Site-specific functional roles of the Factor X activation peptide in the intrinsic tenase-mediated Factor X activation.

The conversion of zymogen Factor X (FX) to an active protease involves the removal of a 52-residue long activation peptide (AP). Through site-directed mutagenesis, we investigate the role of the AP and demonstrate that the high abundance of proline residues is important for efficient proteolysis of FX. Moreover, we identify an essential interaction site for Factor IXa (FIXa) between residues 22 and 30 (AP numbering) and find that the residues between 31 and 41 may provide an important interaction site for the intrinsic tenase complex, composed of Factor IXa (FIXa) and Factor VIIIa (FVIIIa). Finally, we suggest that the carbohydrate chain at Asn-39 restricts the activator specificity, as elimination of this glycosylation site increases the activation rate for activation by FIXa and FXa.

Functional Effects of Receptor-Binding Domain Mutations of SARS-CoV-2 B.1.351 and P.1 Variants.

The recent identification and rise to dominance of the P.1 and B.1.351 SARS-CoV-2 variants have brought international concern because they may confer fitness advantages. The same three positions in the receptor-binding domain (RBD) are affected in both variants, but where the 417 substitution differs, the E484K/N501Y have co-evolved by convergent evolution. Here we characterize the functional and immune evasive consequences of the P.1 and B.1.351 RBD mutations. E484K and N501Y result in gain-of-function with two different outcomes: The N501Y confers a ten-fold affinity increase towards ACE-2, but a modest antibody evasion potential of plasma from convalescent or vaccinated individuals, whereas the E484K displays a significant antibody evasion capacity without a major impact on affinity. On the other hand, the two different 417 substitutions severely impair the RBD/ACE-2 affinity, but in the combined P.1 and B.1.351 RBD variants, this effect is partly counterbalanced by the effect of the E484K and N501Y. Our results suggest that the combination of these three mutations is a two-step forward and one step back in terms of viral fitness.