Structural Mechanics of the Alpha-2-Macroglobulin Transformation

Abstract

Alpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native- and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 Å and 6.4 Å resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 Å-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a "latch-lock" to block futile A2M transformation until its protease-mediated cleavage.

Keywords: Xenopus egg extract; alpha-2-macroglobulin; cryo-EM; innate immunity; protein inhibitor.

Figure 1.. Cryo-EM structure determination of A2Moo

(A) Cryo-EM maps of the native-form A2M family protein tetramer (left), locally refined native-form A2M family protein protomer (center), and intermediate-form A2M family protein tetramer (right). (B) Identification of A2Moo, the A2M family protein in Xenopus egg. Top panel; amino acid sequence alignment of sixteen Xenopus laevis A2M family proteins with reasonable protein length to satisfy the EM map. The representative region used for protein identification is shown. A yellow rectangle indicates the protein that matches to the EM density (LOC431886: named A2Moo). Bottom panels; overlay of the atomic model of A2Moo and cryo-EM density of the locally refined native-form A2M protein protomer. Three other representative regions used for protein identification are shown in Figure S5.

Figure 2.. 2D and 3D structures of the A2Moo tetramers

(A) Representative 2D class averages of the native-form A2Moo tetramer mimic previously proposed depictions of A2M architectures. (B) 3D atomic model of the native-form A2Moo tetramer. Two pairs of “connected mitten”-shaped A2M dimers stack to form a cross-like configuration with D2 symmetry, where each monomer consists of a “bulky finger” module, a “thumb” module, and a “palm” module. (C) 3D atomic model of the intermediate-form A2Moo tetramer. The “bulky finger” module folds toward the “wrist”. (D) Atomic model of the induced-form human A2M (PDB ID: 4ACQ) [7].

Figure 3.. 3D arrangements of the A2Moo domains

(A) Domain organization of A2Moo. (B) 3D arrangements of the domains of the native-form A2Moo (left) and an induced-protomer of the intermediate-form A2Moo (right).

Figure 4.. The path of the bait region in the native-form A2Moo

(A) Tetramer atomic model and cartoon model to depict the location of flexible bait region. (B) High-resolution locally refined map around flexible bait region. The density of the bait region is missing. (C) Low-pass (11Å) filtered map around flexible bait region. The bait region of A2Moo can be traced. (D) The cartoon representation depicting the location of the flexible bait region. The bait region of A2Moo is located beside the MG5 and MG6.

Figure 5. Structural transformation around the “latch hole”

(A) Tetramer atomic model and cartoon model to depict the viewpoint in Figure 5. (B, C) Structures around the latch hole in a protomer of the native-form A2Moo (B) and induced-protomer of the intermediate-form A2Moo (C). Top panels show a protomer in A2Moo tetramers. Middle panels show a zoom-up view around the latch hole. (D) The cartoon representation depicting the structural transformation around the latch hole. In the induced protomer, the bait region is not observed within the latch hole, and MG2 and MG6 are shifted to fill the latch hole.

Figure 6. Structural variations of native-form A2Moo tetramer

(A) The flexible A2M variation expands the 60 Å groove and prey chamber. (B) Structural model of the plasmin serine protease domain accessing the bait region of the 60 Å groove in native-form A2M tetramer. Instead of plasmin, full length plasmin precursor (human plasminogen) structure (PDB: 4DUU) was mapped on the “open” native-form A2Moo tetramer [57].

Figure 7. The model mechanics of the A2M transformation during protease inactivation

(A) Protease entrapment by A2M tetramer by the Venus flytrap mechanism. The cross-like structure and flexible nature of native-form A2M tetramer allow large proteases to access the bait region of A2M inside the 60 Å groove. In the intermediate-form tetramer, an A2M protomer is induced transformation by bait cleavage. In the induced-form tetramer, proteases can be trapped in the prey chamber. (B) Mechanics of A2M tetramer structural transformation by bait cleavage. The intact bait region in the latch hole blocks the shifting of MG2 and MG6, while bait cleavage unlocks this movement. The shifting of MG2 and MG6 pushes TED away from the RBD. The released TED makes a new interaction with MG1 of the adjacent native-form protomer to stabilize the induced-form A2M.