Structural basis for chitin acquisition by marine Vibrio species

Abstract

Chitin, an insoluble polymer of N-acetylglucosamine, is one of the most abundant biopolymers on Earth. By degrading chitin, chitinolytic bacteria such as Vibrio harveyi are critical for chitin recycling and maintenance of carbon and nitrogen cycles in the world's oceans. A decisive step in chitin degradation is the uptake of chito-oligosaccharides by an outer membrane protein channel named chitoporin (ChiP). Here, we report X-ray crystal structures of ChiP from V. harveyi in the presence and absence of chito-oligosaccharides. Structures without bound sugar reveal a trimeric assembly with an unprecedented closing of the transport pore by the N-terminus of a neighboring subunit. Substrate binding ejects the pore plug to open the transport channel. Together with molecular dynamics simulations, electrophysiology and in vitro transport assays our data provide an explanation for the exceptional affinity of ChiP for chito-oligosaccharides and point to an important role of the N-terminal gate in substrate transport.

Fig. 1

X-ray crystal structure of in vitro-folded VhChiP. a SDS-PAGE gel of OM-expressed (native; 1), in vitro-folded (2) and truncated VhChiP (3) loaded both as non-boiled (left lanes) and boiled samples (right lanes). b Side view and c top view cartoon presentations of the in vitro-folded VhChiP trimer. d, e Top view superpositions of in vitro-folded VhChiP (red) with Neisseria meningitidis PorB (green; d) and Delftia acidovorans Omp32 (blue; e). f Superposition of in vitro-folded VhChiP (red) with OM-expressed VhChiP (green). The N-termini have been labeled

Fig. 2

OM-expressed VhChip has N-terminally plugged channels. a, b Surface representations (top view) of in vitro-folded VhChiP (a) and natively expressed VhChiP (b). The barrel lumen of the natively expressed VhChiP is occupied by the N-terminus of a neighbor barrel. c N-terminal insertion mode of natively expressed VhChiP. The N-terminus of one barrel (blue) plugs the lumen of a neighbor barrel (red). d Polar interactions between the inner barrel wall (red) with the N-terminus of a neighbor barrel (blue)

Fig. 3

Behavior of the pore plug in MD simulations. a Average Cα RMSF of the protein dynamics in vitro-folded (red) and OM-expressed (blue) VhChiP. In addition to the differences for the first 14 residues, a difference in the RMSF for residues 143–146 in loop L3 can be clearly observed. b Cumulative total charge as a function of time for a 1 M KCl solution at 200 mV. In addition, the cumulative charges for K⁺ and Cl⁻ ions separately are shown as well. The data was averaged over the three simulations and the slopes of these curves yield the respective currents. c Free-energy surfaces of unbinding of the N-terminus in wild type and mutant channels. The shaded areas indicate the respective error estimates. The arrow represents the position of the N-terminal plug in the crystal structure of OM-expressed VhChiP

Fig. 4

Chitohexaose binding to in vitro-folded VhChiP. a Overview from the extracellular side. b Slabbed view from the side, with substrate residues labeled. c Schematic, showing interactions of key VhChiP residues with chitohexaose

Fig. 5

Alignment of VhChiP orthologs from Vibrio species. Observed secondary structure elements have been indicated (orange cylinders, helices; green arrows, β-sheets). The first helix that is part of the N-terminal plug is presented in red. Key aromatic (purple) and hydrophilic residues (cyan) interacting with chitooligosaccharides are colored. The following orthologs have been aligned: V. harveii (GenBank ID: HF558985.1), V. parahaemolyticus (GenBank ID: CPO12950.1), V. cholera (GenBank ID: DQ774012.1), V. furnissii (GenBank ID: AF129934.1), and V. fischeri (GenBank ID: CP001139.1). Chitoporin from V. harveyi (UniProtKB/TrEMBL entry: L0RVU0) was used as protein template to identify putative chitoporins. The alignment was generated using “CLUSTALW” algorithm in the DNASTAR package and displayed in Genedoc. The secondary structure of VhChiP was generated by ESPript v. 2.2

Fig. 6

Chitotetraose binding to VhChiP displaces the N-terminal plug. Side views in the same orientations, showing the bound chitotetraose in natively expressed VhChiP (left panel) and chitohexaose in in vitro-folded VhChiP (right panel). Residues interacting with the sugars are labeled

Fig. 7

Voltage-induced ejection of the pore plug in VhChiP. a–c Single-channel electrophysiology of OM-folded VhChiP (a), in vitro-folded VhChiP (b) and truncated VhChiP (c). Left panels show typical traces at −100 mV, whereas the center and right panels show the current–voltage profiles. Data represent mean ± s.d., n = 3. d Stepwise voltage-induced closure of OM-folded VhChiP channels at 200 mV. e, f Typical traces observed at ±2.5 mV (e) and at ±10 mV (f). The data shown in d–f are for the same experiment, with protein only added at the start (d). Traces shown are representative of three independent experiments

Fig. 8

Channel blockage by an N-terminal plug peptide. Single-channel current traces at −100 mV (1 M KCl) of truncated VhChiP in the absence (a) and presence of 100 μM (b) of the plug peptide DGANSDAAK. c E. coli OmpF traces in the presence of 200 μM peptide. Traces shown are representative of three independent experiments

Fig. 9

Chitoxexaose binds to VhChiP with high affinity. Single-channel electrophysiology experiments for natively expressed VhChiP (a), refolded VhChiP (b), and N-terminally truncated VhChiP (c). The fully open VhChiP trimeric channels were exposed to different concentrations of chitohexaose. Ion current traces were acquired at −100 mV with sugar added on the cis side. For full trace recordings of 2 min duration and analyses see Supplementary Figs. 3–6

Fig. 10

The N-terminus is important for chito-oligosaccharide substrate transport. Proteoliposome swelling assays in the presence of various mono- and oligosaccharides (a) as well as chito-oligosacharides of different lengths (b). No permeation of any sugars was observed when tested with control liposomes without VhChiP. “Folded” denotes in vitro-folded VhChiP. Data represent mean ± s.d., n = 3. For details see Methods