Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin

Humanα2-macroglobulin (hα2M) is a multidomain protein with a plethoraof essential functions, including transport of signaling molecules and endopeptidaseinhibition in innate immunity. Here, we dissected the molecular mechanism of theinhibitory function of the∼720-kDa hα2M tetramer through eight cryo–electronmicroscopy (cryo-EM) structures of complexes from human plasma. In the native com-plex, the hα2M subunits are organized in twoflexible modules in expanded conforma-tion, which enclose a highly porous cavity in which the proteolytic activity ofcirculating plasma proteins is tested. Cleavage of bait regions exposed inside the cavitytriggers rearrangement to a compact conformation, which closes openings and entrapsthe prey proteinase. After the expanded-to-compact transition, which occurs indepen-dently in the four subunits, the reactive thioester bond triggers covalent linking of theproteinase, and the receptor-binding domain is exposed on the tetramer surface forreceptor-mediated clearance from circulation. These results depict the molecular mecha-nism of a unique suicidal inhibitory trap.Thiswork was supported by grants from the Spanish Ministries of Economy and Com-petitivity (Grant No. BFU2017-88736-R) and of Science and Innovation (GrantNo. PID2020-113287RB-I00) and the Comunidad Aut onoma de Madrid (GrantNo. P2018/NMT-4389) to J.R.C. and by grants from Catalan and Spanish publicand private agencies (Grant Nos. BFU2019-107725-RB-I00 and 2017SGR00003;Fundaci o“La Marat odeTV3”Grant No. 201815) to F.X.G.-R. T.G. acknowledgesa Juan de la Cierva research contract (JCI-2012-13573) from the Ministry ofEconomy (MINECO) and S.R.M. a Ph.D.-fellowship (BES2016-076877) from theMinistry of Science and Innovation. The Structural Biology Unit of the MolecularBiology Institute of Barcelona (IBMB) was a Mar ıa de Maeztu Unit of Excellence(2015 to 2019) and the Centro Nacional de Biotecnolog ıa is a Severo OchoaCenter of Excellence (MINECO award SEV 2017-0712), as awarded by the Span-ish Ministry of Economy, Industry and Competitiveness.Peer reviewe