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. 2017 Nov 8;7:15101. doi: 10.1038/s41598-017-15415-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Dynamic bindings and computational molecular docking between HAI-2’s KDs and matriptase’s protease domain. (A,B) Examination of the dynamic bindings of GST-KD1 (A) and GST-KD2 (B) to matriptase’s protease domain using BIAcore assay. The purified recombinant HAI-2’s KD1 and KD2 GST-fusion proteins were dialyzed and diluted in running buffer to the indicated concentrations. One hundred nanomolar of GST fusion proteins were used as a negative control. To measure the dynamic bindings between HAI-2’s KDs and matriptase’s protease domain, the indicated concentrations of the analytes (HAI-2’s KD GST-fusion proteins) were injected to Trx-MTX PD-immobilized CM5 chip at 30 µl/min for 120 sec. The dissociation was carried out by passing running buffer 30 µl/min for 180 sec. The estimated dissociation constants (K_D) were statistically calculated by the affinity analysis of BIAevaluation software v1.0 (GE Lifescience, CT, USA). (C) The amino acid sequence alignment of the HAI-1’s and HAI-2’s KD1 and KD2 functional regions. The amino acid sequence alignment was performed by the MUltiple Sequence Comparison by Log-Expectation (MUSCLE, EMBL-EBI). Δ indicated the important residues which potentially interacted with matriptase’s protease domain⁴⁰. (D) The neighbor-joining tree of phylogenetic analysis was performed by MUSCLE (EMBL-EBI). The values mean the genetic distances for each KD. (E,F) The computational docking models of HAI-2 KD1 (E) and KD2 (F) with matriptase’s protease domain. The structural models of matriptase’s protease domain (Matriptase PD, PDB ID: 4ISO) and HAI-2 KD1 (PDB ID: 4U32) were downloaded from Protein Data Bank. HAI-2’s KD2 was constructed by the computational docking according to HAI-2’s KD1 (PDB ID: 4U32). The best-fit docking models of HAI-2’s KD1-matriptase PD and HAI-2’s KD2-matriptase PD were generated by ClusPro 2.0 (https://cluspro.bu.edu/)^77–81 and drew by CCP4mg software (MRC Laboratory of Molecular Biology, Cambridge, UK). (G) The superposition of the docking models of KD1 and KD2 with matritpase’s protease domain. (H/I) The interaction residues between the active site in HAI-2’s KD1 (R48) (H) and KD2 (R143) (I) and matriptase’s protease domain (catalytic site S190) in the docking models. (J) The superposition of the residue R48 in HAI-2’s KD1 and the residue R143 in HAI-2’s KD2 with the residue S190 in matriptase’s protease domain in the docking models.