The von Willebrand factor D'D3 assembly and structural principles for factor VIII binding and concatemer biogenesis.

D assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D'D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D'D3 forms the binding site for factor VIII. The crystal structure of monomeric D'D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D' with its TIL' and E' modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL' domain of D' distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry.

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.

Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice.

Anti-factor VIII (FVIII) antibodies is a major complication of FVIII replacement therapy for hemophilia A. We investigated the immune response to recombinant human factor VIII Fc (rFVIIIFc) in comparison to BDD-rFVIII and full-length rFVIII (FL-rFVIII) in hemophilia A mice. Repeated administration of therapeutically relevant doses of rFVIIIFc in these mice resulted in significantly lower antibody responses to rFVIII compared to BDD-rFVIII and FL-rFVIII and reduced antibody production upon subsequent challenge with high doses of rFVIIIFc. The induction of a tolerogenic response by rFVIIIFc was associated with higher percentage of regulatory T-cells, a lower percentage of pro-inflammatory splenic T-cells, and up-regulation of tolerogenic cytokines and markers. Disruption of Fc interactions with either FcRn or Fcγ receptors diminished tolerance induction, suggesting the involvement of these pathways. These results indicate that rFVIIIFc reduces immunogenicity and imparts tolerance to rFVIII demonstrating that recombinant therapeutic proteins may be modified to influence immunogenicity and facilitate tolerance.

The many faces of actin: matching assembly factors with cellular structures.

Actin filaments are major components of at least 15 distinct structures in metazoan cells. These filaments assemble from a common pool of actin monomers, but do so at different times and places, and in response to different stimuli. All of these structures require actin-filament assembly factors. To date, many assembly factors have been identified, including Arp2/3 complex, multiple formin isoforms and spire. Now, a major task is to figure out which factors assemble which actin-based structures. Here, we focus on structures at the plasma membrane, including both sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli). Insights gained from studies of adherens junctions and the immunological synapse are also considered.

INF2 is an endoplasmic reticulum-associated formin protein.

In addition to its ability to accelerate filament assembly, which is common to formins, INF2 is a formin protein with the unique biochemical ability to accelerate actin filament depolymerization. The depolymerization activity of INF2 requires its actin monomer-binding WASP homology 2 (WH2) motif. In this study, we show that INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) in Swiss 3T3 cells. Both endogenous INF2 and GFP-fusion constructs display ER localization. INF2 is post-translationally modified by a C-terminal farnesyl group, and this modification is required for ER interaction. However, farnesylation is not sufficient for ER association, and membrane extraction experiments suggest that ionic interactions are also important. The WH2 motif also serves as a diaphanous autoregulatory domain (DAD), which binds to the N-terminal diaphanous inhibitory domain (DID), with an apparent dissociation constant of 1.1 muM. Surprisingly, the DID-DAD interaction does not inhibit the actin nucleation activity of INF2; however, it does inhibit the depolymerization activity. Point mutations to the DAD/WH2 inhibit both the DID-DAD interaction and depolymerization activity. Expression of GFP-INF2 containing these DAD/WH2 mutations causes the ER to collapse around the nucleus, with accumulation of actin filaments around the collapsed ER. This study is the first to show the association of an actin-assembly factor with the ER.

INF2 Is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization.

Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed "diaphanous formins" are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory domain (DID) and a C-terminal diaphanous autoregulatory domain (DAD). Here, we found several striking features for the mouse formin, INF2. First, INF2 interacted directly with actin through a region C-terminal to the FH2. This second interacting region sequesters actin monomers, an activity that is dependent on a WASP homology 2 (WH2) motif. Second, the combination of the FH2 and C-terminal regions of INF2 resulted in its curious ability to accelerate both polymerization and depolymerization of actin filaments. The mechanism of the depolymerization activity, which is novel for formin proteins, involves both the monomer binding ability of the WH2 and a potent severing activity that is dependent on covalent attachment of the FH2 to the C terminus. Phosphate inhibits both the depolymerization and severing activities of INF2, suggesting that phosphate release from actin subunits in the filament is a trigger for depolymerization. Third, INF2 contains an N-terminal DID, and the WH2 motif likely doubles as a DAD in an autoinhibitory interaction.