Amuc_1102 from Akkermansia muciniphila adopts an immunoglobulin-like fold related to archaeal type IV pilus
Introduction
A large variety of microorganisms reside in the gastrointestinal tract of humans and animals. These microorganisms are called intestinal microorganisms, which are the largest microbial group in humans and play an important role in regulating host metabolism and low-grade inflammation [1,2]. Akkermansia muciniphila is considered as a promising probiotic and is one of the key roles in the gut microbiota, accounting for 1–4% of the fecal microbiota in healthy adults [3,4]. Its abundance is negatively correlated with several intestinal diseases (including inflammatory bowel disease, obesity, diabetes, etc.) and also plays a role in cancer treatment [[5], [6], [7], [8], [9]]. The strain A. muciniphila MucT (ATTC BAA-835) was first isolated and cultured in vitro by Derrien in 2004 [10]. It belongs to Gram-negative strictly anaerobic bacteria and can degrade mucin [10,11].
In Gram-negative bacteria, outer membrane proteins play important roles in the adhesion and information transmission of bacteria to the host [12]. The proteins of the Amuc_1098 to Amuc_1102 gene cluster are highly expressed in the membrane protein component of A. muciniphila [13]. In the genome and phylogenetic analysis of the Akkermansia genus, it was found that the Amuc_1098 to Amuc_1102 gene cluster is conservative in the length, sequence, and direction of genes of most species [14]. It was reported that Amuc_1100 in this gene cluster has been experimentally proven to be located on the outer membrane and is a highly abundant pili-like protein [13]. Although sequence alignments between Amuc_1100 and proteins related to pilus formation presented similarities lower than 20%, it shares a similar fold with PilN and PilO of the type IV pilus (T4P) system by structure alignment [15,16]. Amuc_1098 encodes the channel protein PilQ of the T4P system, which is critical for bacterial adhesion and motility [17,18]. Amuc_1101 encodes a cell division protein FtsA, which is homologous to PilM of the T4P system. Amuc_1099 and Amuc_1102 were marked as uncharacterized proteins, and their functional information could not be obtained by sequence analysis [13].
T4P, like other pili, is a type of filamentous appendage on the surface of bacteria. It has a crucial function in host cell adherence as an initial step in colonization [17]. Among the reported articles, the important proteins in the T4P assembly system of Pseudomonas aeruginosa, Neisseria meningitidis, Myxococcus xanthus, etc. are concentrated in a continuous gene cluster, so it is speculated that Amuc_1099 and Amuc_1102 may also be associated with the pili formation of A. muciniphila [[19], [20], [21]]. In this work, we purified the Amuc_1102 by in vitro overexpression and solved its crystal structure at 1.75 Å resolution. A comparison with the known structure showed that Amuc_1102 shared a similar fold with three archaeal T4P proteins, Pilin, FlaG, and FlaF [[22], [23], [24]]. However, Amuc_1102 has a different polymerization state from these proteins.
Section snippets
Overproduction and purification of Amuc_1102
Full-length Amuc_1102 has 238 amino acids according to the entry B2UR43 in UniProt (https://www.uniprot.org/). The residues 1–23 of Amuc_1102 were predicted as a signal peptide. The DNA sequence encoding residues 24–238 of Amuc_1102 was synthesized by GENERAL BIOSYSTEMS (China) and cloned into a p-28a vector with restriction enzymes Nde I and Xho I. The p-28a-Amuc_1102 expression plasmid was identified by restriction endonuclease digestion and further verified by DNA sequencing (Sangon Biotech,
Purification and structure determination of Amuc_1102
Residues 24–238 of Amuc_1102 were cloned into p-28a vector with N-terminal and C-terminal His6-tags and purified through a Ni-NTA affinity column. The theoretical molecular weight of Amuc_1102 was 26.7 kDa. The target protein was eluted with 300 mM imidazole and was further purified by gel exclusion chromatography (Fig. 1A). As shown by SEC, Amuc_1102 existed in a trimeric state in solution, because the molecular weight of the peak position of Amuc_1102 was ∼70 kDa (Fig. 1A). The protein
Conclusions
We determined the crystal structure of the Amuc_1102, which adopted an Ig-like fold and existed as a trimer both in the crystal and in the solution. A triple mutation of Amuc_1102, F202R/W203R/Y204R, disrupted the trimerization of the protein. Amuc_1102 shared a similar fold to three archaeal proteins related to the T4P-like structure, Pilin, FlaF, and FlaG, indicating a similar function. However, the assembly of Amuc_1102 differed from those of Pilin, FlaF, and FlaG. The structural study of
Acknowledgments
This work was supported by the Key Research Program of the Education Department of Anhui Province (Grant No. KJ2019ZD02 to M.Z.). We thank the staff of the Shanghai Synchrotron Radiation Facility (SSRF) for assistance with X-ray data collection.
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