OC 07.4 - Exploring the conformational landscape of Factor VIII B domain in order to generate an all atom full length structure of the coagulation Factor VIII protein

Saturday, July 9, 2022

1:45 PM – 2:00 PM

Location: ExCel Center, ICC Capital Suite Room 2-4

Presentation Format: In-Person

Presenting Author(s)

Samhitha Urs Ramaraje Urs, MSc

PhD Student
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

SS

Sneha Singh, M.Sc, PhD

Post Doctoral Researcher
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany
Haroon Javed, MSc

PhD student
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany
DF

Daphna Fenel

Technician
Institut de Biologie Structurale, University Grenoble Alpes
Grenoble, Rhone-Alpes, France
JT

Jean Marie Teulon

Engineer
Institut de Biologie Structurale, University Grenoble Alpes
Grenoble, Rhone-Alpes, France
GS

Guy Schoehn

Scientist
Institut de Biologie Structurale, University Grenoble Alpes
Grenoble, Rhone-Alpes, France
JP

Jean Luc Pellequer

Scientist
Institut de Biologie Structurale, University Grenoble Alpes
Grenoble, Rhone-Alpes, France
BM

Boxue Ma

Cryo-EM Application Specialist
Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord
Eindhoven, Noord-Brabant, Netherlands
DU

Deniz Ugurlar

Sales Development Representative
Materials and Structural Analysis Division, Thermo Fisher Scientific, Achtseweg Noord
Eindhoven, Noord-Brabant, Netherlands
DI

Diana Imhof

Professor
University of Bonn
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
Arijit Biswas, PD Dr (Biol)

Scientist
Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany

Background: The B domain of coagulation Factor VIII (FVIII) has a heavily glycosylated and highly disordered structure. Traditional structural characterization methods are unable to resolve the structure of the B domain and hence of full length FVIII (FL-FVIII).

Aims: Our study aims to structurally characterize the FVIII B domain using integrated hybrid methodology [Figure 01]. Bioanalytical techniques such as cross-linking mass spectrometry (XL-MS) are being used on purified FL-FVIII to identify interdomain (between B domain and other domains of FVIII) and intradomain (within B domain) interface residues. The interdomain and intradomain interface residues will be used as modeling restraints for modeling the structure of the B domain and of FL-FVIII. Simultaneously biophysical techniques such as AFM and cryo-EM are being carried out on the purified FL-FVIII. The structural models will be validated by matching/fitting/docking them onto biophysical maps/images generated.

Methods: Plasma concentrates and recombinant FL-FVIII products are further purified by various chromatography methods to remove all excipients. One mM of DSSO crosslinker is used to crosslink 10uM of pure FL-FVIII and the cross-linked product is further purified by SEC (size exclusion chromatography). Glycosyl residues in FL-FVIII were removed by de-glycosylation reaction using 5000 units of PNGase-F enzyme. Negative staining of the purified FL-FVIII followed by 2D classification was done towards future cryo-EM studies. Air and liquid AFM imaging was also performed on the highly pure FL-FVIII. The FL-FVIII model from the alpha-fold database was modified in silico to incorporate all post-translational modification and subjected to all-atom MD simulation.

Results: Pure FL-FVIII was successfully crosslinked with DSSO towards future XL-MS data. Negative staining and AFM of FL-FVIII showed structural heterogeneity in FL-FVIII which is also reflected in the simulation trajectory of the alpha-fold model of FL-FVIII.

Conclusion(s): FL-FVIII is structurally heterogeneous owing to the conformational variability of the B domain.