OC 07.4 - Structural biology of substrate recognition by tissue factor/factor VIIa

Saturday, June 22, 2024

13:45 – 14:00 ICT

Room: 210 A-D

Presenting Author(s)

Josepha C. Sedzro, PhD

Postdoctoral Researcher
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States

Co-Author(s)

FB

Fabienne Birkle

Graduate Student
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
KM

Katarina Meze

Postdoctoral Researcher
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
AP

Amanda Photenhauer

Postdoctoral Researcher
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
XP

Xueyuan Peng

Research Assistant
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
MM

Melanie P. Muller

Graduate Student
Department of Biochemistry, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Ann Arbor, Michigan, United States
SK

Sarah Kearns

Graduate Student
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
MC

Michael A. Cianfrocco

Professor
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
ET

Emad Tajkhorshid

Professor
Department of Biochemistry, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Urbana, Illinois, United States
MO

Melanie D. Ohi

Professor
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA, Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States
JM

James H. Morrissey

Professor
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Ann Arbor, Michigan, United States

Background: Clotting is triggered when factor VIIa (FVIIa) binds to the membrane protein, tissue factor (TF), on cell surfaces, and then activates factors IX (FIX) and X (FX) by limited proteolysis. Mutational studies have implied the existence of a TF “exosite” region near the membrane that helps TF/FVIIa recognize FIX and FX as substrates. However, structures of the membrane-bound TF/FVIIa complex are lacking, impeding progress toward understanding the interactions between these proteins.

Aims: Use cryo-EM analysis on nanoscale membrane bilayers (Nanodiscs) to understand the structural basis of substrate recognition by membrane-bound TF/FVIIa.

Methods: Ternary TF/FVIIa/XK1 complexes were formed by incubating TF/FVIIa on Nanodiscs with XK1, a FX mimetic composed of the FX light chain fused to the first Kunitz domain of TFPI (first created by the Broze lab). Cryo-EM studies were carried out at the U-M Cryo-Electron Microscopy Lab.

Results: Enzyme assays showed that interaction of XK1 with TF/FVIIa required the intact TF exosite, validating TF/FVIIa/XK1 as a model for the TF/FVIIa/FX Michaelis complex. Preliminary cryo-EM images of TF/FVIIa/XK1 lacked the membrane bilayer, but studies employing smaller Nanodiscs and a stabilized version of membrane-associated TF allowed us to determine higher-resolution structures of this membrane-bound ternary complex. In this structure, XK1 interacts with TF/FVIIa at two locations: its Kunitz domain is bound to the FVIIa protease domain, while the membrane-bound GLA domain of XK1 interacts with TF/FVIIa near the membrane surface (TF exosite region). Surprisingly, most of the rest of the FX light chain of XK1 projects into the solvent, having no direct interactions with TF/FVIIa.

Conclusion(s): Cryo-EM analysis of the TF/FVIIa/XK1 complex shows a surprising mode of protein substrate binding to the enzyme complex that triggers blood clotting in normal hemostasis and many thrombotic diseases. (Funded by the National Institutes of Health).