Crystal structure of a bony fish beta2-microglobulin: insights into the evolutionary origin of immunoglobulin superfamily constant molecules

Abstract

Three-dimensional structures of beta(2)-microglobulin (beta2m) from chicken and various mammals have been described previously, but aside from genomic sequences, very little is known about the three-dimensional structures of beta2m in species other than warm-blooded vertebrates. Here, we present the first three-dimensional structure of beta2m from bony fish grass carp (Ctid-beta2m), resolved at 2.1 A. The key structural differences between this new structure and previously published structures are two new hydrogen bonds at positions Ile(37) and Glu(38) in strand C and Lys(66) in strand E, and a hydrophobic pocket around the center of the protein found in Ctid-beta2m. Importantly, Ctid-beta2m has a short D strand and a long loop between stands C and D, rather than the flexible region found in other beta2m structures that serves as a putative binding region for the major histocompatibility complex heavy chain. Comparing the Ctid-beta2m structure with those of bovine and human beta2ms, the Calpha root mean square deviation of the latter are 1.3 A and 1.8 A, respectively. Compared with the constant domains of Lamprey T cell receptor-like receptor (Lamp-TCRLC) and Amphioxus V and C domain-bearing protein (Amphi-VCPC), Ctid-beta2m exhibits very different topology. The three-dimensional structures of domains predicted from Amphi-VCPC/Lamp-TCRLC are distinctly lacking in strand A of beta2ms. There are 18 amino acids at the N terminus of Amphi-VCPC that may have evolved into strand A of beta2ms. A mutation in the BC loops of Amphi-VCPC may have led to the novel topology found in beta2m. Based on these results, Ctid-beta2m may well reflect evolutionary characteristics of ancestral C set molecules.

FIGURE 1.

Overall structure of Ctid-β2m. A, Ctid-β2m is shown in a ribbon representation and colored orange. β-strands are labeled with letters A to G. The CD loop and the intrachain disulfide bridge are labeled in red and purple, respectively. B, the hydrophobic pocket in Ctid-β2m. The hydrophobic residues (Ile⁷, Ile⁹, and Tyr¹⁰ on strand A; Leu²³, Ile²⁴, Ytr²⁶, and Val²⁷ on strand B; Ile³⁷, Leu³⁹, and Leu⁴⁰ on strand C; Phe⁵⁴ on strand D; Trp⁶⁹, Leu⁶⁴, Thr⁶⁵, Val⁶⁸, and Ple⁷⁰ on strand E; Typ⁷⁸ and Val⁸² on strand F; and Thr⁸⁸ and Val⁹² on strand G) are labeled in blue.

FIGURE 2.

Analysis of Ctid-β2m with β2ms from fish, chicken, and mammals. A, sequence alignment of Ctid-β2m with β2ms from fish and mammals. The sources of the sequences are as follows: Ctid-β2m (GenBank^TM accession no. AB190815, PDB code 3GBL), zebrafish (GenBank^TM accession no. L05383), trout (GenBank^TM accession no. L49056), catfish (GenBank^TM accession no. AF016042), salmon (GenBank^TM accession no. AF180488), chicken (GenBank^TM accession no. M84767), bovine (GenBank^TM accession no. NM_173893), mouse (GenBank^TM accession no. NM_009734), and human (GenBank^TM accession no. NM_004048). Numbers over the alignment denote residues that form Ctid-β2m. Black arrows above the alignment indicate β-strand. T, toil. Residues highlighted in red are absolutely conserved, whereas those with blue squares are highly conserved (80%). Green numbers denote residues that form disulfide bonds. The alignment was generated using the program Clustal X and drawn with ESPript. B, structural superpositions of Ctid-β2m with the β2m monomer from chickens. Ctid-β2m and chicken β2m are colored in orange and green, respectively. C, structural superpositions of Ctid-β2m with β2m monomers from humans and bovines. Ctid-β2m, human, and bovine β2ms are colored in orange, green, and red, respectively. The superposition was created by PyMOL, using the C atoms of the globular segment. Ctid-β2m (PDB code 3GBL), chicken (PDB code 3BEW), HLA β2m (PDB code 1LDS), and bovine (PDB code 1BMG).

FIGURE 3.

Details of the Ctid-β2m contacts of the two extra hydrogen bonds and the disulfide bond. Hydrogen bonds are illustrated as dotted lines. The disulfide bond is shown as a yellow stick. Residues forming hydrogen bonds are shown in the stick model and colored by atom types: blue, N; red, O. All of the residues are labeled.

FIGURE 4.

Structural superposition of Ctid-β2m with Human HLA-A*0201. Ctid-β2m is shown in a ribbon representation and colored orange, and the CD loop is indicated by a box. The PyMOL Molecular Graphics System was used to prepare the figure, and the geometry of the refined structure was validated according to Ramachandran plot criteria.

FIGURE 5.

The predicted three-dimensional structures of Lamp-TCRLC, Amphi-VCPC, and a structural overlay with Ctid-β2m. A, sequence alignment of Ctid-β2m with Lamp-TCRLC. Three-dimensional structure of Lamp-TCRLC alone (B) and overlaid with Ctid-β2m (C). Using the first approach mode in SWISS-MODEL, the three-dimensional structure of the Lingo-1 molecule (2ID5A) is found to share 24.68% identity with the IgSF C domain of Lamp-TCRLR at the amino acid level. The best E-value of the constructed Lamp-TCRLC three-dimensional structure is 2.20e⁻⁸. The three-dimensional structure of Lamp-TCRLC is composed of 76 residues (amino acids in the 19–94 region) with a six-stranded β-sandwich fold. D, sequence alignment of Ctid-β2m with Amphi-VCPC. Structure of Amphi-VCPC alone (E) and overlaid with Ctid-β2m (F). The three-dimensional structure of Amphi-VCPC was predicted by amino acid homology modeling, based on part of a CD4 complex (PDB code 2NY4B). Amphi-VCPC shares 16.88% identity with human T cell surface glycoprotein CD4. The E-value of the constructed three-dimensional structure is 1.00e⁻⁸. The predicted three-dimensional structure of the Amphi-VCPC domain is composed of 75 residues (amino acids in the 8–82 region) with a six-stranded β-sandwich fold.