Biological properties of reverse ankyrin engineered for dimer construction to enhance HIV-1 capsid interaction

On-anong Juntit,^1,3 Umpa Yasamut,^1,3,4 Supachai Sakkhachornphop,² Koollawat Chupradit,^1,3 Weeraya Thongkum,^3,4 Chatchawan Srisawat,⁵ Tawan Chokepaichitkool,⁶ Prachya Kongtawelert,⁶ Chatchai Tayapiwatana^1,3,4

¹ Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
² Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand
³ Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
⁴ Center of Innovative Immunodiagnostic Development, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
⁵ Department of Biochemistry, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
⁶ Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Abstract

Background: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity.
Objective: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4_NC-NC) and its inverted form (Ank1D4_NC-CN), with each domain connected by a flexible (G₄S)₄ linker peptide.
Methods: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4_NC-NC and Ank1D4_NC-CN were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4.
Results: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4_NC-CN with CAp24 was significantly greater than that of Ank1D4_NC-NC and Ank1D4 by capture ELISA. Ank1D4_NC-CN also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4_NC-CN, Ank1D4_NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis.
Conclusions: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4_NC-CN may provide an alternative treatment strategy for inhibiting HIV-1 replication.
Key words: Ankyrins, Ankyrin dimer, Binding kinetics, Capsid proteins, HIV-1