Structural evidence proposes that the VWFpp mutations affect the
N-terminal assembly and/or VWF recruitment for anterograde transport
Our VWF N-terminal model is organized into N terminal globular domains
D1 and D2 (VWFpp) connected to the C terminal globular D3 domain by a
filamentous like D´ domain. The D1 and D2 domains are characterized by
several regions of disorder connecting three helix-bundles to beta
sheeted regions in each domain. The disordered regions are most likely
instrumental in the conformational changes occurring in the course of
disulfide exchange within the ER. In the present model apart from 9
cysteines in the D1-D2 domains, all others were observed in an oxidized
form. The D3:D3 domain interaction regions in the model suggest a clear
symmetry within the monomer that serves the purposes of
uni-directionality for the appropriate alignment of the dimeric pro-VWF
subunits and during the multimerization process (Figure 4B). Therefore
the D1, D2 domain surfaces most likely serve one of the two purposes: 1)
as dimeric assembly interfaces or as 2) binding region for recruitment
proteins (like ARF and Sec24) during anterograde transport of VWF from
ER to Golgi. The mutations on the D1, D2 domains could, therefore,
affect either (N-terminal assembly/recruitment process) or both
functional aspects of these domains. The docking of Sec24 and ARF1
protein on the VWF N terminal model strongly supports this idea. The
docking poses/clusters of both Sec24 and ARF1 show strong similarity in
spatial positioning. The majority of the high scoring docking poses for
both proteins occupy the D1 domain surfaces while select few were
concentrated on the D2 domain, the docking surfaces on both domains
facing away from the D3:D3 domain interface (Figures 4B, 4D, and 4F).
The structural alignment of the active and inactive forms of ARF-1 i.e.
GTP and GDP bound structures shows a specific ARF-1 region that
undergoes a conformational change (also called switch region) and
therefore is the part most likely to acts as the protein recruitment
surface/interface for ARF-1 (Supplemental Figure 1). In most of our
observed docks, this region forms a major part of the interface between
ARF 1 and VWF (Supplemental Figure 2). Interestingly, the majority of
our naturally occurring mutations (3 out of 4), are located exactly on
the ARF 1/ or Sec24-VWF-D1 domain interaction interface. One D2 domain
mutation i.e. p.Cys608Trp does not occur directly at the interface but
close to it. Since the Cys608 participates in a disulfide bond, the
mutation will result in an unpaired cysteine which could significantly
influence disulfide bond combinations and also disulfide exchange
(especially since there exist other unpaired Cysteines in close spatial
proximity). Effectively this mutation, therefore, could have potential
allosteric effects on the noncovalent D1-D2 dimer interfaces or affect
interaction with ARF 1 or Sec24.