The crystal structure of a multidomain protease inhibitor (HAI-1) reveals the mechanism of its auto-inhibition

Abstract

Edited by Norma AllewellHepatocyte growth factor activator inhibitor 1 (HAI-1) is a membrane-bound multidomain protein essential to the integrity of the basement membrane during placental development and is also important in maintaining postnatal homeostasis in many tissues. HAI-1 is a Kunitz-type serine protease inhibitor, and soluble fragments of HAI-1 with variable lengths have been identified in vivo. The full-length extracellular portion of HAI-1 (sHAI-1) shows weaker inhibitory activity toward target proteases than the smaller fragments, suggesting auto-inhibition of HAI-1. However, this possible regulatory mechanism has not yet been evaluated. Here, we solved the crystal structure of sHAI-1 and determined the solution structure by small-angle X-ray scattering. These structural analyses revealed that, despite the presence of long linkers, sHAI-1 exists in a compact conformation in which sHAI-1 active sites in Kunitz domain 1 are sterically blocked by neighboring structural elements. We also found that in the presence of target proteases, sHAI-1 adopts an extended conformation that disables the auto-inhibition effect. Our results also reveal the roles of non-inhibitory domains of this multidomain protein and explain the low activity of the full-length protein. The structural insights gained here improve our understanding of the regulation of HAI-1 inhibitory activities and point to new approaches for better controlling these activities.

Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection

“…Here, the HAI-1 ectodomain is shown, according to the reported crystal structure (PDB 5H7V; ref. ( 162 )); the side chain of the P 1 residue, Arg260, is highlighted. Other cellular processes that coregulate membrane fusion and viral uptake are (4) shedding of ACE2, MASPs and/or HAI-1/HAI-2 ectodomains, either by MASPs in trans or by membrane-bound metalloproteinases, and (5) endocytosis, which could be modulated by phosphorylation of cytosolic peptides in the human factors, or by palmitoylation of the Cys-rich endodomain of SARS-CoV-2.…”

Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases

“…The presence of two Kunitz domains in the structures of the closely related Kunitz-type transmembrane serine protease inhibitors HAI-1 and HAI-2 aids inactivation of various TLPs including matriptase, trypsin and prostasin [ 164 , 165 ]. HAI-1 has also been reported as an inhibitor of HAT activation and proteolytic function [ 166 ].…”

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

“…In addition to the LRPs, we also identified a novel O-glycosite in the single LA linker of SPINT1, the matriptase inhibitor also known as HAI-1. In SPINT1, the LA domain affects the Kunitz domain 1 inhibition of matriptase (60), and it is possible that an O-glycan in the LA linker may modulate this effect. Besides glycosylation of LA linkers, increasing GalNAc-T11 expression induced a number of O-glycosites located in cysteine-rich globular domains.…”