. 98). CBM74 sequences were pulled from the CAZy database (http://www.cazy.org; CAZy update, March 2022) and via BLAST against GenBank (https://www.ncbi.nlm.nih.gov/genbank) and/or UniProt (https://www.uniprot.org) databases in March 2022. Native MS data are publicly available in the Deep Blue Data Repository administered by the University of Michigan at https://doi.org/10.7302/5fmh-8f87. The HDX–MS data are publicly available in the Zenodo database under accession number 8371163 (https://zenodo.org/record/8371163). Source data and Supplementary Data files are provided with this paper. All other relevant data supporting the key findings of this study are available within the article, its Supplementary Information or from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

SAXS data collection was performed using the Python-based BioCon software developed at and for the BioCAT beamline, available at https://github.com/biocatiit/beamline-control-user/tree/master/biocon. The UniDec software is available at https://github.com/michaelmarty/UniDec/. Source data are provided with this paper.

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Acknowledgements

This work is primarily supported by a Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship (F31–F31AT011282 to A.L.P.) from the National Center for Complementary and Integrative Health (NCCIH) and a Research Program Project grant (P01-HL149633 to N.M.K.) from the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health (NIH). Next-generation Native MS technologies were supported by the National Institute of General Medical Sciences (NIGMS) of the NIH (R01-GM095832 to B.T.R.). HDX–MS acquisition was supported by the National Science Foundation (NSF) (DBI 2018007 to C.W.V.K.). The structural biology approaches used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The Biophysics Collaborative Access Team is supported by P30-GM138395 from NIGMS-NIH. Use of the Pilatus3 X 1M detector was provided by Grant 1S10OD018090-01 from NIGMS-NIH. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). S.J. and F.M. thank the Slovak Grant Agency VEGA for the financial support by grant no. 2/0146/21. In collaboration with this research, we acknowledge support from the University of Michigan Biomedical Research Core Facilities Light Microscopy Core. For the native MS work, we would like to acknowledge the Biological Mass Spectrometry facility at the University of Michigan Department of Chemistry. The content is solely the responsibility of the authors and does not necessarily represent the official views of VEGA, the National Science Foundation or the NIH.

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Authors and Affiliations

  1. Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI, USA

    Amanda L. Photenhauer, Filipe M. Cerqueira, Krista M. Armbruster & Nicole M. Koropatkin

  2. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    Rosendo C. Villafuerte-Vega & Brandon T. Ruotolo

  3. Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia

    Filip Mareček & Štefan Janeček

  4. Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA

    Tiantian Chen & Craig W. Vander Kooi

  5. Northwestern Synchrotron Research Center-LS-CAT, Northwestern University, Argonne, IL, USA

    Zdzislaw Wawrzak

  6. The Biophysics Collaborative Access Team (BioCAT), Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL, USA

    Jesse B. Hopkins

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  1. Amanda L. Photenhauer
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N.M.K. and A.L.P. conceived the study. A.L.P., F.M.C., R.C.V-V., K.M.A., F.M., T.C., Z.W., J.H., C.W.V.K, S.J., B.T.R. and N.M.K. curated the data. A.L.P., F.M.C., R.C.V-V., K.M.A., F.M., T.C., Z.W., J.H., C.W.V.K., S.J., B.T.R. and N.M.K. conducted formal analysis and data interpretation. N.M.K., A.L.P., C.W.V.K., S.J., B.T.R., Z.W. and J.H. were involved with funding acquisition. A.L.P., F.M.C., R.C.V-V., K.M.A., F.M., T.C., Z.W., J.H., C.W.V.K. and S.J. performed the investigations. A.L.P., F.M.C., R.C.V-V., K.M.A., F.M., T.C., Z.W., J.H., C.W.V.K., S.J., B.T.R. and N.M.K. created the methodology. A.L.P., F.M.C., R.C.V-V., C.W.V.K., S.J. and N.M.K. wrote the original draft of the manuscript. A.L.P., F.M.C., R.C.V-V., F.M., J.H., C.W.V.K., S.J., B.T.R. and N.M.K. were involved with writing, review and editing. N.M.K., J.H., C.W.V.K., S.J. and B.T.R. supervised the study. A.L.P., F.M.C., R.C.V-V., T.C. and S.J. visualized the project.

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Correspondence to Nicole M. Koropatkin.

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Nature Structural & Molecular Biology thanks Stephen Withers and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Small Angle X-Ray Scattering indicates that Sas6 remains mostly compact in solution with minor extension beyond that of the crystal structure.

a. Total subtracted scattering intensity (left y axis) and Rg (right y axis) as a function of time for the SEC-SAXS elution. The elution resolved several peaks, including a single strong monodisperse peak as indicated by the constant radius of gyration (Rg). b. Guinier fit analysis with normalized residual shown in the bottom panel. Rg and I(0) values of 29.44 ± 0.04Å and 0.04 ± 3.65 × 10−5 were obtained and the fit and normalized fit residuals confirmed this peak was monodisperse. The molecular weight of Sas6T from the SAXS data was calculated to be 61.0 kDa (theoretical 68.9 kDa) indicating it is primarily monomeric in solution. c. P(r) versus r normalized by I(0). The Dmax from the P(r) function for Sas6T is 90Å. The overall shape of the P(r) function for Sas6T, calculated by indirect Fourier transform (IFT) using GNOM, has a relatively Gaussian shape that is characteristic of a globular compact particle with the main peak at r = ∼30 Å. There is a small peak at r = 55Å which suggests there are two structurally separate motifs, possibly RbCBM26 and RbCBM74. d. Dimensionless Kratky plot; y = 3/e and x = \(\sqrt{3}\) as dashed gray lines to indicate where a globular protein would peak. The small plateau in the mid to high q region, around qRg = 5 in the dimensionless Kratky plot indicates some extension or disorder in the system. These results suggest the presence of two separate modules with flexibility between them, likely corresponding to the two CBMs. e. FoXS and f. MultiFoXS fits (black) to the Sas6T SAXS data (red) with normalized residual shown in the bottom panel. The FoXS fit had a χ2= 2.46 and showed systematic deviations in the normalized fit residual suggesting significant differences between the lowest energy conformation of Sas6T in the crystal structure and the structure of Sas6T in solution. For MultiFoXS we assigned the linkers between the domains (residues 130-137 and 572-583) as flexible. MultiFoXS gave a best fit with a 1-state solution with a χ2= 0.96 and calculated Rg of 29.2Å which corroborates the Guinier Rg calculation. g. Topology map of BIgA and BIgB domains illustrating the Greek key motif in BIgA and showing the loops that hydrogen bond with one another. h. A surface area analysis of the BIg domains using PISA in CCP4 gives a buried surface area of 353.9Å34. Residues providing hydrogen bonding are represented by stick side chains and the hydrogen bonds are shown by dashed yellow lines.

Extended Data Fig. 2 RbCBM74 is a singular globular domain, most similar to TmCBM9.

a. Structure of RbCBM74 (PDB 7uww) colored from N-terminus (blue) to C-terminus (red). b. Short β-strands leading into and out of RbCBM74 domain are colored in red and blue. c. Overlay of TmCBM9 (gold) (PDB 1i82-A) and RbCBM74 (blue). The DALI server calculated an RMSD of 3.2Å and sequence identity of 17%. d. Close-up view of TmCBM9 binding site showing the two TmCBM9 Trp residues involved in binding cellobiose (gold) and W373 of RbCBM74 (blue) which lies in the same region but is occluded from the surface by a loop containing residues 374-384. e. Zoomed in view of calciums coordinated in the RbCBM74 domain with side chains shown in sticks, main chain shown in lines and Ca2+ ions by yellow spheres. Atomic distances are shown in Å and residues are labeled. Residues are colored by element with oxygen shown in red.

Extended Data Fig. 3 RbCBM26 shares a conserved binding site with other CBM26.

The top structural homologs of RbCBM26 from DALI36,42 are the CBM25 from Bacillus halodurans C-125 (BhCBM25) from α-amylase G-6 (PDB ID: 2C3V-A, Z-score: 12.4, RMSD 1.9Å, identity: 16%) and CBM26 (BhCBM26) from the same enzyme (PDB ID: 6B3P-B, Z-score: 12.1, RMSD 1.9Å, identity: 20%)41. Another top DALI result is ErCBM26b of Amy13K from Eubacterium rectale (PDB ID 2C3H-B, Z-score: 10.8, RMSD 1.7Å, identity: 19%)43. a. Sequence alignment of RbCBM26 (RBL236_00020), ErCBM26 (ERE_20420), BhCBM26 (BH0413), and LaCBM26 (Q48502). Conserved binding site residues are indicated by a red arrow while variable residues are indicated by a blue arrow and provide hydrogen bonding. b. Overlay of RbCBM26 (green) with BhCBM26 (PDB 2c3h, orange), and ErCBM26 (PDB 6b3p, purple). c. Overlay of unliganded RbCBM26 (blue) and ACX-bound RbCBM26 (green) showing that loop 1 does not move upon ligand binding. b-strands are numbered for reference.

Extended Data Fig. 4 Representative ITC graphs of Sas6 domains.

Sas6T, RbCBM26, and BIg-RbCBM74-BIg binding to a. potato amylopectin, b. maltodecaose (G10), and c. α-cyclodextrin (ACX). Note that exothermic heat release is denoted with an upward peak on this machine.

Extended Data Fig. 5 RbCBM74 selects a double helical ligand geometry.

a. Overlay of RbCBM74 from Sas6T structure (PDB 7uww) in blue with RbCBM74 from BIg-RbCBM74-BIg co-crystal structure (PDB 7uwv) in deep teal. b. Loop from G374-G382 demonstrating that the unliganded loop (blue) occludes W373 but moves to allow access to W373 in the ligand-bound structure (deep teal). c. An extended view of the geometry of the G10 ligand. Intramolecular hydrogen bonds (3.6Å cutoff for ideal geometry and 3.2Å with minimal acceptable geometry) within and between G10 chains are shown in slate. Φ (O5-C1-O4′-C4′) and ψ (C1-O4′-C4′-C5′) angles of the Glc linkages in the G10 double helix ligand are labeled with G10A in magenta and G10B in grey. d. The geometry of the G10 ligand more closely resembles that of double helical B starch (cyan)48 than single helical cycloamylose (yellow, 1c58)50. Models were manually aligned in PyMOL to compare the angles, pitch, and period of the helical turns.

Extended Data Fig. 6 HDX-MS analysis of RbCBM74.

a. Heatmap of exchange dynamics of BIg-RbCBM74-BIg. All values are the average of three replicates. b. Representative differential uptake for peptides that both showed no significant difference (upper panels) and those which showed significant differential decreased deuteration (lower panels) in the G10 bound BIg-RbCBM74-BIg. Data points are represented by the mean +/− standard deviation.c. Heatmap of the differential exchange dynamics of BIg-RbCBM74-BIg in the absence and presence of G10. Blue represents lower exchange (protection) in the G10 bound form and red higher exchange in the G10 bound form. All values are the average of three replicates.

Extended Data Fig. 7 Phylogenetic tree of the 99 CBM74 family members.

a. A maximum-likelihood tree covering 99 sequences with emphasis on the two experimentally characterized CBM74s, Sas6 from Ruminococcus bromii (No. 28, blue cluster) and the subfamily GH13_32 α-amylase from Microbacterium aurum (No. 52; cyan cluster)35. The bootstrap values higher than 70% are shown. For details concerning all 99 CBM74 sequences, see Supplementary Table 1.

Extended Data Fig. 8 Representative ITC graphs of RbCBM74 mutations.

BIg-RbCBM74-BIg, H289A, F236A, and W373A mutations binding to a. maltodecaose (G10), and b. potato amylopectin (PAP). Note that exothermic heat release is denoted with an upward peak on this machine.

Extended Data Fig. 9 Mass spectra of Sas6 constructs at different ligand concentrations (0–300µM) and a fixed protein concentration of 5µM.

Charge states for unbound protein are annotated with an orange dashed line. Peaks corresponding to different bound states are observed after each charge state of the unbound protein. Spectra of a. BIg-RbCBM74-BIg or b. Sas6T in equilibrium with G10. Spectra of c. BIg-RbCBM74-BIg or d. Sas6T in equilibrium with G14.

Source data

Extended Data Table 1 Table of Φ (O5-C1-O4′-C4′) and ψ (C1-O4′-C4′-C5′) angles of G10 ligand bound by RbCBM74
Full size table

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figs. 1–5. Legends are with each table or figure.

Reporting Summary

Source data

Source Data Fig. 1

Raw microscopy images and unprocessed gels and blots.

Source Data Fig. 2

Raw data used to generate Fig. 2f.

Source Data Fig. 2

Unprocessed gels for Fig. 2g.

Source Data Fig. 4

All 99 sequences annotated by dbCAN with data used to generate Fig. 4b.

Source Data Fig. 5

Raw data used to generate Fig. 5a.

Source Data Fig. 5

Uncropped gel for Fig. 5c.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Table 1

Phi-psi angle calculations.

Source Data Extended Data Fig. 9

Statistical Source data for relative intensities in Fig. ED9.

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Photenhauer, A.L., Villafuerte-Vega, R.C., Cerqueira, F.M. et al. The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch. Nat Struct Mol Biol 31, 255–265 (2024). https://doi.org/10.1038/s41594-023-01166-6

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