Functional analysis of an unusual porin-like channel that imports chitin for alternative carbon metabolism in Escherichia coli

Abstract

Escherichia coli have the genetic potential to use chitin as a carbon source in the absence of glucose, importing it via the chitin-uptake channel EcChiP for processing by the glucosamine catabolic pathway. The chip gene is usually not expressed when E. coli are grown on glucose-enriched nutrients, providing a general regulatory mechanism for the pathway. EcChiP is unusual in that it is homologous to porins and monomeric instead of trimeric, the typical form of sugar-specific channels, making it unclear how this channel operates. We recently reported that EcChiP could form a stable channel in lipid membranes and that the channel is specific for chitooligosaccharides. This report describes the biophysical nature of sugar-channel interactions and the kinetics of sugar association and dissociation. Titrating EcChiP with chitohexaose resulted in protein fluorescence enhancement in a concentration-dependent manner, yielding a binding constant of 2.9 × 10⁵ m^-1, consistent with the value of 2.5 × 10⁵ m^-1 obtained from isothermal titration microcalorimetry. Analysis of the integrated heat change suggested that the binding process was endothermic and driven by entropy. Single-channel recordings confirmed the voltage dependence of the penetration of chitohexaose molecules into and their release from EcChiP. Once inside the pore, the sugar release rate (k_off) from the affinity site increased with elevated voltage, regardless of the side of sugar addition. Our findings revealed distinct thermodynamic and kinetic features of the activity of sugar-specific EcChiP and advance our knowledge of the physiological possibility of chitin utilization by non-chitinolytic bacteria.

Figure 1.

EcChiP sequence analysis for fluorescence spectroscopy. The amino acid sequence of EcChiP (P75733) was retrieved from the Uniprot database. The Trp residues that are putatively important for fluorescence spectroscopy are indicated in red. The secondary structure of E. coli was constructed by ESPript 3.0, according to the structure of P. aeruginosa OprD (Protein Data Bank code 2odj). β-Strands are marked with gray arrows, and α-helices are marked with gray spirals.

Figure 2.

Protein–ligand binding studied by fluorescence spectroscopy. A and B, surface and cartoon representation of top view of model EcChiP (A) and surface and cartoon representation (B) of EcChiP, showing a top view and a 90° rotation representing a cross-section of the side view. All Trp residues in the EcChiP pore are represented as red stick-and-ball models. β-Barrels, smooth loops, and the surface of EcChiP are shown in gray, and α-helices are shown in yellow. Trp-22, Trp-120, Trp-138, Trp-316, and Trp-317 are positioned inside the pore, whereas Trp-98 (in L2), Trp-176 (in L4), Trp-364 (in L8), and Tyr-417 (in L9) can protrude into the channel from extracellular side. Trp-342 is part of a periplasmic turn. Other tryptophan residues (positions 46, 168, 266, 288, and 357) are located at different positions around the outer surface of the barrel. C, chromatographic profile of EcChiP purification with a HiPrep 16/60 Sephacryl S-200 high-resolution column connected to an ÄKTA Prime plus FPLC system. Bound proteins were eluted with a 20 mm phosphate buffer, pH 7.4, in the presence of 0.05% LDAO. SDS-PAGE analysis of purified EcChiP is shown in an inset (EcChiP samples were heated). D, effects of chitohexaose on the intrinsic fluorescence of EcChiP. Aliquots of chitohexaose were added to 720 ng/ml (final concentration) of EcChiP as shown. E, binding curves for chitohexaose. Binding curves were evaluated with a non-linear regression function available in Prism version 5.0 (GraphPad Software) using a model based on a single binding site. Experiments were performed in triplicate, and the results shown are averages.

Figure 3.

Calorimetric titration of EcChiP with chitohexaose and maltohexaose. A, ITC profile of the binding of chitohexaose to EcChiP, with three different controls (control traces are shown by offsetting −0.06, −0.15, and −0.32 μcal·s⁻¹ for ligand to buffer, buffer to buffer, and buffer to EcChiP, respectively). B, integrated heat of binding of chitohexaose obtained from raw data, after subtracting the controls. The solid line represents the best fit to the experimental data using a one-site model from Microcal PEAQ-ITC. The inset is the signature plot for chitohexaose binding to EcChiP with error bars for three independent experiments. C, ITC profile of the binding of maltohexaose to EcChiP, with control trace maltohexaose to buffer offset by −0.05 μcal·s⁻¹. D, integrated heat of binding of maltohexaose obtained from raw data, indicating no detectable binding.

Figure 4.

Channel activity of purified EcChiP in an artificial lipid bilayer. Lipid bilayers were formed across a 70-μm aperture by the lowering and raising technique, using 5 mg ml⁻¹ 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine in n-pentane and 1 m KCl in 20 mm HEPES, pH 7.4, on both sides of the chamber. The protein was added on the cis side. Ion current fluctuations were monitored for 120 s at applied potentials of ±100 mV with sugars added on either the cis or the trans side. Here, only current traces for 1,000 ms at +100 mV, trans are presented. A, fully open state of EcChiP before sugar addition. B, 20 μm chitohexaose (GlcNAc₆). C, 100 μm chitosan hexamer. D, recording with maltohexaose at a final concentration of 200 μm.

Figure 5.

Typical ion current recordings through a single EcChiP channel at different voltages. Ion current fluctuations were monitored for 120 s at applied potentials of ±100 mV with sugars on either the cis or the trans side in 1 m KCl in 20 mm HEPES, pH 7.4. Here, only current traces for 1,000 ms at cis are presented. A–D, ion current fluctuations in a single EcChiP channel in the presence of 5 μm chitohexaose at +25, +50, +100, and +150 mV, respectively. E, association rates on cis with potentials ranging from ±25 to ±199 mV. F, dissociation rates on cis with potentials ranging from ±25 to ±199 mV. G, association rates on trans with potentials ranging from ±25 to ±199 mV in the presence of 5 μm chitohexaose. H, dissociation rates on trans with potentials ranging from ±25 to ±199 mV in the presence of 5 μm chitohexaose.

Figure 6.

Reduction of single EcChiP channel conductance by increasing concentrations of chitohaxaose. Ion current fluctuations were monitored for 120 s at applied potentials of ±100 mV with sugar addition on either the cis or the trans side. Here only current traces for 1,000 ms are presented (A–D, left panels) for four different chitohexaose concentrations at trans/+100 mV, with the corresponding histograms (A–D, right panels). E, dependence of association rates (k_on) on chitohexaose concentration. F, dependence of dissociation rates (k_off) on chitohexaose concentration.

Figure 7.

Summary of the data obtained from ITC, fluorescence spectroscopy, and electrophysiology experiments, comparing binding constants of chitohexaose with EcChiP in solution and at the single molecule level.