Structure Report
Crystal structure of the 30 K protein from the silkworm Bombyx mori reveals a new member of the β-trefoil superfamily

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Abstract

The hemolymph of the fifth instar larvae of the silkworm Bombyx mori contains a group of homologous proteins with a molecular weight of approximately 30 kDa, termed B. mori low molecular weight lipoproteins (Bmlps), which account for about 5% of the total plasma proteins. These so-called “30 K proteins” have been reported to be involved in the innate immune response and transportation of lipid and/or sugar. To elucidate their molecular functions, we determined the crystal structure of a 30 K protein, Bmlp7, at 1.91 Å. It has two distinct domains: an all-α N-terminal domain (NTD) and an all-β C-terminal domain (CTD) of the β-trefoil fold. Comparative structural analysis indicates that Bmlp7 represents a new family, adding to the 14 families currently identified, of the β-trefoil superfamily. Structural comparison and simulation suggest that the NTD has a putative lipid-binding cavity, whereas the CTD has a potential sugar-binding site. However, we were unable to detect the binding of either lipid or sugar. Therefore, further investigations are needed to characterize the molecular function of this protein.

Introduction

The silkworm Bombyx mori has been domesticated in China as a major economic insect for silk production for 5000 years. Similar to other Lepidoptera, the silkworm has an open circulatory hemolymph system (Vierstraete et al., 2003). The silkworm “30 K” proteins are a group of plasma proteins with a molecular weight of approximately 30 kDa, which are synthesized in fat bodies and secreted into the hemolymph in the later stage of the fourth through to the fifth instar larvae (Mori et al., 1991). 30 K proteins are transported from the hemolymph back to the fat body cells during the larval-pupal transformation (Mine et al., 1983). In sexually mature female moths, these proteins are transported from the fat bodies to yolk granules, where they become the second major yolk protein of the eggs, after vitellin (Maki and Yamashita, 1997). During embryonic development, the 30 K proteins are detectable and eventually vanished after the larval hatching (Zhong et al., 2005).

30 K proteins were previously identified as lipoproteins (Gamo, 1978). However, the lipid content of these proteins and their functional relationships to human lipoproteins were unknown (Chapman, 1980). 30 K proteins had also been shown to bind to glucose, maltose and glucans, suggesting their involvement in insect self-defense systems (Ujita et al., 2005). In addition, 30 K proteins had been found to exhibit anti-apoptotic activity (Kim et al., 2001) and to be involved in translocating chymotrypsin inhibitor-8 to the membrane of the midgut (Ueno et al., 2006). Compared with the classic lipoproteins in the insect hemolymph or human serum, the 30 K proteins exhibit a lower molecular weight, and might, at most, represent protein–fatty acid complexes (Chapman, 1980); therefore, they were classified into the lepidopteran low molecular weight lipoproteins family (Lipoprotein_11 family, PF03260, http://pfam.sanger.ac.uk/).

Based on the analysis of the primary sequences deduced from the genomic sequences (Xia et al., 2004) and expressed sequence tags (ESTs), ten 30 K proteins (Bmlp1–10) of the silkworm were identified and grouped into three subfamilies (Sun et al., 2007). We overexpressed five of them (Bmlp1–3, Bmlp7 and Bmlp8), and determined the crystal structure of Bmlp7, which is the first structure in the lipoprotein_11 family. It reveals a novel β-trefoil family, in addition to the current 14 families of the β-trefoil superfamily (http://pfam.sanger.ac.uk/), which are proposed to arise from a common ancestor, despite sharing no characteristic binding sites or subcellular localization motif (Hazes, 1996). Bmlp7 has an all-α N-terminal domain (NTD) fused to a all-β trefoil C-terminal domain (CTD). Structural analysis in combination with simulation indicated a putative lipid-binding cavity at the NTD and one potential sugar-binding site at the CTD; however, the in vitro binding for neither lipid nor sugar was detected.

Section snippets

Expression, purification, crystallization and data collection

Considering an N-terminal signal peptide (residues from 1–45) was predicted by SignalP 3.0 server, we cloned the truncated Bmlp7 (Silkworm Knowledgebase, http://silkdb.genomics.org.cn/silkworm/index.jsp, Accession code: Bmb021422, residues from 46 to 284) into the NcoI/NotI digested pET28b expression vector with a hexahistidine tag (AAALEHHHHHH) at the C-terminus. The non-labeled protein was expressed in Escherichia coli BL-21 RIL (DE3) with LB medium. The seleno-methionine (Se-Met) labeled

Structure determination

Before overexpressing Bmlp7 in E. coli, we have successfully purified the natural Bmlp7 from silkworm hemolymph for crystallization and collected the diffraction data at 1.60 Å at BSRF. However, the dataset of natural Bmlp7 crystal was rather poor (results not shown). Therefore, we cloned eight of the ten 30 K genes, purified five of them (those encoding Bmlp1–3, Bmlp7 and Bmlp8) and obtained the crystal of Bmlp7. After optimization, the crystal of non-labeled Bmlp7 was diffracted to 1.91 Å at

Overall structure of Bmlp7

Each asymmetric unit of the Bmlp7 crystal contains two identical molecules with a root mean square deviation (RMSD) of 0.31 Å over 229 Cα atoms. We could trace the electron density from Val50 to Phe284 for chain A, and Val50 to Ala285 for chain B. The dimeric interface in the asymmetric unit predominantly consists of electrostatic interactions via an interface adding up to 1600 Å2. This dimeric form could be observed in both crystals of space group P1 and C2. Results from PISA server (Krissinel

The all-α NTD and the putative lipid binding cavity

Structural comparison using the DALI server (Holm and Sander, 1998) showed that, although the arrangement of the NTD of Bmlp7 is similar to that of a monomer of the cleavage and polyadenylation factor Pcf11 (Meinhart and Cramer, 2004) from Saccharomyces cerevisiae (PDB: 1SZA, RMSD = 2.20 Å, over 69 Cα atoms), residues involved in hydrophobic-core packing or binding sites are totally different. Another hit of the DALI output is the barley lipid transfer protein (Lerche and Poulsen, 1998) (LTP, PDB:

The all-β CTD and the putative sugar-binding sites

In addition to the all-α NTD, Bmlp7 has an all-β CTD which belongs to the β-trefoil fold. Several hydrophobic residues (Leu137, Leu146, Trp174, Ile189, Leu198, Trp225, Ile240, Leu249 and Trp279) form the hydrophobic core of the β-trefoil barrel (Fig. 3A). According to the Pfam database, the β-trefoil fold has been found in 14 families (CL0066, http://pfam.sanger.ac.uk/), including fibroblast growth factors, Kunitz soybean trypsin inhibitors, and ricin-like toxins. The relationship between these

All 30K proteins have a similar organization

Based on the complete genome sequence draft of the silkworm (Xia et al., 2004) and ESTs, Sun et al. systematically analyzed and renamed ten isoforms of 30 K proteins which shared a sequence similarity above 85% (Sun et al., 2007). In order to further analyze the 30 K proteins, we submitted the sequence of Bmlp7 to BLAST (Altschul et al., 1990) against the non-redundant protein sequences database. Among the 32 top hits, 29 are silkworm proteins, whereas three proteins are from two other species (

Putative structure–function relationship of 30K proteins

In all innate immune response systems, the most important step is that pattern-recognition proteins bind sugar molecules (e.g. β-1,3-glucan) on the surfaces of invading pathogens (Yu et al., 2002). A recombinant B. mori 30 K protein 6G1 (Bmlp1) was reported to bind glucans and participate in the activation of prophenoloxidase cascade. Also, Bmlp1 could interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes (Ujita et al., 2005). Recently, several anti-apoptotic

Acknowledgements

We would like to thank Dr. Sheng-Peng Wang at the Sericultural Research Institute, Chinese Academy of Agricultural Sciences for offering the cDNA of B. mori P50 strain. We also would like to thank the staff at BSRF and SSRF for their help. This work was supported by the National Natural Science Foundation of China (Program 90608027) and the Ministry of Science and Technology of China (Projects 2005CB121002 and 2006AA10A119).

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