Crystal Structure of a Mucus-binding Protein Repeat Reveals an Unexpected Functional Immunoglobulin Binding Activity

Donald A Mackenzie,Louise E Tailford,Andrew M Hemmings,Nathalie Juge

doi:10.1074/jbc.m109.040907

Donald A Mackenzie, Louise E Tailford + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.040907

Copy DOI

Abstract

Lactobacillus reuteri mucus-binding protein (MUB) is a cell-surface protein that is involved in bacterial interaction with mucus and colonization of the digestive tract. The 353-kDa mature protein is representative of a broadly important class of adhesins that have remained relatively poorly characterized due to their large size and highly modular nature. MUB contains two different types of repeats (Mub1 and Mub2) present in six and eight copies, respectively, and shown to be responsible for the adherence to intestinal mucus. Here we report the 1.8-A resolution crystal structure of a type 2 Mub repeat (184 amino acids) comprising two structurally related domains resembling the functional repeat found in a family of immunoglobulin (Ig)-binding proteins. The N-terminal domain bears striking structural similarity to the repeat unit of Protein L (PpL) from Peptostreptococcus magnus, suggesting binding in a non-immune Fab-dependent manner. A distorted PpL-like fold is also seen in the C-terminal domain. As with PpL, Mub repeats were able to interact in vitro with a large repertoire of mammalian Igs, including secretory IgA. This hitherto undetected activity is consistent with the current model that antibody responses against commensal flora are of broad specificity and low affinity.

Highlights

Plex mixture of large, highly glycosylated proteins (3), covers the epithelial cells of the intestine and offers an attachment site for the colonizing bacteria
mucus-binding repeats (Mub) repeat-containing proteins are most abundant in lactic acid bacteria (LAB), with the highest abundance in lactobacilli of the gastrointestinal tract (GIT), strongly suggesting that the Mub repeat is a functional unit specific to LAB that could fulfill an important function in host-microbe interactions
After the production of SeMet-labeled proteins, ESI-MS analysis revealed that SeMet incorporation was essentially complete (92–98%) at the two or three methionine residues present in the recombinant 185-amino acid wild-type Mub-R5 and L48M proteins, respectively

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification of Mub Repeats—L. reuteri 1063 was obtained from the American Type Culture Collection (strain ATCC 53608). Protein Binding by Slot-blot Analysis—Recombinant Mubs were labeled with fluorescein isothiocyanate at pH 9.3 using an adapted standard protocol (see supplemental materials). Crystals were cryoprotected by increasing the concentration of polyethylene glycol 3350 in the drops to 30% (v/v) and a native diffraction dataset was subsequently collected to 1.8-Å resolution at 100 K. These crystals were of space group P212121 and contained two copies of the protein in the asymmetric unit with a solvent content of 48% (v/v). Refinement at 1.8-Å resolution resulted in a final structural model lacking only the C-terminal alanine residue in both independent molecular copies of Mub-R5 in the crystallographic asymmetric unit. Analysis of functional regions via evaluation of residue evolutionary conservation scores was performed with CONSURF (42) using sequence alignments generated using T-COFFEE (43) and visualized with ESPript (44)

RESULTS

Most favored

DISCUSSION