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Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.

In the absence of arabinose, the dimeric Escherichia coli regulatory protein of the l-arabinose operon, AraC, represses expression by looping the DNA between distant half-sites. Binding of arabinose to the dimerization domains forces AraC to preferentially bind two adjacent DNA half-sites, which stimulates RNA polymerase transcription of the araBAD catabolism genes. Prior genetic and biochemical studies hypothesized that arabinose allosterically induces a helix-coil transition of a linker between the dimerization and DNA binding domains that switches the AraC conformation to an inducing state [Brown, M. J., and Schleif, R. F. (2019) Biochemistry, preceding paper in this issue (DOI: 10.1021/acs.biochem.9b00234)]. To test this hypothesis, hydrogen-deuterium exchange mass spectrometry was utilized to identify structural regions involved in the conformational activation of AraC by arabinose. Comparison of the hydrogen-deuterium exchange kinetics of individual dimeric dimerization domains and the full-length dimeric AraC protein in the presence and absence of arabinose reveals a prominent arabinose-induced destabilization of the amide hydrogen-bonded structure of linker residues (I167 and N168). This destabilization is demonstrated to result from an increased probability to form a helix capping motif at the C-terminal end of the dimerizing α-helix of the dimerization domain that preceeds the interdomain linker. These conformational changes could allow for quaternary repositioning of the DNA binding domains required for induction of the araBAD promoter through rotation of peptide backbone dihedral angles of just a couple of residues. Subtle changes in exchange rates are also visible around the arabinose binding pocket and in the DNA binding domain.

The frequent overexpression of CD46 in malignant tumors has provided a basis to use vaccine-lineage measles virus (MeV) as an oncolytic virotherapy platform. However, widespread measles seropositivity limits the systemic deployment of oncolytic MeV for the treatment of metastatic neoplasia. Here, we report the development of MeV-Stealth, a modified vaccine MeV strain that exhibits oncolytic properties and escapes antimeasles antibodies in vivo. We engineered this virus using homologous envelope glycoproteins from the closely-related but serologically non-cross reactive canine distemper virus (CDV). By fusing a high-affinity CD46 specific single-chain antibody fragment (scFv) to the CDV-Hemagglutinin (H), ablating its tropism for human nectin-4 and modifying the CDV-Fusion (F) signal peptide we achieved efficient retargeting to CD46. A receptor binding affinity of ~20 nM was required to trigger CD46-dependent intercellular fusion at levels comparable to the original MeV H/F complex and to achieve similar antitumor efficacy in myeloma and ovarian tumor-bearing mice models. In mice passively immunized with measles-immune serum, treatment of ovarian tumors with MeV-Stealth significantly increased overall survival compared with treatment with vaccine-lineage MeV. Our results show that MeV-Stealth effectively targets and lyses CD46-expressing cancer cells in mouse models of ovarian cancer and myeloma, and evades inhibition by human measles-immune serum. MeV-Stealth could therefore represent a strong alternative to current oncolytic MeV strains for treatment of measles-immune cancer patients., Author summary Vaccine strains of the measles virus (MeV) have been shown to be promising anti-cancer agents because of the frequent overexpression of the host-cell receptor CD46 in human malignancies. However, anti-MeV antibodies in the human population severely restrict the use of MeV as an oncolytic agent. Here, we engineered a neutralization-resistant MeV vaccine, MeV-Stealth, by replacing its envelope glycoproteins with receptor-targeted glycoproteins from wild-type canine distemper virus. By fully-retargeting the new envelope to the receptor CD46, we found that in mouse models of ovarian cancer and myeloma MeV-Stealth displayed oncolytic properties similar to the parental MeV vaccine. Furthermore, we found that passive immunization with measles-immune human serum did not eliminate the oncolytic potency of the MeV-Stealth, whereas it did destroy the potency of the parental MeV strain. The virus we here report may be considered a suitable oncolytic agent for the treatment of MeV-immune patients.

In circulation, plasma glycoprotein von Willebrand Factor plays an important role in hemostasis and in pathological thrombosis under hydrodynamic forces. Mutations in the A1 domain of von Willebrand factor cause the hereditary types 2B and 2M von Willebrand disease that either enhance (2B) or inhibit (2M) the interaction of von Willebrand factor with the platelet receptor glycoprotein Ibalpha. To understand how type 2B and 2M mutations cause clinically opposite phenotypes, we use a combination of protein unfolding thermodynamics and atomic force microscopy to assess the effects of two type 2B mutations (R1306Q and I1309V) and a type 2M mutation (G1324S) on the conformational stability of the A1 domain and the single bond dissociation kinetics of the A1-GPIbalpha interaction. At physiological temperature, the type 2B mutations destabilize the structure of the A1 domain and shift the A1-GPIbalpha catch to slip bonding to lower forces. Conversely, the type 2M mutation stabilizes the structure of the A1 domain and shifts the A1-GPIbalpha catch to slip bonding to higher forces. As a function of increasing A1 domain stability, the bond lifetime at low force decreases and the critical force required for maximal bond lifetime increases. Our results are able to distinguish the clinical phenotypes of these naturally occurring mutations from a thermodynamic and biophysical perspective that provides a quantitative description of the allosteric coupling of A1 conformational stability with the force dependent catch to slip bonding between A1 and GPIbalpha.

The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3. We investigate the extent to which structural elements contributing to intermediate conformations can be identified using a residue-specific implementation of the structure-energy-equivalence-of-domains algorithm (SEED), which parses proteins of known structure into their constituent thermodynamically cooperative components using protein-group-specific, transfer free energies. The structural elements computed to contribute to the non-two-state character coincide with regions where Von Willebrand disease mutations induce misfolded molten globule conformations of the A1 domain. This suggests a mechanism for the regulation of rheological platelet adhesion to A1 based on cooperative flexibility of the α2 and α3 helices flanking the platelet GPIbα receptor binding interface.

The binding of Von Willebrand Factor to platelets is dependent on the conformation of the A1 domain which binds to platelet GPIbalpha. This interaction initiates the adherence of platelets to the subendothelial vasculature under the high shear that occurs in pathological thrombosis. We have developed a thermodynamic strategy that defines the A1:GPIbalpha interaction in terms of the free energies (DeltaG values) of A1 unfolding from the native to intermediate state and the binding of these conformational states to GPIbalpha. We have isolated the intermediate conformation of A1 under nondenaturing conditions by reduction and carboxyamidation of the disulfide bond. The circular dichroism spectrum of reduction and carboxyamidation A1 indicates that the intermediate has approximately 10% less alpha-helical structure that the native conformation. The loss of alpha-helical secondary structure increases the GPIbalpha binding affinity of the A1 domain approximately 20-fold relative to the native conformation. Knowledge of these DeltaG values illustrates that the A1:GPIbalpha complex exists in equilibrium between these two thermodynamically distinct conformations. Using this thermodynamic foundation, we have developed a quantitative allosteric model of the force-dependent catch-to-slip bonding that occurs between Von Willebrand Factor and platelets under elevated shear stress. Forced dissociation of GPIbalpha from A1 shifts the equilibrium from the low affinity native conformation to the high affinity intermediate conformation. Our results demonstrate that A1 binding to GPIbalpha is thermodynamically coupled to A1 unfolding and catch-to-slip bonding is a manifestation of this coupling. Our analysis unites thermodynamics of protein unfolding and conformation-specific binding with the force dependence of biological catch bonds and it encompasses the effects of two subtypes of mutations that cause Von Willebrand Disease.