OC 26.2 - Exploring structural dynamics in the ADAMTS13 CUB domains upon antibody-mediated ADAMTS13 activation

Sunday, June 25, 2023

15:00 – 15:15 ET

Room: 510

Session Available As: On-Demand

Presenting Author(s)

Quintijn Bonnez, MA

PhD researcher
Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak Kortrijk
Kortrijk, West-Vlaanderen, Belgium

Co-Author(s)

IP

Inge Pareyn

BSc.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
EL

Emily Legan

PhD
Emory University, Department of Pediatrics, Aflac Cancer & Blood Disorder Center
Atlanta, Georgia, United States
SM

Sarah Mansour

PhD researcher
Emory University, Department of Pediatrics, Aflac Cancer & Blood Disorder Center
Atlanta, Georgia, United States
CT

Claudia Tersteeg

Prof., Dr.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
SD

Simon F. De Meyer

Prof., Dr., Eng.
Laboratory for Thrombosis Research, KU Leuven Campus Kulak Kortrijk, Kortrijk, Belgium
Kortrijk, West-Vlaanderen, Belgium
Renhao Li, PhD

Professor of Pediatrics
Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Department of Pediatrics, Emory University School of Medicine
Atlanta, Georgia, United States
Karen Vanhoorelbeke, PhD

Prof., Dr.
KU Leuven
Kortrijk, West-Vlaanderen, Belgium

Background: The mouse monoclonal antibody 17G2 disrupts the ADAMTS13 spacer-CUB interaction and induces conformational changes that allosterically activate the metalloprotease domain. Docking models identified interaction sites of CUB1 and CUB2 with the spacer domain, but the mechanism for antibody-mediated spacer-CUB interdomain disruption remains elusive.

Aims: To explore the molecular dynamics in the ADAMTS13 CUB1 and CUB2 domains upon 17G2 activation using hydrogen/deuterium exchange mass spectrometry (HDX-MS).

Methods: HDX-MS analysis was performed in triplicate using a fusion protein of the truncated ADAMTS13 C-terminal tail: Albumin domain 1 (AD1)-T2C2. In absence of 17G2, AD1-T2C2 was analyzed undeuterated as well as after 10, 100, 500 and 1000 seconds of deuterium exchange. An equimolar complex of AD1-T2C2 and 17G2 was evaluated at identical deuterium exchange timepoints.

Results: With an average residue redundancy of 9.52, the ADAMTS13 CUB domains were fully covered by 202 peptides in presence as well as in absence of the 17G2 antibody. Relative fractional deuterium uptake was mapped onto the CUB1-CUB2 crystal structure (PDB ID: 7B01), which identified 2 loops in the CUB1 domain to contribute to the conformational epitope of 17G2. HDX rate differences revealed structural dynamics on CUB1, CUB2 and the CUB1-CUB2 interface, whereas antibody binding showed minimal HDX difference at the suggested spacer-CUB interaction site.

Conclusion(s): HDX-MS localized the 17G2 binding epitope in 2 loops of the CUB1 domain. HDX rates of the CUB domains at the suggested spacer-CUB interaction site remained unaffected upon binding of 17G2, illustrating marginal effect of 17G2 binding to this interface. Interestingly, 17G2 induced HDX rate differences at various CUB1 and CUB2 regions and at the CUB1-CUB2 interface. These data suggest that structural flexibility of the CUB1-CUB2 complex could explain spacer-CUB disruption upon antibody-mediated activation. Mutagenesis studies are ongoing to further support these data.

Learning Objectives:

Interpret presented data obtained by hydrogen deuterium exchange mass spectrometry.
Recognize structural rearrangements in the ADAMTS13 C-terminal CUB1 and CUB2 domains.
Describe the molecular mechanism of ADAMTS13 opening by activating antibodies.