The plasma protease inhibitor ITIH4 forms oligomeric complexes and senses broader proteolytic activity

In self-perpetuating proteolytic cascades within the body, tight regulation by an excess of inhibitors is essential to maintain homoeostasis. The plasma protein inter-α-trypsin inhibitor heavy chain 4 (ITIH4) inhibits extracellular proteolytic pathways, including the contact system and the complement cascade, by a mechanism requiring cleavage of ITIH4 but without direct binding to the active site of the cleaving protease. The structural and mechanistic details of the cleavage-induced changes in ITIH4 that lead to protease inhibition remain elusive. Here, we show that ITIH4 undergoes an unexpectedly large, conformational change upon cleavage involving complete unfolding of an immunoglobulin domain and a reorganisation of the N-terminal F3 domain. The cleaved N-terminal fragment of ITIH4 (ITIH4N) forms ultra stable homo-oligomers with as many as seven ITIH4N molecules attached to the inhibited protease. This homo-interaction mimics the initial binding event with the cleaving protease. Mutation-induced disruption of homo-oligomerization leads to direct attachment of multiple ITIH4N molecules to different residues on the same protease and does not compromise inhibition, pointing to a function of ITIH4N oligomerization beyond protease inhibition. While proteolysis of ITIH4 may lead to inhibition of the cleaving protease, we show that nascent ITIH4N may associate with a molecule different from the cleaving protease or an existing protease-ITIH4N complex. Our data provide a comprehensive structural foundation for establishing a mechanistic framework for the function of ITIH4 and drive the future analysis of its in vivo function. Using electron microscopy, the authors describe the conformational changes induced when the plasma protease inhibitor ITIH4 is cleaved and activated. Moreover, oligomerization of cleaved ITIH4 is shown not to impact protease inhibition.