Low resolution cryo-EM maps and AFM analysis combined with alpha fold model of full-length coagulation Factor VIII sheds light on the conformational positioning of the Factor VIII B domain

Introduction The coagulation factor VIII protein (FVIII) is a structurally well- characterized protein with almost 26 crystal structures corresponding to it deposited in the protein structure database. However, in all these structures, the central large glycosylated B domain remains structurally unresolved and therefore structural information on the full length (FL-FVIII) is currently missing. In the current study, we have analyzed the structure of the FL-FVIII using a combination of atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) and the recently released alpha fold model.

Method The FL-FVIII was purified from two commercial sources of FL-FVIII, one a recombinant (Kovaltry) and the other one a plasma-derived concentrate (Haemoctin) using gel filtration chromatography. The highly pure protein was subjected to AFM and cryo-EM analysis. Both Kovaltry and Haemoctin were processed on Krios G4 platform. The data processing was performed using the cryoSPARC Software. Air and Liquid AFM was performed for Kovaltry on the AFM platform of IBS, Grenoble, France. Computational studies were performed on the alpha fold model of FL-FVIII. The model was treated in two different settings one where the model was glycosylated, and its heavy and light chains separated at the furin cleavage site and subjected to all atomic molecular dynamic simulation studies while in the other setting the non-glycosylated model subjected to MD simulation studies. The simulation-equilibrated models were analyzed with respect to the cryo-EM and AFM data.[1] [2] [3]

Results Cryo-EM and AFM analysis of FL-FVIII showed extensive conformational and particle heterogeneity for the sample purified from Kovaltry unlike Haemoctin which showed relative particle homogenity. Low-resolution maps were obtained from both Kovaltry and Haemoctin samples (6.3 Å and 8.3 Å respectively). Owing to the low resolution of these maps, the B domain was not resolved at an atomic level. However, by rigid fitting of specific domains of FL-FVIII we could identify spatial positioning of parts of the B domain. Computational studies showed that glycosylation has a significant impact in determining the final orientation of the B domain since the non-glycosylated model post equilibration showed an unfolded C terminal while in the glycosylated model, the B domain appeared to stable at a restrictive conformation guided by the formation of select intermediary secondary structures. The conformational variability observed in the cryo-EM and AFM studies was reflected well in the thermal motion observed within the simulation trajectory of the glycosylated alpha fold model of FVIII ([Fig. 1]).

Fig. 1  Full-length coagulation Factor VIII structure; Side by side images of a 8.3 Å cryo-EM density map, liquid AFM and a simulation equilibrated glycosylated and furin cleaved structure of full-length coagulation FVIII (Left to Right).

Conclusion The FL-FVIII shows a significant amount conformational variability, most of which is attributed to the disordered B domain. Glycosylation plays a key role in the folding and stabilization of the B domain with respect to the rest of the FL-FVIII.

This work has been funded by grants from Takeda-Shire under the IIR Program awarded to PD Dr. Arijit Biswas and Prof. Dr. med. Johannes Oldenburg

Publikationsverlauf

Artikel online veröffentlicht:
20. Februar 2023

• References

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