Samhitha Urs Ramaraje Urs (Bonn/ DE), Deniz Ugurlar (Eindhoven/ NL), Boxue Ma (Eindhoven/ NL), Jean-Luc Pellequer (Grenoble/ FR), Jean-Marie Teulon (Grenoble/ FR), Daphna Fenel (Grenoble/ FR), Anna Pepanian (Bonn/ DE), Haroon Javed (Bonn/ DE), Muhammed Mubassherhul Islam (Bonn/ DE), Sneha Singh (Bonn/ DE), Diana Imhof (Bonn/ DE), Prof. Dr. Johannes Oldenburg (Bonn/ DE), Arijit Biswas (Bonn/ DE)
Background
Full-length factor VIII protein (FL-FVIII) is structurally well characterized containing a heavily glycosylated B domain. Current structural information is skewed toward the B domain deleted FVIII (BDD-FVIII), leaving most of the B domain to be structurally unresolved. We aim at the structural importance of the B domain by applying biophysical techniques on FL-FVIII and BDD-FVIII and correlating the experimental observations to computational studies performed on FVIII structural models.
Methods
Our sources of FL-FVIII protein were recombinant and plasma-derived concentrates which were subjected to processing on the Krios G4 platform. The data was processed using cryoSPARC software. Air and liquid AFM was performed on high pure rFVIII protein. Comparative CD spectrometry for rFL-FVIII and rBDD-FVIII was performed. Computational studies were performed by glycosylating and cleaving at the furin cleavage site of the alpha fold model of FL-FVIII and BDD-FVIII. The models were subjected to all atomic molecular dynamic simulations and the simulation-equilibrated models were analyzed with the cryo-EM and AFM data. Comparative studies for the binding of interaction partners with FL-FVIII and BDD-FVIII computational models were performed.
Results
Cryo-EM analysis of rFVIII showed conformational and particle heterogeneity whereas pdFVIII showed particle homogeneity thus yielding low-resolution cryo-EM maps. Spatial positioning of the B domain identified by rigid fitting appeared to be scattered around the central core of the FVIII protein. Similar observations of the B domain wrapping around the core globular domains were made in the AFM images. Similar orientation and conformational variability of the B domain were reflected well in our simulation equilibrated models. Qualitative and quantitative differences in secondary structure between FL-FVIII and BDD-FVIII were observed. Surface area accessibility for the binding of interacting partners differed between FL-FVIII and BDD-FVIII.
Conclusion
Owing to the low resolution of the maps, the B domain was not resolved to an atomic level. Biophysical analysis suggests conformational variability and heterogeneity in the FL-FVIII structure indicating high disorderness of the B domain. Glycosylation stabilizes the orientation of the B domain. Differences in secondary structures between FL-FVIII and BDD-FVIII indicated that the binding of FVIII interacting partners or neutralizing antibodies might structurally, and functionally differ.
Offenlegung Interessenkonflikt:
The authors declare no conflicts of interest. This work is funded by IIR grant awarded to PD Dr. Arijit Biswas and Prof. Johannes Oldenburg from Takeda (Shire)