Autoinhibitory Module Research Articles

Nanobody activator improves sensitivity of the von Willebrand factor activity assay to multimer size

BackgroundThe activity of von Willebrand factor (VWF) in facilitating platelet adhesion and aggregation correlates with its multimer size. Traditional ristocetin-dependent functional assays lack sensitivity to multimer sizes. Recently, nanobodies targeting the autoinhibitory module and activating VWF were identified. ObjectivesTo develop an assay that can differentiate the platelet-binding activity of VWF multimers. MethodsA novel enzyme-linked immunosorbent assay (nanobody-triggered glycoprotein Ib binding assay [VWF:GPIbNab]) utilizing a VWF-activating nanobody was developed. Recombinant VWF, plasma-derived VWF (pdVWF), and selected gel-filtrated fractions of pdVWF were evaluated for VWF antigen and activity levels. A linear regression model was developed to estimate the specific activity of VWF multimers. ResultsOf the 3 activating nanobodies tested, 6C11 with the lowest activation effect exhibited the highest sensitivity for high-molecular-weight multimers (HMWMs) of VWF. VWF:GPIbNab utilizing 6C11 (VWF:GPIbNab6C11) produced significantly higher activity/antigen ratios for recombinant VWF (>2.0) and HMWM-enriched pdVWF fractions (>2.0) than for pdVWF (∼1.0) or fractions enriched with shorter multimers (<1.0). The differences were much larger than those produced by VWF:GPIbNab utilizing other nanobodies, VWF:GPIbM, VWF:GPIbR, or VWF:CB assays. Linear regression analysis of 5 pdVWF fractions of various multimer sizes produced an estimated specific activity of 2.7 for HMWMs. The analysis attributed >90% of the VWF activity measured by VWF:GPIbNab6C11 to that of HMWMs, which is significantly higher than all other activity assays tested. ConclusionThe VWF:GPIbNab6C11 assay exhibits higher sensitivity to HMWMs than ristocetin-based and collagen-binding assays. Future studies examining the application of this assay in clinical settings and any associated therapeutic benefit of doing so are warranted.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

A novel role of acellular hemoglobin in hemolytic thrombosis

BackgroundHemolytic thrombosis has been associated with acellular hemoglobin released from damaged red blood cells during hemolysis. However, the precise molecular mechanism underlying acellular hemoglobin-induced thrombosis remains arguable. In this study, we examined the interaction between hemoglobin and the A1 domain of von Willebrand factor (VWF), which is a critical mediator of platelet activation. MethodsPrevious studies have suggested that the interaction between hemoglobin and the A1 domain of VWF enhances VWF's hemostatic activity. We employed a multidisciplinary investigation to re-examine this interaction, and identified significant differences in binding affinity between the active and inactive forms of A1. ResultsWe found that hemoglobin binds more strongly to the active A1 than the inactive form. Using hydrogen‑deuterium exchange mass spectrometry, we identified the specific residues involved in this interaction, which are located on the α1-β2 and β3-α2 loops that are typically covered by the “autoinhibitory module” in the inactive A1. This observation provides a structural explanation for the differential binding affinity between the active and inactive forms of A1. We demonstrated that the binding of hemoglobin to A1 blocks the interaction between GPIbα and VWF, and inhibits VWF-mediated thrombosis in vivo. Furthermore, we found that administration of hemoglobin led to similar levels of thrombocytopenia and microthrombosis in both wildtype and VWF-deficient mice, indicating that the mechanism underlying acellular hemoglobin-induced thrombosis is VWF-independent. ConclusionsThese findings challenge the previous theory that hemoglobin-induced thrombosis occurs solely through binding with VWF, and provide evidence supporting a novel role for hemoglobin in hemolytic thrombosis.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain

AbstractType 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain’s thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.

Autoinhibitory module underlies species difference in shear activation of von Willebrand factor

BackgroundVon Willebrand factor (VWF) is a multimeric plasma protein that bridges the gap between vessel injury and platelet capture at high shear rates. Under high shear or tension, VWF can become activated upon the unfolding of its autoinhibitory module (AIM). AIM unfolding exposes the A1 domain, allowing for binding to platelet glycoprotein (GP)Ibα to initiate primary hemostasis. The characteristics of the AIM and its inhibitory properties within mouse VWF are unknown. ObjectivesTo determine and characterize the autoinhibitory properties of mouse VWF. MethodsRecombinant mouse VWF A1 fragments containing or lacking the flanking regions around the A1 domain were generated. We tested the ability of these fragments to bind to human or mouse GPIbα and platelets. We compared the unfolding of mouse AIM‐A1 to human AIM‐A1 by single‐molecule force spectroscopy. ResultsRecombinant mouse AIM‐A1 binds with higher affinity to GPIbα than its human counterpart. Recombinant mouse proteins lacking part of the AIM show increased binding to GPIbα. Activated A1 fragments lacking the AIM can effectively agglutinate platelets across the species barrier. Using single‐molecule force spectroscopy, we determined that the mouse AIM unfolds under forces similar to the human AIM. Additionally, the human AIM paired with mouse A1 largely recapitulates the behavior of human AIM‐A1. ConclusionsOur results suggest that the regulation of VWF‐GPIbα binding has been specifically tuned to work optimally in different rheological architectures. Differences in the AIM sequence may contribute to the difference in VWF shear response between human and mice.

Hydrogen-deuterium exchange of plasma-derived von Willebrand factor reveals similar dynamics to isolated A1 domain and autoinhibitory module

Von Willebrand factor (VWF) is a large multimeric, multidomain glycoprotein that circulates in the bloodstream and is essential for mediating platelet adhesion at sites of vascular injury through its A1 domain. Our lab has recently provided detailed characterizations of the autoinhibitory module (AIM), the discontinuous flanking regions that cooperatively shield the A1 domain and regulate its binding to cognate platelet receptor glycoprotein (GP) Ibα. While our recent analyses have described the mechanical and biochemical properties of AIM-A1 as an independent functional unit, the impact of the neighboring D’D3 and A2 domains on AIM dynamics remain unclear.

Desialylation of O‐glycans activates von Willebrand factor by destabilizing its autoinhibitory module

BackgroundThe binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O‐glycans in VWF are located around the A1 domain. Removing certain O‐glycans in the flanking sequences by site‐directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. ObjectivesTo understand if and how desialylation of O‐glycans in the flanking sequences increases A1 activity. MethodsA recombinant AIM‐A1 fragment encompassing VWF residues 1238–1493 and only O‐glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. ResultsAsialo‐AIM‐A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen–deuterium exchange rates at residues near the secondary GPIbα binding site and the N‐terminal flanking sequence. Asialo‐AIM‐A1 is less mechanically stable than sialo‐AIM‐A1, with its unstressed unfolding rate approximately 3‐fold greater than the latter. ConclusionsDesialylation of O‐glycans around A1 increases its activity by destabilizing the AIM.

The soluble N-terminal autoinhibitory module of the A1 domain in von Willebrand factor partially suppresses its catch bond with glycoprotein Ibα in a sandwich complex.

von Willebrand factor (VWF) senses and responds to the hemodynamic forces to interact with the circulatory system and platelets in hemostasis and thrombosis. The dark side of this mechanobiology is implicated in atherothrombosis, stroke, and, more recently, the COVID-19 thrombotic symptoms. The force-responsive element controlling VWF activation predominantly resides in the N terminal auto-inhibitory module (N-AIM) flanking its A1 domain. Nevertheless, the detailed mechano-chemistry of soluble VWF N-AIM is poorly understood at the sub-molecular level as it is assumed to be unstructured loops. Using the free molecular dynamics (MD) simulations, we first predicted a hairpin-like structure of the soluble A1 N-AIM derived polypeptide (Lp; sequences Q1238-E1260). Then we combined molecular docking and steered molecular dynamics (SMD) simulations to examine how Lp regulates the A1-GPIbα interaction under tensile forces. Our simulation results indicate that Lp suppresses the catch bond in a sandwich complex of A1-Lp-GPIbα yet contributes an additional catch-bond residue D1249. To experimentally benchmark the binding kinetics for A1-GPIbα in the absence or presence of Lp, we conducted the force spectroscopy-biomembrane force probe (BFP) assays. We found similar suppression on the A1-GPIbα catch bond with soluble Lp in presence. Clinically, as more and more therapeutic candidates targeting the A1-GPIbα axis have entered clinical trials to treat patients with TTP and acute coronary syndrome, our work represents an endeavor further towards an effective anti-thrombotic approach without severe bleeding side effects as most existing drugs suffer.

Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library.Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin.Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267).When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12.Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Autoinhibition of Mouse Von Willebrand Factor: Addressing the Species Compatibility between VWF and Gpibα in Humans and Mice

Introduction: The usage of mouse models to investigate thrombosis and hemostasis has now spanned several decades, with the advent of genetic manipulation elucidating the roles of platelet receptors, secondary messengers, and coagulation factors to name a few. While there are many similarities between humans and mice, the vast differences in physical parameters of blood circulation and coagulation should not be ignored. The incompatibility of mouse platelet receptor glycoprotein (GP)Ibα with human von Willebrand factor (VWF) has been well documented, yet human GPIbα can interact with mouse plasma VWF under shear. In light of the recently discovered autoinhibitory module (AIM) in modulating the activity of human VWF, here we seek to address the role of the AIM in species compatibility between these two interacting proteins.Methods: Recombinant human and mouse VWF fragments containing the A1 domain and either intact or truncated AIM, followed by a poly-histidine tag, were expressed from stably transfected mammalian cells. Recombinant human and mouse GPIbα ligand-binding domain (LBD) (residues 1-290, mature protein numbering) were similarly expressed bearing a biotin acceptor sequence and poly-histidine tag. Stable cell lines co-expressing E. coli biotin ligase were used to express and biotinylate the LBD protein before purification. All proteins were purified by nickel-affinity chromatography followed by size exclusion chromatography. Bio-layer interferometry was performed on an Octet QK e using streptavidin biosensors to capture biotinylated GPIbα LBD fragments. Data was analyzed using a 2:1 heterogeneous ligand binding model for kinetic parameters, and steady state affinity (K D) was calculated either from binding responses at the end of the association phase, or from predicted equilibria from the aforementioned model. Human or mouse washed platelets were isolated by centrifugation, followed by pelleting the PRP in the presence of PGE-1. Platelets were resuspended to 150,000/ μL in modified Tyrode's buffer, and aggregation was monitored in a light transmission aggregometer.Results: Consistent with earlier reports, human AIM-A1 protein (VWF residues 1238-1493), at concentrations below 1 μM, showed no appreciable binding to human GPIbα LBD. Conversely, human truncated AIM-A1 protein (tAIM-A1, residues 1261-1472) exhibited a K D of 348 nM towards the LBD. Mouse tAIM-A1 proteins showed enhanced binding to mouse LBD (K D = 214 nM), but mouse AIM-A1 is able to bind to mouse LBD with a K D of 716 nM, which is considerably higher than the affinity of the human-human pair of tens of μM (Figure 1).Given the increased affinity of mouse AIM-A1 to mouse LBD, we reasoned that the mouse AIM may be less stable than the human counterpart. A chimeric AIM-A1 protein, in which human AIM sequences flank mouse A1 (human residues 1238-1271, mouse 1272-1458, and human 1459-1493), was generated. The chimeric AIM-A1 showed little binding to mouse LBD. However, this protein can still aggregate washed platelets from both species and bind to gain-of-function human GPIbα, indicating it still is a folded domain capable to recognizing its cognate receptor, mouse GPIbα (Figure 2).The isolated mouse AIM-A1 fragments are capable of inducing robust mouse and human platelet aggregation for washed platelets in a dose dependent manner. It had been previously demonstrated that in platelet rich plasma, ristocetin has no effect on mouse VWF. However, washed mouse platelets incubated with 60 nM of mouse AIM-A1 followed by addition of 1.5 mg/mL ristocetin was able to agglutinate the platelets (Figure 2). These results suggest that these proteins may be used to help elucidate the signaling pathways of GPIb-IX-V complex in mice.Conclusion: In agreement with previous reports, we observe that in general mouse AIM-A1 is more active than human AIM-A1 in its propensity to bind to GPIbα. On the aspect that human VWF in incompatible with mouse platelets, we observed that mouse LBD has lower affinity for human tAIM-A1 than human LBD does for mouse tAIM-A1. These findings altogether suggest that there are likely differences in activation forces for each species AIM, and that the GPIbα-A1 interactions, in terms of their mechanical and thermodynamic properties, are somewhat unique, and perhaps optimized, for each species. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mapping the Masked Region of the A1 Domain in Full-Length VWF

Von Willebrand factor (VWF) is the largest protein in plasma and promotes the early hemostatic response following vascular injury. The hemostatic response of VWF relies on the exposure of its A1 domain, which recognizes its receptor GPIbα on the platelet surface. To avoid binding and activating platelets in the absence of injury, the A1 domain is dormant in full-length VWF through a mechanism that is not completely understood. We recently characterized two recombinant A1 fragments (longA1, residues 1238-1472; and shortA1, residues 1261-1472) with differential GPIbα-binding affinities by hydrogen-deuterium exchange (HDX) mass spectrometry. We found that N- and C-terminal residues flanking the A1 domain (residues 1238-1271 and 1459-1472) cooperatively constitute an autoinhibitory module (AIM) that masks a region in A1 and impedes its binding to GPIbα[1]. To determine if the A1 domain in full-length VWF is “masked” in the same manner, we have characterized human plasma-derived VWF using the same HDX approach.On-column peptic fragmentation was performed following HDX from 10 to 10,000 sec. We obtained 946 sequenced peptides covering 96.4% of the VWF sequence with an average redundancy of 6 per residue. Among them, 113 peptides covered the A1 domain, allowing HDX analysis of A1 at high resolution. We found that residues around the 1272-1458 disulfide bond, helix α1, loops α1β2 and β3α2 in the A1 domain, which are protected by the AIM in the longA1 fragment (residues 1238-1472), are deeply protected in full-length VWF, suggesting that AIM in full-length VWF likely masks A1 in a similar pattern. Additionally, sheet β3, helix α3, and loops β4α3 in the A1 domain of full-length VWF reported lower HDX rates than their counterparts in the shortA1 fragment. These regions, along with those protected by AIM, form a contiguous surface that significantly overlaps with the GPIbα-binding interface (Fig. 1). Residues in loop β6α6, helix α6 and helix α1 also reported lower HDX rates, but they are separate from the regions protected by AIM and the GPIbα-binding interface.In summary, our results suggest that AIM and likely other domains in full-length VWF are in direct contact with the A1 domain and regulate the hemostatic properties of VWF.Figure 1: Difference in HDX between isolated VWF A1 domains and full length VWF.1 Deng W, Wang Y, Druzak SA, Healey JF, Syed AK, Lollar P, Li R. A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor. Journal of thrombosis and haemostasis : JTH . 2017. 10.1111/jth.13775. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activation of von Willebrand factor via mechanical unfolding of its discontinuous autoinhibitory module

Von Willebrand factor (VWF) activates in response to shear flow to initiate hemostasis, while aberrant activation could lead to thrombosis. Above a critical shear force, the A1 domain of VWF becomes activated and captures platelets via the GPIb-IX complex. Here we show that the shear-responsive element controlling VWF activation resides in the discontinuous autoinhibitory module (AIM) flanking A1. Application of tensile force in a single-molecule setting induces cooperative unfolding of the AIM to expose A1. The AIM-unfolding force is lowered by truncating either N- or C-terminal AIM region, type 2B VWD mutations, or binding of a ristocetin-mimicking monoclonal antibody, all of which could activate A1. Furthermore, the AIM is mechanically stabilized by the nanobody that comprises caplacizumab, the only FDA-approved anti-thrombotic drug to-date that targets VWF. Thus, the AIM is a mechano-regulator of VWF activity. Its conformational dynamics may define the extent of VWF autoinhibition and subsequent activation under force.

Delimiting the autoinhibitory module of von Willebrand factor

Background The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods ELISA, flow cytometry, a thermal stability assay and hydrogen-deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results The longest A1 fragment (rVWF1238-1493 ) showed higher inactivity in binding the platelet receptor glycoprotein (GP)Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459-1478 at the C-terminus of rVWF1238-1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479-1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238-1461 , or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271-1493 , showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion The AIM is probably composed of residues 1238-1271 (N-AIM) and 1459-1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.

A discontinuous autoinhibitory module masks the A1 domain of von Willebrand factor

Essentials The mechanism for the auto-inhibition of von Willebrand factor (VWF) remains unclear. Hydrogen exchange of two VWF A1 fragments with disparate activities was measured and compared. Discontinuous residues flanking A1 form a structural module that blocks A1 binding to the platelet. Our results suggest a potentially unified model of VWF activation. Click to hear an ISTH Academy presentation on the domain architecture of VWF and activation by elongational flow by Dr Springer SUMMARY: Background How von Willebrand factor (VWF) senses and responds to shear flow remains unclear. In the absence of shear flow, VWF or its fragments can be induced to bind spontaneously to platelet GPIbα. Objectives To elucidate the auto-inhibition mechanism of VWF. Methods Hydrogen-deuterium exchange (HDX) of two recombinant VWF fragments expressed from baby hamster kidney cells were measured and compared. Results The shortA1 protein contains VWF residues 1261-1472 and binds GPIbα with a significantly higher affinity than the longA1 protein that contains VWF residues 1238-1472. Both proteins contain the VWF A1 domain (residues 1272-1458). Many residues in longA1, particularly those in the N- and C-terminal sequences flanking the A1 domain, and in helix α1, loops α1β2 and β3α2, demonstrated markedly reduced HDX compared with their counterparts in shortA1. The HDX-protected region in longA1 overlaps with the GPIbα-binding interface and is clustered with type 2B von Willebrand disease (VWD) mutations. Additional comparison with the HDX of denatured longA1 and ristocetin-bound longA1 indicates the N- and C-terminal sequences flanking the A1 domain form cooperatively an integrated autoinhibitory module (AIM) that interacts with the HDX-protected region. Binding of ristocetin to the C-terminal part of the AIM desorbs the AIM from A1 and enables longA1 binding to GPIbα. Conclusion The discontinuous AIM binds the A1 domain and prevents it from binding to GPIbα, which has significant implications for the pathogenesis of type 2B VWD and the shear-induced activation of VWF activity.

Structural Insights into the Autoregulation and Cooperativity of the Human Transcription Factor Ets-2

Ets-2, like its closely related homologue Ets-1, is a member of the Ets family of DNA binding transcription factors. Both proteins are subject to multiple levels of regulation of their DNA binding and transactivation properties. One such regulatory mechanism is the presence of an autoinhibitory module, which in Ets-1 allosterically inhibits the DNA binding activity. This inhibition can be relieved by interaction with protein partners or cooperative binding to closely separated Ets binding sites in a palindromic arrangement. In this study we describe the 2.5 Å resolution crystal structure of a DNA complex of the Ets-2 Ets domain. The Ets domain crystallized with two distinct species in the asymmetric unit, which closely resemble the autoinhibited and DNA bound forms of Ets-1. This discovery prompted us to re-evaluate the current model for the autoinhibitory mechanism and the structural basis for cooperative DNA binding. In contrast to Ets-1, in which the autoinhibition is caused by a combination of allosteric and steric mechanisms, we were unable to find clear evidence for the allosteric mechanism in Ets-2. We also demonstrated two possibly distinct types of cooperative binding to substrates with Ets binding motifs separated by four and six base pairs and suggest possible molecular mechanisms for this behavior.

Structural basis of Ets1 activation by Runx1

Runx1 is required for definitive hematopoiesis and is well-known for its frequent chromosomal translocations and point mutations in leukemia. Runx1 regulates a variety of genes via Ets1 activation on an Ets1•Runx1 composite DNA sequence. The structural basis of such regulation remains unresolved. To address this problem, we determined the crystal structure of the ternary complex containing Runx11-242 and Ets1296-441 bound to T cell receptor alpha (TCRα) enhancer DNA. In the crystal, an Ets1-interacting domain of Runx1 is bound to the Ets1 DNA-binding domain and displaced an entire autoinhibitory module of Ets1, revealing a novel mechanism of Ets1 activation. The DNA binding and transcriptional studies with a variety of structure-guided Runx1 mutants confirmed a critical role of direct Ets1•Runx1 interaction in Ets1 activation. More importantly, the discovered mechanism provides a plausible explanation for how the Ets1•Runx1 interaction effectively activates not only a wild-type Ets1, but also a highly inhibited phosphorylated form of Ets1.

Structure nor stability of the transmembrane spanning 6/7 domain of presenilin I correlates with pathogenicity

Since its cloning in 1995, missense point mutations in presenilin I (PS-I) have been shown to be responsible for greater than 70% of the cases of early onset familial Alzheimer’s disease (EOFAD), which can affect individuals as early as age 18. PS-I is known to be a component of γ-secretase, the enzyme responsible for cleavage of the amyloid precursor protein (APP) into 42 amino acid peptides that aggregate to form the plaques surrounding neurons of Alzheimer’s patients. It has recently been hypothesized that wild-type (wt) PS-I contains an autoinhibitory module that prevents γ-secretase cleavage of the APP, while pathogenic PS-I point mutants lack a structure necessary for this inhibition. In this work, spectroscopic data is presented that does not correlate structure or stability of the proposed PS-I autoinhibitory module with pathogenicity.

Asef2 Functions as a Cdc42 Exchange Factor and Is Stimulated by the Release of an Autoinhibitory Module from a Concealed C-Terminal Activation Element

Asef (herein called Asef1) was identified as a Rac1-specific exchange factor stimulated by adenomatous polyposis coli (APC), contributing to colorectal cancer cell metastasis. We investigated Asef2, an Asef1 homologue having a similar N-terminal APC binding region (ABR) and Src-homology 3 (SH3) domain. Contrary to previous reports, we found that Asef1 and Asef2 exchange activity is Cdc42 specific. Moreover, the ABR of Asef2 did not function independently but acted in tandem with the SH3 domain to bind APC. The ABRSH3 also bound the C-terminal tail of Asef2, allowing it to function as an autoinhibitory module within the protein. Deletion of the C-terminal tail did not constitutively activate Asef2 as predicted; rather, a conserved C-terminal segment was required for augmented Cdc42 GDP/GTP exchange. Thus, Asef2 activation involves APC releasing the ABRSH3 from the C-terminal tail, resulting in Cdc42 exchange. These results highlight a novel exchange factor regulatory mechanism and establish Asef1 and Asef2 as Cdc42 exchange factors, providing a more appropriate context for understanding the contribution of APC in establishing cell polarity and migration.

Different Domains of the Transcription Factor ELF3 Are Required in a Promoter-specific Manner and Multiple Domains Control Its Binding to DNA

Elf3 is an epithelially restricted member of the ETS transcription factor family, which is involved in a wide range of normal cellular processes. Elf3 is also aberrantly expressed in several cancers, including breast cancer. To better understand the molecular mechanisms by which Elf3 regulates these processes, we created a large series of Elf3 mutant proteins with specific domains deleted or targeted by point mutations. The modified forms of Elf3 were used to analyze the contribution of each domain to DNA binding and the activation of gene expression. Our work demonstrates that three regions of Elf3, in addition to its DNA binding domain (ETS domain), influence Elf3 binding to DNA, including the transactivation domain that behaves as an autoinhibitory domain. Interestingly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA. On the basis of these studies, we suggest a model for autoinhibition of Elf3 involving intramolecular interactions. Importantly, this model is consistent with our finding that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C terminus, which contains the ETS domain. In parallel studies, we demonstrate that residues flanking the N- and C-terminal sides of the ETS domain of Elf3 are crucial for its binding to DNA. Our studies also show that an AT-hook domain, as well as the serine- and aspartic acid-rich domain but not the pointed domain, is necessary for Elf3 activation of promoter activity. Unexpectedly, we determined that one of the AT-hook domains is required in a promoter-specific manner.

Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.