Molecular insights into the regulation of complement protease C1s by the multi-specific SERPIN C1 esterase inhibitor

Abstract

Functioning as a sentinel of the innate immune response, the complement system is an evolutionarily ancient proteolytic cascade that is involved in the recognition, opsonization, and lysis of foreign pathogens and apoptotic cells. Initiation of this system is paramount to the potency of the immune response but must be carefully controlled by negative regulators to mitigate aberrant activation. One such regulator, known as C1 esterase inhibitor (C1-INH), is the only canonical complement regulator that directly inhibits the initiating proteases of the classical pathway of complement C1r and C1s. C1-INH is part of a superfamily of serine protease inhibitors (SERPIN), which covalently react with target proteases through a substrate-like reactive center loop (RCL). In addition to its regulatory role in complement, C1-INH is multi-specific targeting a dozen proteases of the coagulation, contact, and fibrinolytic systems. Dysregulation of C1-INH is most apparent in congenital loss-of-function mutations of its encoding gene SERPING1 which causes a chronic debilitating disease known as hereditary angioedema (HAE). However, the molecular basis for C1-INH recognition of any of its cognate proteases including C1s has yet to be elucidated. Towards this end we aimed to characterize the SERPIN domain of C1-INH using a bacterial expression system and determine the molecular basis for C1s interactions. Utilizing structure-guided site-directed mutagenesis coupled to a novel surface plasmon based C1s binding assay we show that C1-INH relies on an extensive binding surface outside of the RCL for full affinity. The implications of this structure to the fields of SERPIN biology are discussed.