The conformational change of the protease inhibitor α2-macroglobulin is triggered by the retraction of the cleaved bait region from a central channel

Seandean Lykke Harwood, Khang Diep, Nadia Sukusu Nielsen, Kathrine Tejlgård Jensen, Jan J. Enghild*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Abstract

The protease inhibitor α2-macroglobulin (A2M) is a member of the ancient α2-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel's subsequent collapse triggers the conformational change of A2M and other A2MF proteins.

Original languageEnglish
Article number102230
JournalJournal of Biological Chemistry
Volume298
Issue8
Number of pages11
ISSN0021-9258
DOIs
Publication statusPublished - Aug 2022

Keywords

  • inhibition mechanism
  • mutagenesis in vitro
  • protease
  • protease inhibition
  • protein engineering
  • proteinase
  • proteolysis
  • thiol ester
  • α-macroglobulin

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