The ultra-long length of the blood plasma protein von Willebrand factor (VWF) enables its activation by hydrodynamic forces at sites of hemorrhage (Springer, 2014; Figure 1A, B). VWF, a long concatemer of monomers linked head-to-head and tail-to-tail, undergoes a change of its multimeric superstructure from a random coil form (Parker and Lollar, 2021) to a thread-like, extended form when exposed to elongational flow (Bonazza et al., 2015; Fu et al., 2017). Hydrodynamic flow activates VWF to bind to glycoprotein Ib (GPIb) on platelets, the essential interaction required for hemostasis and thrombosis in the arteriolar circulation. Fluorescent imaging of single VWF concatemers tethered in a flow cell showed that binding of GPIbα not only requires elongation of VWF but also a half-maximal tensile force of ~20 pN transmitted through the backbone of VWF concatemers (Fu et al., 2017). Activation could be well fit to a model of transition from a low-affinity state 1 to a high-affinity state 2 (Figure 1B). Measuring A1–GPIbα binding and unbinding with laser tweezers also revealed two states, with a force dependent switch between two states with different on- and off-rates (Kim et al., 2015; Kim et al., 2010). In addition to such flex-bond measurements, A1 and GPIbα are also reported to form catch bonds, although these measurements lack single molecule fiduciary markers or fits to models from which on- and off-rates could be extracted (Ju et al., 2013). Despite multiple crystal structures of the A1 domain bound to GPIb, distinct conformational states of A1 that explain force-dependent regulation of A1 affinity for GPIbα have yet to emerge (Blenner et al., 2014; Dumas et al., 2004).

Figure 1

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The force-responsive A1 domain is unusual among protein domains in being linked to neighboring domains through O-glycosylated, mucin-like segments (Figure 1A, blue hashed lines). Tensile force exerted on A1 is transmitted through these linkers. It has been suggested that A1-flanking sequences (Auton et al., 2012; Interlandi et al., 2017; Ju et al., 2013; Miyata and Ruggeri, 1999; Sugimoto et al., 1993) or domains (Aponte-Santamaría et al., 2015; Ulrichts et al., 2006) may shield the GPIbα-binding epitope and thereby inhibit otherwise tight binding to GPIb. It was also proposed that the N- and C-linkers interact with each other and form an autoinhibitory module that masks A1 (Deng et al., 2017). A force-dependent signature for breakage of this module has been reported that correlates with the combined expected extension of the N- and C-linkers (Arce et al., 2021). An alternative mechanism has also been proposed by which the N-linker could regulate A1 affinity, that is, by regulating the relative stability of the A1 native and intermediate states. A1 has been found to have three states, native (N), intermediate (I), and denatured (D), while the homologous A2 and A3 domains in VWF have only two states, native and denatured (Auton et al., 2007). Lower stability of the A1 native state correlates with higher affinity (Auton et al., 2012; Auton et al., 2010a). This anticorrelation between A1 affinity and stability is also seen with von Willebrand disease type 2B gain-of-function mutations, which increase A1 affinity and decrease stability (Auton et al., 2010b; Tischer et al., 2017). Conversely, the G1342S decrease-in-function type 2M von Willebrand disease mutation decreases A1 affinity for GpIbα and increases A1 stability (Auton et al., 2009).

Mucin-like regions in proteins have unique characteristics distinct from both intrinsically disordered polypeptide segments and folded domains. The bulky O-linked glycans which include sialic acid that are attached to threonine and serine residues in mucins are highly solvated, repel one another, and make mucins extended, with an average length of ~2 Å/residue (Clemetson, 1983; Woollett et al., 1985). Electron microscopy showed that D3 and A1 in VWF, which are connected by the N-linker, have many possible orientations, and an interdomain distance as long as ~7 nm (Zhou et al., 2011). A1 is mechanically stabilized by a long-range disulfide bond between Cys1272 and Cys1458 (Figure 1B, C). Under tension, the core of A1 between these two cysteines, residues 1273–1457, is protected from complete unfolding. Only the portions of A1 external to this disulfide bond, residues 1238–1272 and 1458–1489, are directly subjected to the mechanical force transmitted through the spine of the VWF concatemer. However, multiple internal residues are noncovalently associated with folded external residues, and thus the stability of internal residues is expected to be affected by elongation of external residues by tensile force.

Here, to understand how binding of the VWF A1 domain to platelet GPIbα is regulated by its N- and C-terminal mucin-like linkers, we have measured the effects of both the glycan and polypeptide moieties of these linkers on stability, thermodynamics, and ligand-binding affinity and kinetics of A1. These linkers include the multiple serine and threonine residues that were found to be O-glycosylated during protein sequencing of native plasma VWF, including Thr-1248, Thr-1255, Thr-1256, and Ser-1263 in the N-linker (Titani et al., 1986). Mass spectrometry verified these assignments and showed a total of eight sialic acids over the four sites (Solecka et al., 2016). We show that both the N- and C-linkers affect affinity, that both their polypeptide and O-glycan moieties are important, and that the N-linker affects stability and hydrogen deuterium exchange (HDX) of A1 including in specific regions internal to its disulfide.

Among domains in proteins, the A1 domain of VWF is highly unusual in being separated from neighboring domains by mucin-like linkers and being activated by tensile force transmitted through those linkers that originates from hydrodynamic force on VWF concatemers and bound platelets. Here, we have investigated the effects of these linkers on A1 thermodynamics and function in binding to its ligand, GPIbα. Previous studies have examined the effect of the N-terminal linker or both linkers on various types of quantitative or nonquantitative binding assays and thermodynamics and found inhibitory effects on GPIbα binding from adding the N-linker or both linkers to A1 (Arce et al., 2021; Auton et al., 2012; Deng et al., 2018; Deng et al., 2017; Interlandi et al., 2017; Ju et al., 2013; Miyata and Ruggeri, 1999; Nakayama et al., 2002; Tischer et al., 2017; Tischer et al., 2014). We found that both the N- and C-linker decreased A1 affinity for GPIbα and that for both the N- and C-linker, O-glycosylated linkers decreased affinity more than polypeptide linkers. For the C-linker, both the O-glycan and polypeptide moieties were similar in importance, giving ~2.2-fold decreases in affinity each, while for the N-linker, the O-glycan component was more influential (9.5-fold) than the polypeptide alone (4.3-fold). While adding O-glycosylated linkers on the N- and C-terminal ends of A1 lowered affinity substantially (51- and 6-fold, respectively), the presence of both O-glycosylated linkers lowered binding little compared to the presence of the N-linker alone (1.2-fold). The separate contributions of the polypeptide and glycan moieties of the A1 N- and C-linkers had not previously been examined; however, O-glycosylation of both linkers together was reported to decrease affinity from 3.9 to 32 µM (Tischer et al., 2020). While these reported affinities differ from ours by two- to threefold, they agree in terms of the affinity difference caused by O-glycosylation. Kinetic measurements showed that the effect of both the polypeptide and O-glycan components of the linkers was primarily on-rate driven. Overall, among the seven constructs, on-rates varied by up to 80-fold (A1 short versus A1+OGly+N+C) while off-rates varied up to 10-fold (A1+OGly+C versus A1+N).

Rapid binding is especially important for proteins that function in hemostasis; electrostatics can help orient proteins for proper binding before they contact one another, as previously demonstrated using the ionic strength dependence of binding of soluble GPIbα to VWF tethered to the wall of a flow chamber (Fu et al., 2017). Our kinetic measurements in 10 mM compared to 150 mM NaCl showed a 20-fold increase in affinity for A1+OGly+N+C largely driven by a sevenfold increase in k_on, while VWF tethered to a flow chamber showed a 130-fold increase in affinity driven by an 11-fold increase in k_on.

In 150 mM NaCl, the high-affinity state of tethered, mechanically tensioned VWF had an affinity of 80 nM for GPIbα, compared to the K_d values of 180 nM for A1 short and 9200 nM for A1+OGly+N+C measured here. Introduction of two VWD mutations into A1 was shown to increase affinity for GPIbα by 10-fold (Blenner et al., 2014). However, even though such a mutant would be expected to increase the affinity of the A1 short construct to a higher level than found for mechanically tensioned A1 in intact VWF, a crystal structure of A1 short with the two VWD mutations bound to GPIbα revealed only minor differences from a WT-GPIbα crystal structure (Blenner et al., 2014). Mechanical tension on the one hand and introduction of VWD mutations in the absence of tension and removal of the N- and C-linkers on the other hand may have different effects on the structure of the A1 domain, consistent with the hypothesis that A1 short with two VWD mutations bound to GPIbα did not reveal an A1 high-affinity state, despite an affinity for GPIbα comparable to that seen with mechanically tensioned VWF (Blenner et al., 2014).

We buttressed BLI affinity measurements here with ITC measurements of affinity and binding energies. While affinity measurements in the literature on essentially identical A1 fragments can differ up to 15-fold from those reported here (Huizinga et al., 2002), affinity measurements here on seven different A1 constructs by BLI and ITC were all within 1.6- to 1.04-fold of one another. We found it was necessary to remove aggregates from A1 preparations by gel filtration immediately prior to BLI measurements to avoid heterogeneous kinetics that required two on- and off-rates to fit, which appear in some of the literature on A1 binding to GPIbα (Arce et al., 2021; Tischer et al., 2017). In general, measurements in the liquid phase are more accurate than those in solid/liquid phases such as SPR and BLI, where a small amount of an avid aggregate can dominate binding kinetics.

We are unaware of previous thermodynamic measurements of A1 and GPIbα binding. ITC results show that the interaction of GPIbα with all seven A1 constructs was endothermic with a large entropic term and ΔG values that ranged from −8.2 to −14.7 kcal/mol at 22°C. Among all seven A1 constructs, the enthalpy and entropy terms were highly correlated, so that their rank orders were identical. Entropy-driven binding of proteins is usually attributed to the increase in water entropy when waters are released from hydrophobic binding interfaces and is often counterbalanced by an increase in enthalpy (Liu and Guo, 2001; Richards, 1977; Starikov and Nordén, 2007).

Our HDX studies revealed that the O-glycosylated N-linker has a significant effect in stabilizing the α1–β2, β3–α2, and the α2–α3 loops, as shown by less deuterium incorporation. Previous HDX studies have shown that destabilizing A1 by reducing and alkylating its long-range disulfide, introducing VWD type 2B mutations, or omitting N-linker residues 1238–1260 increase HDX in the same regions (Deng et al., 2018; Deng et al., 2017; Tischer et al., 2017; Tischer et al., 2014). The finding that stands out from previous reports, which did not study the C-linker individually in HDX, is that in contrast to the N-linker, the C-linker did not significantly affect HDX in any of these regions.

We directly measured the stability of A1 by measuring its unfolding by heat and urea. We found evidence for three states by both techniques, consistent with a previous finding of an intermediate state that is hypothesized to represent a state with high affinity for GPIbα (Auton et al., 2010a; Tischer et al., 2017; Tischer et al., 2014). A plateau between the first and second transition clearly demonstrated the existence of the intermediate state for three of our four constructs, in contrast to the decrease in slope seen previously. This difference was likely due to measurement here of the fluorescence of the single tryptophan residue, Trp-1313 in A1, as opposed to circular dichroism, which measures contributions from all A1 secondary structures. Trp-1313 is in the α1–β2 loop that has HDX dynamics that are sensitive to the presence of the N-linker, locates near the GPIbα β-finger, is mutated in VWD type 2B, and although it may report denaturation of only a portion of A1, Trp-1313 discriminates between the native, intermediate, and denatured states of A1 similarly to circular dichroism.

Heat and urea denaturation had similar linker-dependent effects on stability of A1. A1+OGly+N and A1+OGly+N+C were each ~3°C more stable than A1 short and A1+OGly+C. Likewise, A1+OGly+N and A1+OGly+N+C each had native (N) states that were relatively more stable than their intermediate (I) states, by ~2 kcal/mol, than found for A1 short and A1+OGly+C. Additionally, the N-linker was sufficient to completely protect A1 against heat-induced aggregation up to 85°C. Since physiological tensile force on A1 may induce the I state, which could make A1 and with it an entire VWF concatemer susceptible to aggregation, one of the several important physiological roles of the N-linker and its O-glycosylation may be to protect VWF from aggregation. The neighboring A2 domain in VWF can be completely and reversibly unfolded by tensile force and can also be reversibly heated to 90°C without aggregation in the presence of its O-linked N-linker (the C-linker of A1) and its C-linker (Xu and Springer, 2013; Zhang et al., 2009), which may have a similar protective role physiologically to prevent aggregation during physiological partial (A1) and full (A2) unfolding.

Our results on the effects of the O-linked N- and C-linkers on affinity and stability differ. The C-linker decreased A1 affinity for GPIbα in two different measures, BLI and ITC, but had no effect on A1 stability as measured by HDX, melting temperature, and the relative stability of the A1 N and I states in urea denaturation experiments. These results suggest that the C-linker decreases affinity by direct mechanisms such as by steric or electrostatic repulsion. O-Glycans are decorated with sialic acid, which would repel negatively charged GPIbα. In contrast to the C-linker, the N-linker not only decreased affinity but also increased A1 stability as shown by significantly decreased HDX, increased melting temperature, and increased stability of the N state relative to the I state. The ~2 kcal/mol difference in ΔG between the N and I state for A1+OGly+N compared to A1 short corresponds to a ~30-fold difference in population of the I state. Thus, if the I state was the high-affinity state, much of the 40-fold difference in affinity between the I and N states could be accounted for by the shift in equilibrium toward the native state caused by the N-linker, and the remainder could be caused by steric and electrostatic repulsion of GPIbα. In VWF concatemers, the ΔG required for activation by mechanical tension of the high-affinity state was measured as 1.9 kcal/mol (Fu et al., 2017), considerably lower than the difference in energy between the I and N states measured for A1+OGly+N+C here of 4.7 kcal/mol. These differences suggest that the directional nature of energy input by mechanical tension allows for more efficient activation of A1 than urea denaturation and may induce a distinct and less disordered intermediate state.

Three different mechanisms have been proposed for how the linkers affect A1 binding to GPIbα. The proposal that the N- and C-linker associate with one another to form an autoinhibitory module (Deng et al., 2018; Deng et al., 2017) is not supported by our data that show that the N-linker alone is sufficient to inhibit binding of A1 to GPIbα. Furthermore, we find that the N- and C-linker are not synergistic and to the contrary, have much less than additive effects on lowering binding affinity. It also has been proposed that residue Asp-1261 in the N-linker interacts with basic residues in the A1 domain to stabilize a low-affinity state of A1 (Interlandi et al., 2017). While our results do not bear on this mechanism, they do demonstrate that the O-linked glycans within the N-linker are sufficient to strongly inhibit binding of A1 to GPIbα. Finally, Auton et al. have described an intermediate state of A1 that is proposed to correspond to an activated high-affinity state of A1 (Auton et al., 2007; Auton et al., 2012; Tischer et al., 2014). Our data are consistent with and provide further evidence in support of this hypothesis.

The association of the N-linker but not the C-linker with A1 stability is consistent with structural analysis of A1 complexes with GPIbα that demonstrate that their association strains the N-terminal portion of A1 (reviewed in Blenner et al., 2014). The main site of GPIbα association is at the β-switch in its C-terminal cap (Figure 5D). The flexible β-finger in the N-terminal cap of GPIbα associates with A1 over a smaller region, including the α1–β2 loop, with few specific contacts. Furthermore, these contacts vary among GPIbα complexes. Strain in A1 upon binding GPIbα causes shifts in some residues in the α1–β2 loop and Cys-1272 away from GPIbα. Furthermore, the hydrogen bond between the backbone of Cys-1272 and the backbone of Arg-1308 (a residue often mutated in VWD), which is conserved in all structures of the isolated A1 domain, is lost in all A1–GPIbα complex structures. This loss admits a water molecule to the hydrophobic core of A1. These structural observations begin to define a pathway for conformational communication between the N-linker and regions of A1 that change upon GPIbα binding, including the α1–β2 loop, which showed increased HDX dynamics in the absence of the N-linker. Crystal structures of A1 diverge prior to residue 1270, suggesting that the O-glycosylated N-linker of A1 (defined in our experiments as residues 1238–1263) is natively disordered. It is now well established that natively disordered regions in proteins frequently have important roles in regulating protein allostery (Motlagh et al., 2014). We speculate that in the intermediate state, A1 would reshape to enable closer approach to the concave, acidic surface of GPIbα of more A1 basic residues, such as those in its β3–α2 loop shown in stick in Figure 5D.

In summary, our results show that the A1 N-linker, but not the C-linker, increases the stability of the A1 native state relative to the intermediate state, thus regulating conformational change. The previous paragraph discussed structural evidence for a pathway for communication from the N-linker to regions that are known to change structurally upon GPIbα binding and also have backbone motions that are influenced by the N-linker as shown by HDX. Additionally, our results suggest that the C-linker influences affinity for GPIbα by steric and possibly charge repulsion. The N-linker may repel GPIbα as well, in addition to regulating A1 conformation.

Figure 4—source data 1–4 contain the numerical data used to generate the figures. All HDX MS data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with dataset identifier PXD029942.

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Decision letter after peer review:

Thank you for submitting your article "Von Willebrand factor A1 domain affinity for GPIbα and stability are differentially regulated by its O-glycosylated N-linker and C-linker" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: P. Clint Spiegel (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

In this paper, the authors use several biophysical tools, including biolayer interferometry, isothermal calorimetry, protein denaturation, and hydrogen-deuterium exchange mass spectrometry, to systemically dissect the influence of the N- and C-terminal polypeptide linkers and O-glycan moieties of the VWF A1 domain on A1's activation state and binding affinity to platelet receptor GPIbα.

Through a series of elegant experiments, the authors concluded that the N-terminal linker together with its O-linked glycans stabilize the A1 in an inactive, native conformation. Removal of the N-linker or its O-linked glycans destabilizes the A1 structure and may activate the A1 into an intermediate, higher affinity state for GPIbα binding. The authors also concluded that the C-terminal linker and its O-linked glycans do not contribute to stabilizing the A1 structure, though they prevent GPIbα binding through steric hindrance and electrostatic repulsion. The data and the figure presented in the manuscript are overall clear, and the conclusions are consistently supported by the data from multiple experimental techniques. The reported results are highly interesting for the hematology and biophysics communities.

1) Define the A1 domain in terms of the sequence that delimits it and the sequences that define the N- and C- linkers and provide justification for the selection.

2) Show the standard deviation of the fit instead of showing the average of two experiments in figure 2 and provide additional description for the methods used to purify protein constructs in the gel images as the bands appear differentially pure, in different amounts in figure 1.

Reviewer #1 (Recommendations for the authors):

In this paper, the authors use several biophysical tools, including biolayer interferometry, isothermal calorimetry, protein denaturation, and hydrogen-deuterium exchange mass spectrometry, to systemically dissect the influence of the N- and C-terminal polypeptide linkers and O-glycan moieties of the VWF A1 domain on A1's activation state and binding affinity to platelet receptor GPIbα.

Through a series of elegant experiments, the authors concluded that the N-terminal linker together with its O-linked glycans stabilize the A1 in an inactive, native conformation. Removal of the N-linker or its O-linked glycans destabilizes the A1 structure and may activate the A1 into an intermediate, higher affinity state for GPIbα binding. The authors also concluded that the C-terminal linker and its O-linked glycans do not contribute to stabilizing the A1 structure, though they prevent GPIbα binding through steric hindrance and electrostatic repulsion. The data and the figure presented in the manuscript are overall clear, and the conclusions are consistently supported by the data from multiple experimental techniques. The reported results are highly interesting for the hematology and biophysics communities

I have only a few suggestions for revision and improvement, detailed below.

1) The length of the long C-linker is inconsistent among the constructs. While the constructs expressed from E. coli end at residue 1486, the mammalian constructs end at residue 1489. No further justification was given by the authors. Moreover, since the A2 domain starts at residue 1494, why didn't the authors include residues 1490-1493?

2) In regard to Figure 2, some of the global fits are not of high quality (e.g., A1+C with 150 mM NaCl). The authors should show the standard deviation of the fit instead of showing the average of two experiments.

3) It would be worth discussing the difference between the intermediate state identified by the thermal/chemical denaturation experiments and the more physiological mechanically tensioned structure. The intermediate state may or may not be the same high-affinity conformation induced by tensile pulling, since heat/urea destabilizes the entire A1 structure and tensile force/tension is not expected to spread into the structures protected by the long-ranged disulfide.

4. Lines 223-224: It would be nice to specify the exact constructs and conditions that account for the 80-fold and 10-fold differences.

Reviewer #2 (Recommendations for the authors):

Bonazza, Klaus et al. investigate the molecular details for how von Willebrand factor (VWF) binds to Glycoprotein Ib (GPIb) on platelets. Previous studies have demonstrated that VWF binds to GPIb only under conditions of high tensile force through an unfolding event that occurs for the A1 domain of VWF. This study emphasizes the role(s) of the unstructured linkers, along with their associated glycosylation states, that are N- and C-terminal to the A1 domain. Their data demonstrate that both linkers decrease the affinity of VWF A1 for GPIb; the N-terminal linker decreases the affinity more strongly whilst stabilizing the A1 fold, and glycosylation of the linkers contributes more to the decreased affinity than the peptide component. With biolayer interferometry (BLI), they present association and dissociation curves for this intermolecular interaction that fit well to a one-site binding model to calculate on- and off-rates with high confidence, showing a decrease in affinity for various constructs containing the N- and/or C- terminal linkers (with and without glycosylation). Additionally, they use BLI to show that the interaction is largely electrostatic by calculating higher affinities at lower ionic strength conditions. They further support the BLI data with measuring equilibrium binding thermodynamics with isothermal titration calorimetry (ITC). This experimental approach allows them to demonstrate similar affinity values to the BLI results, but also provides a deeper understanding for the thermodynamic basis for the VWF/GPIb interaction; the binding event is endothermic (entropically driven). In order to investigate how the N- and C-linkers affect protein stability, they employed both thermal and chemical denaturation measured with intrinsic tryptophan fluorescence. These data suggest that the A1 domain adopts an intermediate, partially unfolded state, which has differential stability dependent on the presence of the linkers; the N-terminal linker stabilizes the 'native' state, thus restricting the partially unfolded state that is hypothesized to be the GPIb binding state. Following back scattering intensity as a function of temperature in the same experiment, they also show that the N-linker prevents aggregation of the A1 domain. Lastly, the authors employ H/D exchange measured by mass spectrometry (HDX-MS) to show that the presence of the N-linker specifically increases protection patterns (decreased H/D exchange) for regions of the A1 domain that are adjacent to the GPIb binding interface and the internal disulfide bond. Taken together, what emerges from this study is a model for VWF A1/GPIb association that is regulated by glycosylated linkers whereby the N-linker stabilizes a 'native' conformation, which is only perturbed to an intermediate, partially unfolded state under high tensile force, leading to the activation of a high affinity binding state for GPIb. The data presented in this study are clearly communicated and appropriately controlled and interpreted.

Some perceived weaknesses are as follows:

1. The authors appropriately report a gel image of their purified protein constructs. The bands on the gel appear differentially pure, in different amounts, and there is no description in the manuscript about the relative 'diffuse' nature of the bands for glycosylated protein constructs.

2. The study describes the rigorous treatment of their thermal and chemical denaturation curves, and they briefly address the difference between measuring protein unfolding via intrinsic tryptophan fluorescence vs. circular dichroism. None of the curves presented illustrate error bars, but they do provide a measure of error in the associated table. Their measurements in this experiment are exclusively dependent on the environment of a single tryptophan residue in the A1 domain structure; their interpretations of the unfolding curves are warranted, but not without the caveat that it is only measuring the local environment of the tryptophan and not the entire protein structure.

My recommendations for the authors concern the gel image in figure 1 and the Materials and methods section. For figure 1, the protein bands are underwhelming. The reader would have more confidence in the study if this gel demonstrated a more uniform concentration of the protein constructs used in the study. Some of the bands are quite large and dominate the figure while some are comparatively weaker (+N+C in particular). For the Materials and methods section, I question the accuracy of a couple statements: it is stated that you have NiCl in the purification buffers. Ni is divalent, so this should be more accurately reported as NiCl2. In the section on non-glycosylated proteins, the authors state that SHuffle cells are grown at 37C, but these cells are typically grown at 30C due to their thermolability (if this is correct, that is fine, but it diverges from the manufacturer's protocol). Lastly, please define for Equation 1 what b and s are.

Reviewer #3 (Recommendations for the authors):

The authors examined the effect of N- and C-linkers of the von Willebrand factor (VWF) A1 domain on binding to platelet glycoprotein Ibalpha (GPIbalpha). They made recombinant A1 proteins with and without the linkers and with and without O-glycans. They measured the interaction of the proteins by biolayer interferometry (BLI), isothermal titration calorimetry (ITC). Additionally, they measured the thermal and urea-induced denaturation and the hydrogen deuterium exchange (HDX) kinetics of the proteins. They found that both the N-and C-linkers lower A1 affinity, the effects of which are increased by O-glycosylation. The N-linker, but not the C-linker, stabilized the A1 domain and affected HDX exchange. They conclude that the A1 domain is regulated allosterically by the N-linker.

Overall, the conclusions of the study appear to be supported by carefully conceived and conducted experiments. The manuscript is well-written and nicely illustrated. The dissociation constants measured by BLI differ significantly from those measured by ITC, indicating systematic errors in one or both of the methods. However, the linker and glycan effects are qualitatively the same. Thus, the authors' conclusions are supported by the results of two independent physical methods.

The findings support and extend previously published findings by several groups that N- and C-linkers of the A1 domain regulate its interaction with GPIbalpha. However, the authors choose to interpret their results as being inconsistent with the proposal that N-linker and C-linker associate with one another to form an autoinhibitory module because they find that the N-linker alone is sufficient to inhibit binding of A1 to GPIbalpha. However, it is possible that the C-linker also contributes to the regulation of A1 binding to GPIbalpha, which, in fact, is supported by the authors' own the BLI and ITC results.

It would be useful for the authors to define the A1 domain in terms of the sequence that delimits it and the sequences that define the N- and C- linkers. Is the A1 delimited by residues 1272 and 1458 or by residues 1264 – 1469 (Figure 1C)?

The organization at the end of the Introduction perhaps could be improved. The last paragraph begins with "Here, to understand … we have made fundamental measurements …" preparing the reader for what the authors did. However, the authors proceed in the next few sentences to continue to cite observations from the literature.

The authors examined the effect of N- and C-linkers of the von Willebrand factor (VWF) A1 domain on binding to platelet glycoprotein Ibalpha (GPIbalpha). They made recombinant A1 proteins with and without the linkers and with and without O-glycans. They measured the interaction of the proteins by biolayer interferometry (BLI), isothermal titration calorimetry (ITC). Additionally, they measured the thermal and urea-induced denaturation and the hydrogen deuterium exchange (HDX) kinetics of the proteins. They found that both the N-and C-linkers lower A1 affinity, the effects of which are increased by O-glycosylation. The N-linker, but not the C-linker, stabilized the A1 domain and affected HDX exchange. They conclude that the A1 domain is regulated allosterically by the N-linker.

Overall, the conclusions of the study appear to be supported by carefully conceived and conducted experiments. The manuscript is well-written and nicely illustrated. The dissociation constants measured by BLI differ significantly from those measured by ITC, indicating systematic errors in one or both of the methods. However, the linker and glycan effects are qualitatively the same. Thus, the authors' conclusions are supported by the results of two independent physical methods.

The findings support and extend previously published findings by several groups that N- and C-linkers of the A1 domain regulate its interaction with GPIbalpha. However, the authors choose to interpret their results as being inconsistent with the proposal that N-linker and C-linker associate with one another to form an autoinhibitory module because they find that the N-linker alone is sufficient to inhibit binding of A1 to GPIbalpha. However, it is possible that the C-linker also contributes to the regulation of A1 binding to GPIbalpha, which, in fact, is supported by the authors' own the BLI and ITC results.

It would be useful for the authors to define the A1 domain in terms of the sequence that delimits it and the sequences that define the N- and C- linkers. Is the A1 delimited by residues 1272 and 1458 or by residues 1264 – 1469 (Figure 1C)?

The organization at the end of the Introduction perhaps could be improved. The last paragraph begins with "Here, to understand … we have made fundamental measurements …" preparing the reader for what the authors did. However, the authors proceed in the next few sentences to continue to cite observations from the literature.

Essential revisions:

In this paper, the authors use several biophysical tools, including biolayer interferometry, isothermal calorimetry, protein denaturation, and hydrogen-deuterium exchange mass spectrometry, to systemically dissect the influence of the N- and C-terminal polypeptide linkers and O-glycan moieties of the VWF A1 domain on A1's activation state and binding affinity to platelet receptor GPIbα.

Through a series of elegant experiments, the authors concluded that the N-terminal linker together with its O-linked glycans stabilize the A1 in an inactive, native conformation. Removal of the N-linker or its O-linked glycans destabilizes the A1 structure and may activate the A1 into an intermediate, higher affinity state for GPIbα binding. The authors also concluded that the C-terminal linker and its O-linked glycans do not contribute to stabilizing the A1 structure, though they prevent GPIbα binding through steric hindrance and electrostatic repulsion. The data and the figure presented in the manuscript are overall clear, and the conclusions are consistently supported by the data from multiple experimental techniques. The reported results are highly interesting for the hematology and biophysics communities.

1) Define the A1 domain in terms of the sequence that delimits it and the sequences that define the N- and C- linkers and provide justification for the selection.

The justification is summarized in Results in lines 107-110: "The N and C-terminal boundaries of the constructs were based on allowing comparability to previous studies; references to the previous literature and a structural rationale for defining the minimal size of A1 as residues 1270-1463 and the maximal size of A1 plus linkers as residues 1240 to 1494 are described in Methods.” A new section in Methods, "Boundaries of A1 constructs" appears on lines 341-357.

2) Show the standard deviation of the fit instead of showing the average of two experiments in figure 2 and provide additional description for the methods used to purify protein constructs in the gel images as the bands appear differentially pure, in different amounts in figure 1.

Global non-linear least square fitting the traces at all analyte concentrations, we obtained standard fitting errors ranging from 1% to 5% of fit values. These fitting errors were smaller than the difference from the mean of 2 independent experiments shown in Figure 2; therefore, we have reported these results in the Figure 2 legend.

The proteins may have varied from 90 to 99% pure but that would not affect the measured quantities. The proteins were at different concentrations and were not adjusted to identical amounts for SDS-PAGE, which is explained in the legend. More details are given in Methods on purification and a summary has been added to the Results.

Reviewer #1 (Recommendations for the authors):

In this paper, the authors use several biophysical tools, including biolayer interferometry, isothermal calorimetry, protein denaturation, and hydrogen-deuterium exchange mass spectrometry, to systemically dissect the influence of the N- and C-terminal polypeptide linkers and O-glycan moieties of the VWF A1 domain on A1's activation state and binding affinity to platelet receptor GPIbα.

Through a series of elegant experiments, the authors concluded that the N-terminal linker together with its O-linked glycans stabilize the A1 in an inactive, native conformation. Removal of the N-linker or its O-linked glycans destabilizes the A1 structure and may activate the A1 into an intermediate, higher affinity state for GPIbα binding. The authors also concluded that the C-terminal linker and its O-linked glycans do not contribute to stabilizing the A1 structure, though they prevent GPIbα binding through steric hindrance and electrostatic repulsion. The data and the figure presented in the manuscript are overall clear, and the conclusions are consistently supported by the data from multiple experimental techniques. The reported results are highly interesting for the hematology and biophysics communities

I have only a few suggestions for revision and improvement, detailed below.

1) The length of the long C-linker is inconsistent among the constructs. While the constructs expressed from E. coli end at residue 1486, the mammalian constructs end at residue 1489. No further justification was given by the authors. Moreover, since the A2 domain starts at residue 1494, why didn't the authors include residues 1490-1493?

We have explained this above.

2) In regard to Figure 2, some of the global fits are not of high quality (e.g., A1+C with 150 mM NaCl). The authors should show the standard deviation of the fit instead of showing the average of two experiments.

Please see above.

3) It would be worth discussing the difference between the intermediate state identified by the thermal/chemical denaturation experiments and the more physiological mechanically tensioned structure. The intermediate state may or may not be the same high-affinity conformation induced by tensile pulling, since heat/urea destabilizes the entire A1 structure and tensile force/tension is not expected to spread into the structures protected by the long-ranged disulfide.

This was included in the original manusucript,: "In VWF concatemers, the ΔG required for activation by mechanical tension of the high affinity state was measured as 1.9 kcal/mol {Fu, 2017 #24942}, considerably lower than the difference in energy between the I and N states measured for A1+OGly+N+C here of 4.7 kcal/mol. These differences suggest that the directional nature of energy input by mechanical tension allows for more efficient activation of A1 than urea denaturation and may induce a distinct and less disordered intermediate state."

There are many interactions between residues internal and external to the disulfide bond, including hydrogen bonds, van der Waals contacts, burial from water. Therefore, we expect that force will have a significant indirect effect on the structure and stability of regions internal to the disulfide. This is mentioned in the Discussion.

4. Lines 223-224: It would be nice to specify the exact constructs and conditions that account for the 80-fold and 10-fold differences.

Done.

Reviewer #2 (Recommendations for the authors):

[…]

Some perceived weaknesses are as follows:

1. The authors appropriately report a gel image of their purified protein constructs. The bands on the gel appear differentially pure, in different amounts, and there is no description in the manuscript about the relative 'diffuse' nature of the bands for glycosylated protein constructs.

This information has been added to Results.

2. The study describes the rigorous treatment of their thermal and chemical denaturation curves, and they briefly address the difference between measuring protein unfolding via intrinsic tryptophan fluorescence vs. circular dichroism. None of the curves presented illustrate error bars, but they do provide a measure of error in the associated table. Their measurements in this experiment are exclusively dependent on the environment of a single tryptophan residue in the A1 domain structure; their interpretations of the unfolding curves are warranted, but not without the caveat that it is only measuring the local environment of the tryptophan and not the entire protein structure.

We have revised " Trp-1313 is in the α1-β2 loop that has HDX dynamics that are sensitive to the presence of the N-linker, locates near the GPIbα β-finger, is mutated in VWD type 2B, and appears to be a more discrete reporter of A1 conformational state than circular dichroism."

to read

"Trp-1313 is in the α1-β2 loop that has HDX dynamics that are sensitive to the presence of the N-linker, locates near the GPIbα β-finger, is mutated in VWD type 2B, and although it may report denaturation of only a portion of A1, Trp-1313 discriminates between the native, intermediate, and denatured states of A1 similarly to circular dichroism."

My recommendations for the authors concern the gel image in figure 1 and the Materials and methods section. For figure 1, the protein bands are underwhelming. The reader would have more confidence in the study if this gel demonstrated a more uniform concentration of the protein constructs used in the study. Some of the bands are quite large and dominate the figure while some are comparatively weaker (+N+C in particular).

This has been addressed as discussed above.

For the Materials and methods section, I question the accuracy of a couple statements: it is stated that you have NiCl in the purification buffers. Ni is divalent, so this should be more accurately reported as NiCl2.

Thanks. Done.

In the section on non-glycosylated proteins, the authors state that SHuffle cells are grown at 37C, but these cells are typically grown at 30C due to their thermolability (if this is correct, that is fine, but it diverges from the manufacturer's protocol).

"SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm Julie Lobstein, Charlie A Emrich, Chris Jeans, Melinda Faulkner, Paul Riggs and Mehmet Berkmen" describes multiple growth temperatures. We used 30°C after induction.

Lastly, please define for Equation 1 what b and s are.

Thanks, they were defined many lines above the equation and are now defined again immediately before Equation 1.

Reviewer #3 (Recommendations for the authors):

[…]

It would be useful for the authors to define the A1 domain in terms of the sequence that delimits it and the sequences that define the N- and C- linkers. Is the A1 delimited by residues 1272 and 1458 or by residues 1264 – 1469 (Figure 1C)?

Sections on this have been added as described above.

The organization at the end of the Introduction perhaps could be improved. The last paragraph begins with "Here, to understand … we have made fundamental measurements …" preparing the reader for what the authors did. However, the authors proceed in the next few sentences to continue to cite observations from the literature.

Good point. We have broken this up into two paragraphs.

Author details

1. Klaus Bonazza

   Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   Formal analysis, Funding acquisition, Investigation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Roxana E Iacob

   Department of Chemistry and Chemical Biology, Northeastern University, Boston, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Nathan E Hudson

   Department of Physics, East Carolina University, Greenville, United States

   Contribution

   Funding acquisition, Investigation

   Competing interests

   No competing interests declared

4. Jing Li

   Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

5. Chafen Lu

   Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. John R Engen

   Department of Chemistry and Chemical Biology, Northeastern University, Boston, United States

   Contribution

   Formal analysis, Funding acquisition, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6918-9476

7. Timothy A Springer

   Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Funding acquisition, Methodology, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   springer@crystal.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6627-2904

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