Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Abstract

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

Figure 1. Schematic representation of the domain organization of the α₂M monomer, based on alignment with the structure of C3 [17]

MG designates a macroglobulin domain; CUB, a domain found in a number of developmentally regulated proteins [32]; ‘bait’, bait region domain. Intramolecular disulfide bonds are shown as solid black rectangular loops. The two inter-chain disulfides (Cys²⁵⁵ and Cys⁴⁰⁸) [33,34] are shown as straight black lines and asterisked. The position of possible N-linked glycosylation sites are marked with black diamonds. Note that both MG6 and CUB are split domains whose primary structures are interrupted by other domains, the bait domain and TED respectively.

Figure 2. CD spectrum of TED, showing exclusive α-helical secondary structure

The spectrum is of 1 μM TED in 20 mM potassium phosphate buffer (pH 7.4).

Figure 3. DSC-monitored unfolding of TED

TED unfolds as a single domain with T_m of 45 °C (grey line). The fit to a single two-state unfolding is shown as a continuous black line. C949A TED (1 mg/ml) was in 20 mM sodium phosphate buffer (pH 7.4). 1 kcal=4.184 kJ.

Figure 4. Fluorescence spectra of TED species

Fluorescence emission spectra of C949A and Q952C (oxidized) TED in 20 mM sodium phosphate buffer (pH 7.4) showing very similar environments for the five tryptophan residues present.

Figure 5. ¹H-¹⁵N HSQC spectra of MG2

(A) Uniformly ¹⁵N-labelled MG2 (100 μM) alone; (B) uniformly ¹⁵N-labelled MG2 (100 μM) after addition of 150 μM of unlabelled TED. Samples were in 20 mM sodium phosphate buffer (pH 7.4). Spectra were recorded at 900 MHz.

Figure 6. X-ray crystal structure of MG2 and comparison with MG2 from C3

(A) Ribbon diagram of MG2 from α₂M determined in the present study (Protein Data Bank accession code 2P9R). (B) Super-positioning of MG2 from α₂M (blue) with MG2 from bovine C3 (red).

Figure 7. ¹H NMR spectrum of α₂M CUB domain

Residues 885–930 were joined to residues 1251–1311 to generate a spliced, single-chain CUB domain. Low- and high-field regions of the one-dimensional spectrum of CUB are shown. Evidence for this spliced domain being well folded are the good spread of amide protons in the region from 10 to 8 p.p.m. and a number of upfield-shifted resonances between 0.7 and −0.2 p.p.m.

Figure 8. Relationship of MG2, TED, CUB and MG8 to one another in C3 and C3b

(A) Domains in intact C3 [17]. (B) Domains following activation of C3 to C3b [15]. MG2 is in red, TED in green, CUB in blue and MG8(RBD) in cyan. The thiol ester-forming residues are shown in black and the conserved tyrosine pair in magenta.