Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization

Abstract

Serratia marcescens is an opportunistic pathogen that commonly causes hospital-acquired infections and can utilize chitin-enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Å resolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X-rays to 2.70 Å resolution. Preliminary crystallographic analysis shows that both SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P4₂2₁2 and P4₁2₁2, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = β = γ = 90°, while the crystal of SmChiP in complex with chitohexaose has unit-cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = β = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.

Keywords: Serratia marcescens; chitin; chitoligosaccharides; chitoporin; outer membrane proteins; sugar transport; sugar-specific porins.

Figure 1

Multiple sequence alignment of SmChiP from S. marcescens (UniProtKB entry A0A0P0QBS3, this study) with EcChiP from E. coli (UniProtKB entry P75733) and VhChiP from V. campbellii (UniProtKB entry L0RVU0). The sequence alignment was generated by Clustal 2.1 and displayed by Jalview version 2.11.1.0. Blue shades indicate strictly conserved amino acids and red circles the amino-acid residues of VhChiP that bind to chitohexaose (Aunkham et al., 2018 ▸).

Figure 2

Preparation of SmChiP expressed in E. coli. (a) A representative elution profile of SmChiP eluted from a HiPrep 16/100 Sephacryl S-200 HR column with 10 mM HEPES pH 7.4, 100 mM LiCl, 0.1% LDAO (upper panel). SDS–PAGE showed that a single protein peak with an elution volume of 65–100 ml contained SmChiP as the major component (lower panel). (b) SDS–polyacrylamide gel showing migration of the purified SmChiP after heating at 373 K for 10 min (lane +) and without heat treatment (lane −). Lane Std contains molecular-weight markers (labelled in kDa). (c) Isotopic ionization pattern of SmChiP as determined by electrospray mass spectrometry. The inset shows the peak deconvolution.

Figure 3

Single crystals of SmChiP. (a) Crystals of SmChiP obtained from the initial screen condition MG1_D6. (b) Crystals of SmChiP obtained from the optimized condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0. (c) Crystals of SmChiP co-crystallized with chitohexaose from the optimized condition 19%(w/v) PEG 1500, 0.2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0.

Figure 4

High-resolution images of X-ray data from (a) SmChiP and (b) SmChiP in complex with chitohexaose.