Structure determination of Sa-A2M

Sa-A2M is a single chain, 1,644 residue protein that includes an N-terminal 17 residue signal peptide and an LAGC lipobox. A clone expressing residues 19–1,644 of Sa-A2M with a 6xHis-tag and a thrombin site fused to the N terminus was expressed and purified, but only a single and irreproducible crystal was produced. We thus employed the surface entropy reduction approach34 to search for patches of surface residues whose mutation could potentially promote local stability. Residues 98 and 99 were thus mutated into alanines. This variant labelled with selenomethionine was purified in the ‘native’ or preactivated form and produced crystals that diffracted X-rays to 2.95 Å. The crystal structure of Sa-A2M was solved by performing a single-wavelength anomalous diffraction experiment at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France (Table 1). The crystal contained one molecule per asymmetric unit; the Lys98Ala/Lys99Ala mutations promoted crystal contacts between the backbone carbonyl of residue 101 and the side chain of residue 462 from a symmetry-related molecule (Supplementary Fig. 1a), which most probably stabilized the monomers enough to generate diffraction-quality crystals.

Table 1 Data collection and refinement statistics. Full size table

Model building of native Sa-A2M was challenging due to the large number of domains and the unexpected positions of macroglobulin-like domain (MG)1 and MG2. These two domains were found closely packed against other symmetry-related molecules, resulting in an intertwined configuration (Supplementary Fig. 1b). Subsequent to solving the structure of the native form of Sa-A2M, we solved the structure of a methylamine-treated form by incubating Sa-A2M with methylamine for 2 h at 20 °C before crystallization. Methylamine was present in both the crystallization and cryoprotectant-soaking steps to prevent reformation of the thioester. The structures of both the methylamine-treated Sa-A2M and a thioester pocket mutant (Tyr1175Gly) were solved by performing molecular replacement experiments using the structure of native Sa-A2M as a model.

Crystal structure of Sa-A2M

Sa-A2M is composed of 13 domains (Fig. 1), all of which fold as variants of beta sandwiches with the exception of the TED, which consists of 14 alpha helices (Fig. 1b). Most of the beta sandwich domains appear to serve a structural role and are referred to as the MG domains. Residues 57–281 form MG1 and MG2, which are linked by a flexible loop. MG1 is the domain which is the farthest from the body of the structure; since the N terminus of Sa-A2M is expected to be anchored to the periplasmic inner membrane, as observed for ECAM32, MG1–MG2 could play the role of a linker associating the main body of Sa-A2M to the bilayer. Residues 282–1,010 fold into the six subsequent MG domains (MG 3–8), which together form 1.5 turns of a helical coil to generate what resembles a distorted ‘key ring’ (Fig. 1c) in an arrangement that is highly reminiscent of that of proteins of the eukaryotic A2M/complement/TEP superfamily. The key ring scaffold, also referred to as the beta-ring in TEP1 and complement C3, C4 and C5, provides a frame for a highly flexible stretch of amino acids in the bait region domain (BRD). The first half of the BRD extends from MG8 to MG3 and is adjacent to MG4 and MG7 on the concave side of the key ring scaffold. Interestingly, a segment of the BRD contributes one strand to the MG3 beta sheet before folding back towards MG8 through the key ring cavity. It is precisely this region that contains the bait site for protease cleavage (residues 925–950), and its high flexibility is evidenced from the fact that it can only be partly traced in the electron density map.

Figure 1: Crystal structure of S. typhimurium alpha-2-macroglobulin (Sa-A2M). (a) Schematic of the 13 domains in Sa-A2M. The N-terminal 17-residue signal peptide and the LAGC lipobox sequence were absent in the clone used for crystallization. The domains are coloured identically in a–c. (b) The overall structure of Sa-A2M displayed is the preactivated form before reaction with proteases. The thioester site is buried in the interface between the TED and MG10 domains. MG1 and MG2 are only observed in bacterial A2Ms. MG1, normally anchored to the inner membrane in vivo, is connected to MG2 by a flexible linker. (c) MG3–MG8 form a coiled arrangement that resembles a distorted key ring. The cavity formed by the key ring scaffold houses the partially observed bait region. Full size image

MG9 follows the key ring scaffold, and it is connected through the CUB (complement C1r/C1s, Uegf, Bmp1) domain to the TED domain. The TED domain is in a preactivated conformation that maintains the thioester site buried against MG10. In this conformation, it also interacts with MG4 and CUB, the latter of which is also connected at its C terminus to MG10. MG10 is markedly different from the other MG domains in that it has more beta strands and an alpha helix. The position of MG10 is stabilized by, in addition to other hydrogen bonds, the formation of a beta sheet with MG9. Notably, a hydrogen bond is observed between Tyr1626 of MG10 and Glu1181 of the thioester site in TED (Supplementary Fig. 2) and could serve a local stabilizing role (see below). It is of note that MG10 is structurally reminiscent of the C-terminal, receptor-binding domain of eukaryotic alpha-macroglobulin35,36 (r.m.s.d.=3.54 Å over 120 C-alpha atoms), but its involvement in protease clearance is to date unclear.

Protection of the thioester by a tyrosine lock

The thioester bond of Sa-A2M, which is formed between Cys1179 and Gln1182, is intact in the native structure (Fig. 2a) and buried between the TED and MG10 domains (Fig. 2b). Similarly to the structures of complement components C3, C4 and TEP1, the thioester site is found in a pocket surrounded by aromatic and hydrophobic residues. In Sa-A2M, the pocket is surrounded by Tyr1175, Tyr1177 and Trp1235 from the TED domain, as well as Met1625 and Tyr1626 from MG10. Notably, all of these residues are conserved in bacterial and eukaryotic A2M variants, with the exception of Tyr1175, which is present only in bacterial species (Supplementary Fig. 3a) and points directly towards the interior of the CXEQ pocket in the native structure (Fig. 2). Despite being protected from hydrolysis by its location in the hydrophobic pocket, the thioester bond in Sa-A2M is located near the surface of the molecule (Fig. 1b).

Figure 2: Thioester site of Sa-A2M, methylamine-inactivated Sa-A2M and Sa-A2M Tyr1175Gly. (a), (c) and (e): The 2F o -F c electron density contoured at 1 σ is shown. (b), (d) and (f): The surface representation is shown after rotating (a,c,f) ~180°. (a) In the untreated protein, a thioester bond between Cys1179 and Gln1182 is observed, as evidenced by the electron density connecting these two residues. (b) Before reaction with methylamine, the thioester, which is hidden from view, is buried between the TED and MG10 domain. Tyr1175 contributes to keeping the thioester buried and protected from hydrolysis. (c) Reaction with methylamine breaks the thioester bond between Cys1179 and Gln1182, and causes a conformational change of Tyr1175. (d) After the conformational change, the inactivated thioester is no longer buried in the TED domain. (e) Mutation of Tyr1175 to glycine results in loss of the thioester bond between Cys1179 and Gln1182. (f) Similarly to the methylamine-treated sample, the thioester in the Tyr1175Gly variant is no longer buried in the TED domain. Full size image

Previous studies with human and E. coli A2M have shown that reaction with methylamine inactivates the thioester and causes a major conformational change in the eukaryotic variant5,19,32,33,37,38,39. To address the issue of a potential conformational change in a bacterial A2M on activation, we solved the crystal structure of methylamine-treated Sa-A2M. Notably, the fold of methylamine-treated Sa-A2M is highly reminiscent of the native form, since it does not indicate any major differences in domain positions, and a structural alignment of the two molecules results in an r.m.s.d. of 0.35 Å (1,536 C-alpha atoms). Small-angle X-ray scattering (SAXS) experiments undertaken to explore potential conformational modifications on activation yielded curves that are nearly identical for native and methylamine-treated Sa-A2M (Supplementary Fig. 4). These observations are thus supportive of fluorescence, analytical ultracentrifugation and native polyacrylamide gel electrophoresis (PAGE) studies of E. coli A2M that suggested that thioester cleavage did not lead to major conformational differences32, but differ from SAXS results obtained on the latter molecule, which indicated some degree of conformational modification on methylamine treatment33. Despite the fact that a major shift in domain positions could not be detected for Sa-A2M, reaction with methylamine caused cleavage of the thioester bond (Fig. 2c), causing Tyr1175 to be pushed away from the thioester site and the methylated Gln1182 to move towards its original position. Notably, Gln1182 is exposed to solvent when Tyr1175 is in the ‘open’ conformation (Fig. 2d).

Since Tyr1175 is highly conserved among bacterial A2Ms and reaction with methylamine causes a drastic modification in its position, we postulated that this residue could be crucial for the formation and maintenance of the thioester bond (Supplementary Fig. 3a). To investigate thioester site stability in the absence of the Tyr1175 side chain, we solved the crystal structure of a Sa-A2M Tyr1175Gly point mutant. In this structure, the thioester bond is no longer shielded and is cleaved, potentially through hydrolysis by a water molecule from solvent, and Gln1182 moves into the position normally occupied by Tyr1175 (Fig. 2e). This observation strongly indicates that Tyr1175 is an essential component of a ‘locking mechanism’ that maintains the stability of the thioester bond in the native form. In the Tyr1175Gly variant, the electron density around residues 1,174–1,176 is weak, indicating that this region is more flexible. The flexibility of this loop removes its ability to protect the thioester site, and results in residue 1,182 becoming exposed to solvent (Fig. 2f), indicating that the lock is ‘open’.

Mechanism of activation of Sa-A2M

Following our discovery that methylamine treatment of Sa-A2M does not trigger a conformational change as it does in human A2M, we focused on deciphering the physiological mechanism of activation. In the native form of Sa-A2AM, the thioester site remains buried between the TED and MG10 domains. Cleavage at the bait site by a protease should activate Sa-A2M and trigger a conformational change by a yet undetermined mechanism that exposes the thioester site to the protease. However, in the crystal structure of Sa-A2M, the second half of the bait region is not observable due to the weak electron density for these flexible residues. The entire bait region could be observed to stretch across the concave pocket formed by the key ring scaffold after filling in the missing residues (939–955) using MODELLER (Supplementary Fig. 5a). Thus, to facilitate the investigation of the protease-trapping process after cleavage at the bait site, an artificial bait site for TEV protease was inserted into the centre of the bait region which, based on the structure of this model, should be easily accessible. Reaction products following proteolytic activation by TEV protease were characterized by mass spectrometry and denaturing gel electrophoresis.

Sa-A2M containing the TEV-specific bait site (Sa-A2M-TB), methylamine-treated Sa-A2M-TB (Sa-A2M-TB-MA), native Sa-A2M and Sa-A2AM-TB Tyr1175Gly were incubated alone or with TEV at a molar ratio of 1:2 (Fig. 3, Table 2). Protease cleavage of Sa-A2M-TB at the bait region (residues 940/941) is expected to yield fragments of 102 kDa and 77 kDa, corresponding to the N-terminal fragment and the thioester-containing C-terminal fragment (CTF), respectively. Since TEV has a mass of 29 kDa, its covalent association to the TED domain is expected to yield a protease-bound C-terminal fragment (CTF-TEVP) of 106 kDa (Fig. 3b). This is confirmed both by SDS–PAGE (Fig. 3a) and mass spectrometry measurements (Table 2). Interestingly, the CTF-TEVP band migrates anomalously and slower than expected.

Figure 3: TEV protease digestion profiles of Sa-A2M and Sa-A2M-TB. (a) In total, 40 μM of Sa-A2M-TB, Sa-A2M-TB-MA, Sa-A2M and Sa-A2M-TB Tyr1175Gly were incubated with 0 μM or 80 μM TEV protease. Reaction of TEV protease with Sa-A2M-TB and Sa-A2M-TB-MA shows the different products for samples with an active or inactive thioester, respectively. Sa-A2M without a TEV site is unaffected by the protease. TEV digestion of Sa-A2M-TB Tyr1175Gly resembles methylamine-treated Sa-A2M-TB, which indicates an inactive thioester. Heat degradation products (*) were observed in Sa-A2M-TB and Sa-A2M, a characteristic of samples with an active thioester. Such degradation products have also been observed in other thioester-containing proteins such as human A2M, C3 and C4, and E. coli A2M32,44,61. (b) A schematic diagram of the expected reaction products between TEV and Sa-A2M-TB with an active or inactive thioester. Full size image

Table 2 Electrospray ionisation–time of flight mass spectrometry analyses of reaction products between Sa-A2M variants and tobacco etch virus protease. Full size table

Incubation of Sa-A2M-TB with methylamine before interaction with TEV should inactivate the thioester and preclude protease association. On incubation of a Sa-A2M-TB-MA with TEV, two bands of ~110 kDa and ~80 kDa are visualized on SDS–PAGE; mass spectrometry confirms that they correspond to N-terminal fragment and CTF without bound TEV (Table 2 and Fig. 3a). These observations thus confirm that in bacterial A2Ms, protease entrapment is also dependent on an intact thioester.

To verify that the effects observed in the reactions of Sa-A2M-TB with TEV were caused by cleavage at the bait site, we incubated the protease with native Sa-A2M. As native Sa-A2M does not contain a TEV recognition site, cleavage products are not expected after reaction with the protease, which is what was observed (Table 2 and Fig. 3a). This confirms that cleavage at the bait site must occur prior to covalent linkage of TEV by Sa-A2M, even when the thioester is intact.

The crystal structure of the Sa-A2M Tyr1175Gly variant (described above) shows that the thioester site is disrupted by the Tyr1175Gly mutation. Sa-A2M-TB Tyr1175Gly was thus incubated with TEV to examine the effects of this mutation on its activity. Two major bands, corresponding to the N-terminal and C-terminal regions of the molecule, appear after digestion, similarly to those identified on reacting TEV with Sa-A2M-TB-MA (Table 2 and Fig. 3a). This indicates that, as predicted from the crystal structure of Sa-A2M Tyr1175Gly, Tyr1175 is a key element of the locking mechanism which protects the thioester from hydrolysis; its mutation to glycine inactivates the thioester, preventing TEV from covalently binding Sa-A2M even in the presence of a TEV-specific bait region.