Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism

TY - JOUR

T1 - Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism

AU - Harwood, Seandean Lykke

AU - Lyngsø, Jeppe

AU - Zarantonello, Alessandra

AU - Kjøge, Katarzyna

AU - Nielsen, Peter Kresten

AU - Andersen, Gregers Rom

AU - Pedersen, Jan Skov

AU - Enghild, Jan J

PY - 2021/5

Y1 - 2021/5

N2 - Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

AB - Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

KW - BAIT REGION

KW - COMPLEMENT

KW - COMPONENT C4

KW - CRYSTAL

KW - FAST FORMS

KW - HUMAN ALPHA(2)-MACROGLOBULIN

KW - HUMAN ALPHA-2-MACROGLOBULIN

KW - PROTEINASE BINDING

KW - SMALL-ANGLE SCATTERING

KW - X-RAY-SCATTERING

UR - http://www.scopus.com/inward/record.url?scp=85107290580&partnerID=8YFLogxK

U2 - 10.1016/J.MCPRO.2021.100090

DO - 10.1016/J.MCPRO.2021.100090

M3 - Journal article

C2 - 33964423

SN - 1535-9476

VL - 20

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

M1 - 100090

ER -