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. 2019 Jul 23;14(7):e0220045.
doi: 10.1371/journal.pone.0220045. eCollection 2019.

Structure and Functional Analysis of a Bacterial Adhesin Sugar-Binding Domain

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Free PMC article

Structure and Functional Analysis of a Bacterial Adhesin Sugar-Binding Domain

Tyler D R Vance et al. PLoS One. .
Free PMC article

Erratum in

Abstract

Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-Å resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar's two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein's ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-μM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Organization of adhesins with and without PA14 domains.
Domain architectures are shown for several RTX adhesins, oriented with N termini on the left. Colour scheme: cyan = bacterial membrane anchoring domains, blue = region of tandem repeat domains, yellow = PA14 domain, burgundy = von Willebrand Factor A-like domain, pink = Vibrio-like peptide-binding domain, orange = ice-binding domain, red = Type 1 Secretion Signal and RTX repeats, white = regions of unknown/unpredicted composition.
Fig 2
Fig 2. Sequence alignment of PA14 domains.
Sequences of PA14 domains from Marinobacter hydrocarbonoclasticus (MhLap), Marinomonas primoryensis (MpIBP), Pseudomonas mendocina and resinovorans (PmRTX and PrRTX, respectively), Vibrio cholerae (VcRTX), Candida glabrata (EpA1), and Bacillus anthracis (PA20) were aligned using the MergeAlign software [45]. Sequences with alignment scores above 60% are coloured dark blue, while sequences with alignment scores between 30 and 40% are shown in light blue. Proposed Ca2+-binding residues are boxed with red squares. Residue conservation is indicated as follows: * = 100%,: = ~80%,. = ~70%. The conserved residues are bolded. Residue numbers for the MhPA14 construct are given. Secondary structure taken from the solved structure of MpIBP PA14 domain is shown above the alignment, with beta-strands coloured dark blue, alpha-helices coloured green, and loops coloured black. Bold lines sit above the sequence for three loops that make up the supposed sugar-binding site of the bacterial PA14 domains. Note: sequences with previously solved protein structures are marked with an asterisk.
Fig 3
Fig 3. MhPA14 purification and affinity for Superdex 200 size-exclusion resin.
A) SDS-PAGE tracking of MhPA14 purification through induction, extraction, and nickel-affinity chromatography. Lane 1 = pre-induction, Lane 2 = post-induction, Lane 3 = lysate insoluble fraction, Lane 4 = lysate soluble fraction, Lane 5 = elution from nickel-NTA agarose column. B) SDS-PAGE showing secondary purification of MhPA14 following nickel-affinity via anion-exchange chromatography (QSeph Lane), as well as the pooled fractions following S200-affinity elution with glucose and EDTA, respectively. C) Elution profiles of MhPA14 interacting with a Superdex 200 size-exclusion column under varying gradient conditions. The absolute absorbance at 280 nm (primary y-axis) was measured over 65 mL of buffer flow, during which an glucose gradient (top, red), an EDTA gradient (middle, green), or no gradient (bottom) was run.
Fig 4
Fig 4. Circular dichroism spectra assaying calcium dependence and thermal denaturation of MhPA14 structure.
Overlaid CD spectra of MhPA14 (25 μM) during a Ca2+ titration (top), and increasing temperature in the presence of Ca2+ (middle) or EDTA (bottom).
Fig 5
Fig 5. MhPA14 structure with glucose bound.
The structure for MhPA14 presented as a cartoon diagram from two viewpoints, 180° rotated. The protein is coloured by progression of primary structure from the N terminus in blue, to the C terminus in red. Calcium ions are shown as grey spheres, coordinated water molecules are shown as smaller cyan spheres, and the glucose molecule is shown as a stick structure with white for carbon and red for oxygen. Inset panels detailing the coordination for each calcium ion are shown, with red dashed lines indicating coordinate bonds between the calcium and the labelled residue or water molecule.
Fig 6
Fig 6. Sugar-binding site of MhPA14.
A) Side view of the sugar-binding cleft, produced by loops 1, 2, and 3. Calcium 1 and its coordinated glucose molecule are shown as a grey sphere and white ring, respectively. B) Important residues of the sugar-binding site. Residues are labelled and coloured the same as for Fig 5. Calcium 1 is reduced in size to improve visibility. Red dashed lines are contacts between sugar and calcium, while black dashed lines are between sugar and protein. The 2 Fo−Fc map (σ = 1.5) of the glucose molecule and bound calcium are shown as a grey mesh with carbon atoms of the hexose numbered.
Fig 7
Fig 7. Structural comparison of MhPA14 to other PA14 domains.
Structural alignment of MhPA14 (orange) to either A) MpIBP PA14 (blue) or B) EpA1 A domain (green). Close-up views of the sugar-binding site for both alignments are shown in boxes. C) Sugar-binding pockets of these three PA14 structures. Protein topology is coloured grey, sugars are coloured with oxygen in red, nitrogen in blue, and the carbon atoms in the respective colours of their structures shown in A and B. D) The orientation of these same sugars as they coordinate to the calcium ions in their respective structures. E) Orientation of fucose (purple) from LecB.
Fig 8
Fig 8. Dextran-based competitive binding assay to determine relative affinity of MhPA14 for sugars.
The progressive removal of GFP_MhPA14 from dextran-based resin–using a series of monosaccharide hexoses (top) and disaccharides (bottom)–is shown. The presence of released protein was measured using absorbance at 280 nm, normalized to the maximum released protein value (Bmax). Data points were done in triplicate, with standard deviation shown via error bars. Data were fit with non-linear regression.
Fig 9
Fig 9. Isothermal titration calorimetry of MhPA14 binding to select sugars.
ITC of the interaction between MhPA14 and fucose (A) glucose (B) 2-Deoxy-glucose (C) and galactose (D). A combination of the raw reads over the duration of the titration (top) and the fitted curve (bottom) are shown, along with the calculated Kd value for each sugar and the calculated stoichiometry. The fucose and glucose values are averages from triplicate runs.
Fig 10
Fig 10. Binding of MhPA14 to Glycan arrays.
A) Fluorescent measurements of an array of 585 glycans of varying complexity following incubation with GFP_MhPA14 (200 μg/mL). The fluorescence measured for each glycan is an average of four replicate spots. The four glycan spots that fluoresced above 8000 RFU are labelled, and their structures are presented on the right. Blue squares = N-acetylglucosamine, blue circles = glucose, yellow squares = N-acetylgalactosamine, yellow circles = galactose, green circles = mannose, red triangle = fucose. Terminal sugars proposed to be strong-binders via the competitive assay are outlined in red. B) Fluorescent measurements from a second array, containing eighteen glycans extracted from biological sources following incubation with GFP_MhPA14 (50 ug/mL) and detected through anti-GFP antibody. The fluorescence measured for each glycan is an average of two replicate spots. The highest fluorescing glycan is coloured blue, and its repeating structure is shown on the right using the same colour scheme as in A).

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Grant support

This project was funded by a Natural Science and Engineering Research Council (NSERC, http://www.nserc-crsng.gc.ca/index_eng.asp) Discovery Grant (RGPIN-2016-04810). PLD holds the Canadian Research Chair in Protein Engineering, and TDRV holds a Canadian Graduate Scholarship through NSERC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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