A1 Domain Research Articles

A Mutant Complement Factor H (W1183R) Enhances Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Under Shear.

A loss-of-functional mutation (W1183R) in human complement factor H (CFH) is associated with complement-associated hemolytic uremic syndrome; mice carrying a similar mutation (W1206R) in CFH also develop thrombotic microangiopathy but its plasma von Willebrand factor (VWF) multimer sizes were dramatically reduced. The mechanism underlying such a dramatic change in plasma VWF multimer distribution in these mice is not fully understood. To determine the VWF and CFH interaction and how CFH proteins affect VWF multimer distribution, we employed recombinant protein expression, purification, and various biochemical and biophysical tools. Purified recombinant W1183R-CFH but not wild-type (WT) CFH protein enhanced the proteolytic cleavage of both peptidyl and multimeric VWF substrates by recombinant ADAMTS13 in a concentration-dependent manner. Microscale thermophoresis assay demonstrated that both W1183R-CFH and WT-CFH proteins bound various VWF fragments (e.g., AIM-A1, A1-A2-A3, D'D3, D'D3-A1, and D'D3-A1-A2) with high affinities. Optical tweezer experiments further showed a concentration-dependent alteration in the contour length (Lc) and the persistent length (Lp) following pulling VWF-A2 domain in the presence of W1183R-CFH or WT-CFH protein. AlphaFold experiments revealed conformational changes in the VWF-A2, particularly the central region where the cleavage bond resides following addition of W1183R-CFH or WT-CFH protein. These results demonstrate for the first time that W1183R-CFH but not WT-CFH protein enhances the proteolytic cleavage of VWF by ADAMTS13 under shear. This may be achieved by mechanic-induced conformational changes of the central A2 domain, leading to an altered cleavage of Tyr1605-Met1606 bond by ADAMTS13 under pathophysiological conditions.

Structure resolved dynamics of type 2M Von Willebrand Disease.

Genetically determined amino acid substitutions in the platelet adhesive A1 domain alter von Willebrand factor's platelet agglutination competence resulting in both gain- (Type 2B) and loss-of-function (Type 2M) phenotypes of Von Willebrand disease. Prior studies of variants in both phenotypes revealed defects in secondary structure that altered stability and folding of the domain. An intriguing observation was that loss of function arose from both misfolding of A1 and, in a few cases, hyper-stabilization of the native structure. To fully understand the 2M phenotype, we thoroughly investigated the structure/function relationships of fifteen additional type 2M variants and two polymorphisms in the A1 domain. These variants were characterized using circular dichroism, fluorescence, calorimetry, hydrogen deuterium exchange mass spectrometry, surface plasmon resonance, and platelet adhesion under shear flow. Six variants were natively folded with four being hyper-stabilized. Nine variants disordered A1, causing a loss in α-helical structure and unfolding enthalpy. GPIbα binding affinity and platelet adhesion dynamics were highly correlated to helical structure. Hydrogen deuterium exchange resolved specific C-terminal secondary structure elements that differentially diminish the GPIbα binding affinity of A1. These localized structural perturbations were highly correlated to GPIbα binding affinity and shear-dependent platelet adhesion. While hyper-stabilized dynamics in A1 do impair stable platelet attachment to VWF under flow, variant-induced localized disorder in specific regions of the domain misfolds A1 and abrogates platelet adhesion. These two opposing conformational properties represent two structural classes of VWF that drive the loss-of-function phenotype that is type 2M VWD.

Aptamer-based biotherapeutic conjugate for shear responsive release of Von Willebrand factor A1 domain.

Smart polymers that mimic and even surpass the functionality of natural responsive materials have been actively researched. This study explores the design and characterization of a Single-MOlecule-based material REsponsive to Shear (SMORES) for the targeted release of A1, the platelet binding domain of the blood clotting protein von Willebrand factor (VWF). Each SMORES construct employs an aptamer molecule as the flow transducer and a microparticle to sense and amplify the hydrodynamic force. Within the construct, the aptamer, ARC1172, undergoes conformational changes beyond a shear stress threshold, mimicking the shear-responsive behavior of VWF. This conformational alteration modulates the bioavailability of its target, the VWF-A1 domain, ultimately releasing it at elevated shear. Through optical tweezer-based single-molecule force measurement, ARC1172s role as a force transducer was assessed by examining its unfolding under constant pulling force. We also investigated its refolding rate as a function of force under varied relaxation periods. These analyses revealed a narrow range of threshold forces (3-7 pN) governing the transition between folded and unfolded states. We subsequently constructed the SMORES material by conjugating ARC1172 and a microbead, and immobilizing the other end of the aptamer on a substrate. Single-molecule flow experiments on immobilized SMORES constructs revealed a peak A1 domain release within a flow rate range of (40-70 μL min-1). A COMSOL Multiphysics model translated these flow rates to total forces of 3.10 pN-5.63 pN experienced by the aptamers, aligning with single-molecule force microscopy predictions. Evaluation under variable flow conditions showed a peak binding of A1 to the platelet glycoprotein Ib (GPIB) within the same force range, confirming released payload functionality. Building on knowledge of aptamer biomechanics, this study presents a new strategy to create shear-stimulated biomaterials based on single biomolecules.

NMR resonance assignment of a ligand-binding domain of ephrin receptor A2.

Ephrin receptors regulate intercellular communication and are thus involved in tumor development. Ephrin receptor A2 (EphA2), in particular, is overexpressed in a variety of cancers and is a proven target for anti-cancer drugs. The N-terminal ligand-binding domain of ephrin receptors is responsible for the recognition of their ligands, ephrins, and is directly involved in receptor activation. Here, we report on the complete 1H, 15N and 13C NMR chemical shift assignment of EphA2 ligand binding domain that provides the basis for NMR-assisted drug design.

FVIII peptides presented on HLA-DP and identification of an A3 domain peptide binding with high affinity to the commonly expressed HLA-DP4.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

A Novel P-III Metalloproteinase from Bothrops barnetti Venom Degrades Extracellular Matrix Proteins, Inhibits Platelet Aggregation, and Disrupts Endothelial Cell Adhesion via α5β1 Integrin Receptors to Arginine-Glycine-Aspartic Acid (RGD)-Containing Molecules.

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca2+ ions and inhibited by an excess of Zn2+. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics.

Synthesis of a Truncated Microcystin Tetrapeptide Molecule from a Partial Mcy Gene Cluster in Microcystis Cultures and Blooms.

Microcystis spp. threaten freshwater ecosystems through the proliferation of cyanobacterial harmful algal blooms (cyanoHABs) and production of the hepatotoxin, microcystin. While microcystin and its biosynthesis pathway, encoded by the mcy genes, have been well studied for over 50 years, a recent study found that Microcystis populations in western Lake Erie contain a transcriptionally active partial mcy operon, in which the A2 domain of mcyA and mcyB-C are present but the mcyD-J genes are absent. Here, we investigate the potential biosynthetic products and the evolutionary history of this partial operon. Our results reveal two candidate tetrapeptide constructs, with an X variable position, to be produced by strains with the partial operon. The partial operon appears necessary and sufficient for tetrapeptide biosynthesis and likely evolved from a single ancestor hundreds to tens of thousands of years ago. Bioactivity screens using Hep3B cells indicate a mild elevation of some markers of hepatotoxicity and inflammation, suggesting the need to further assess the effects of these novel secondary metabolites on freshwater ecosystems and public health. The need to assess these effects is even more pressing given the detection of tetrapeptides in both culture and western Lake Erie, which is a vital source of fresh water. Results from this study emphasize previous findings in which novel bacterial secondary metabolites may be derived from the molecular evolution of existing biosynthetic machinery under different environmental forcings.

Dynamic conformational response of von Willebrand factor to varying shear stress: A hybrid computational approach

This computational study examines the mechanobiological responses of von Willebrand Factor (vWF) to different shear stress levels, emphasizing the unique structural dynamics of its various domains. We introduce a novel coarse-grained model, representing each cluster of six amino acids as a bead, to synthesize the Lattice Boltzmann method with Brownian dynamics. This approach captures the vWF molecule’s conformational adaptability across a spectrum of shear rates, ranging from 5 to 5e + 7s−1, encompassing normal physiological flows to those resembling the high-shear environment of arterial stenosis.Our results reveal domain-specific responses of vWF, particularly in the A2 and A3 domains, which demonstrate significant elongation under elevated shear—reflecting their pivotal roles in clotting processes. Moreover, the study underscores the critical influence of random thermal forces on the molecule’s conformation, with simulations including these forces showing substantially greater conformational variability compared to those without.As shear rates increase, we observe a notable reorganization in the spatial arrangement of vWF domains. This reconfiguration is most evident in the increased separation between the A1 and D’D3 domains under high shear conditions, potentially enhancing the accessibility of platelets to key binding sites on vWF. These findings are paramount for a deeper understanding of vWF’s mechanical behavior in blood clotting and thrombosis, particularly the delicate balance between facilitating platelet adhesion and avoiding excessive stretching that could lead to thrombosis.Our work provides a foundation for future studies investigating vWF behavior in complex flow geometries and its interactions with other blood components. Such insights pave the way for more informed therapeutic strategies targeting vWF function in vascular health and disease, potentially leading to improved treatments for thrombotic disorders.

Structure-based discovery of first inhibitors targeting the helicase activity of human PIF1.

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

Persistent splenic-derived IgMs preferentially recognize factor VIII A2 and C2 domain epitopes but do not alter antibody production

BackgroundThe most significant treatment complication for patients with hemophilia A is the development of neutralizing immunoglobins (Igs) G, termed inhibitors, against factor VIII (FVIII), which prevent FVIII replacement therapy. Low titers of FVIII-specific IgMs have been identified in hemophilia A patients with and without inhibitors, as well as in healthy individuals. However, the duration and influence of IgMs on the immune response to FVIII remains unclear. ObjectivesTo characterize the binding interactions of persistently secreted FVIII-specific IgMs in hemophilia A mice and assess their effect on IgG antibody development. MethodsSplenic-derived monoclonal antibodies (mAbs) from immunized FVIII knockout mice were isolated and purified using hybridoma technology. Binding interactions were assessed utilizing a novel fluid-phase enzyme-linked immunosorbent assay and computational modeling with High Ambiguity-Driven protein-protein DOCKing to account for weak IgM binding. ResultsSixteen porcine cross-reactive and noninhibitory FVIII-specific IgM mAbs were identified. RNA sequencing of FVIII-specific IgMs revealed 13 unique variable, diversity, and joining (VDJ)/variable and joining (VJ) sequences indicating derivation from 13 unique B cell clones. The IgMs demonstrated polyclonal and polyreactive binding to FVIII in vitro and in silico. Molecular docking studies with reconstructed IgM variable, diversity, and joining/variable and joining regions identified frequent IgM interactions with amino acid residues K376, T381, K437, R2215, or K2249 within the FVIII A2 and C2 domains. Injections of individual IgMs prior to FVIII exposure and co-injection of FVIII/IgM immune complexes did not affect de novo FVIII antibody production. ConclusionPersistent FVIII-specific IgMs are polyclonal but preferentially bind the A2 and C2 domains. FVIII/IgM immune complex formation does not significantly alter inhibitor development.

Cross-Effects in Folding and Phase Transitions of hnRNP A1 and C9Orf72 RNA G4 In Vitro.

Abnormal intracellular phase transitions in mutant hnRNP A1 may underlie the development of several neurodegenerative diseases. The risk of these diseases increases upon C9Orf72 repeat expansion and the accumulation of the corresponding G-quadruplex (G4)-forming RNA, but the link between this RNA and the disruption of hnRNP A1 homeostasis has not been fully explored so far. Our aim was to clarify the mutual effects of hnRNP A1 and C9Orf72 G4 in vitro. Using various optical methods and atomic force microscopy, we investigated the influence of the G4 on the formation of cross-beta fibrils by the mutant prion-like domain (PLD) of hnRNP A1 and on the co-separation of the non-mutant protein with a typical SR-rich fragment of a splicing factor (SRSF), which normally drives the assembly of nuclear speckles. The G4 was shown to act in a holdase-like manner, i.e., to restrict the fibrillation of the hnRNP A1 PLD, presumably through interactions with the PLD-flanking RGG motif. These interactions resulted in partial unwinding of the G4, suggesting a helicase-like activity of hnRNP A1 RGG. At the same time, the G4 was shown to disrupt hnRNP A1 co-separation with SRSF, suggesting its possible contribution to pathology through interference with splicing regulation.

Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

The aptamer BT200 blocks interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1

AbstractRondaptivon pegol (previously BT200) is a pegylated RNA aptamer that binds to the A1 domain of von Willebrand factor (VWF). Recent clinical trials demonstrated that BT200 significantly increased plasma VWF–factor VIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF has not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full-length and VWF-A1A2A3 binding to macrophages and VWF-A1 domain binding to lipoprotein receptor–related protein 1 (LRP1) cluster II and cluster IV were concentration-dependently inhibited by BT200. Additionally, full-length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of 4 lysine residues (K1405, K1406, K1407, and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (P < .001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (P < .001) reduced than that of wild-type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-K1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represents a novel therapeutic approach for von Willebrand disease and hemophilia A.

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge

G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5′ single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5′ most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

R1205H (Vicenza) causes conformational changes in the von Willebrand factor D′D3 domains and enhances von Willebrand factor binding to clearance receptors LRP1 and SR-AI

Backgroundvon Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. ObjectivesThis study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. MethodsIn vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF−/− mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-AI), low-density lipoprotein receptor–related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin. ResultsIn full-length VWF missing the A1 domain, introduction of R1205H led to significantly enhanced clearance in VWF−/− mice compared with WT-VWF missing the A1 domain. Importantly, R1205H in a truncated VWF-D′D3 fragment also triggered increased clearance compared with WT-VWF-D′D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-AI compared with WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (P < .001) and less marked enhanced binding to LRP1 cluster II (P = .034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to macrophage galactose-type lectin. ConclusionDisruption of the positive charge at amino acid R1205 causes conformational changes in the VWF-D′D3 domains and triggers enhanced LRP1-mediated and SR-AI–mediated clearance.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we investigate the determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by the prion-like low-complexity domain of the protein hnRNP A1 and its designed variants. We find that the dominantly viscous forms of the condensates are metastable Maxwell fluids. A Rouse-Zimm model that accounts for the network-like organization of proteins within condensates reproduces the measured viscoelastic moduli. We show that the strengths of aromatic inter-sticker interactions determine sequence-specific amplitudes of elastic and viscous moduli, and the timescales over which elastic properties dominate. These condensates undergo physical ageing on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of dominantly viscous phases. The ageing of condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to amino acid identities of stickers versus spacers in prion-like low-complexity domains, have evolved to afford control over metastabilities of dominantly viscous fluid phases of condensates. This selection is likely to render barriers for conversion from metastable fluids to globally stable solids insurmountable on functionally relevant timescales.

Cryptic Extensibility in von Willebrand Factor Revealed by Molecular Nanodissection.

Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.

Immune Thrombotic Thrombocytopenic Purpura: Pathophysiology, Diagnosis, Therapy and Open Issues.

Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ischemic end-organ injury due to microvascular platelet-rich thrombi. iTTP pathophysiology is based on a severe ADAMTS13 deficiency, the specific von Willebrand factor (vWF)-cleaving protease, due to anti-ADAMTS13 autoantibodies. Early diagnosis and treatment reduce the mortality. Frontline therapy includes daily plasma exchange (PEX) with fresh frozen plasma replacement and immunosuppression with corticosteroids. Caplacizumab has recently been added to frontline therapy. Caplacizumab is a nanobody that binds to the A1 domain of vWF, blocking the interaction of ultra-large vWF multimers with the platelet and thereby preventing the formation of platelet-rich thrombi. Caplacizumab reduces mortality due to ischemic events, refractoriness, and exacerbations after PEX discontinuation. Until now, the criteria for response to treatment mainly took into account the normalization of platelet count and discontinuation of PEX; with the use of caplacizumab leading to rapid normalization of platelet count, it has been necessary to redefine the response criteria, taking into account also the underlying autoimmune disease. Monitoring of ADAMTS13 activity is important to identify cases with a low value of activity (<10IU/L), requiring the optimization of immunosuppressive therapy with the addition of Rituximab. Rituximab is effective in patients with refractory disease or relapsing disease. Currently, the use of Rituximab has expanded, both in frontline treatment and during follow-up, as a pre-emptive approach. Some patients do not achieve ADAMTS13 remission following the acute phase despite steroids and rituximab treatment, requiring an individualized immunosuppressive approach to prevent clinical relapse. In iTTP, there is an increased risk of venous thrombotic events (VTEs) as well as arterial thrombotic events, and most occur after platelet normalization. Until now, there has been no consensus on the use of pharmacological thromboprophylaxis in patients on caplacizumab because the drug is known to increase bleeding risk.