Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N‐terminal apple domains to recruit substrates. A high‐resolution 1.3 Å structure of full‐length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four‐apple domain disc organization is 180° rotated compared to FXI. Summary BackgroundPlasma prekallikrein (PK) and factor XI (FXI) are apple domain‐containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. ObjectiveTo investigate the three‐dimensional structure of full‐length PKa and perform a comparison with FXI. MethodsA series of recombinant full‐length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusionsA 1.3 Å structure of full‐length PKa reveals the protease domain positioned above a disc‐shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen‐deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.

Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF ) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.