Publications by Year: 2022

2022
A general chemical principle for creating closure-stabilizing integrin inhibitors
Lin, F.-Y., et al. A general chemical principle for creating closure-stabilizing integrin inhibitors. Cell 185, 19, 3533-3550.e27 (2022).Abstract
Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbβ3 and α4β1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbβ3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4β1.
lin_2022_38059.pdf
Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging
Bergal, H.T., Jiang, Y., Yang, D., Springer, T.A. & Wong, W.P. Conformation of von Willebrand factor in shear flow revealed with stroboscopic single-molecule imaging. Blood (2022).Abstract
von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our labs had shown using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically we found that the required shear stress was higher than reported for free-in-flow VWF-an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF's molecular mechanism, we measured free VWF extension in shear flow using PULSIS-Pulsed Laser Stroboscopic Imaging of Single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame, enabling accurate length measurements in the presence of motion blur. At high shear stresses, we observed a mean shift in VWF extension of less than 200 nm, much shorter than the multiple-micron extensions previously reported with no evidence for the predicted sharp globule-stretch conformational transition. Modeling VWF with a Brownian dynamics simulation, our results are consistent with VWF behaving as an uncollapsed polymer rather than the theorized compact ball. The muted response of free VWF to high shear rates implies that 1) the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports and 2) that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis.
bergal_2022_38108.pdf
Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change
Su, Y., Iacob, R.E., Li, J., Engen, J.R. & Springer, T.A. Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change. J Biol Chem 102323 (2022).Abstract
Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.
su_2022_38109.pdf
Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state
Feng, J., Dong, X., Su, Y., Lu, C. & Springer, T.A. Monomeric prefusion structure of an extremophile gamete fusogen and stepwise formation of the postfusion trimeric state. Nat Commun 13, 1, 4064 (2022).Abstract
Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.
feng-2022-38065.pdf
Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange
Anderson, J.R., Li, J., Springer, T.A. & Brown, A. Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange. Blood (2022).Abstract
von Willebrand Factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excess bleeding of type 2A von Willebrand (VW) disease. Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report cryo-EM structures of complete VWF tubules before and after intermolecular disulfide-bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states prior to and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VW disease-causing mutations.
anderson-2022-38064.pdf
Loss of LRRC33-dependent TGFβ1 activation enhances antitumor immunity and checkpoint blockade therapy
Jiang, A., Qin, Y. & Springer, T.A. Loss of LRRC33-dependent TGFβ1 activation enhances antitumor immunity and checkpoint blockade therapy. Cancer Immunol Res 10, 4, 453-467 (2022).Abstract
TGFβ has multiple roles and gene products (TGFβ1, -β2, and -β3), which make global targeting of TGFβ undesirable. Expression of TGFβ requires association with milieu molecules, which localize TGFβ to the surface of specific cells or extracellular matrices. Here, we found that LRRC33 was specifically associated with TGFβ1, not TGFβ2 and TGFβ3, and was required for surface display and activation of TGFβ1 on tumor-infiltrating myeloid cells. Loss of LRRC33-dependent TGFβ1 activation slowed tumor growth and metastasis by enhancing innate and adaptive antitumor immunity in multiple mouse syngeneic tumor models. LRRC33 loss resulted in a more immunogenic microenvironment, with decreased myeloid-derived suppressor cells, more active CD8+ T and NK cells, and more skewing toward tumor-suppressive M1 macrophages. LRRC33 loss and PD-1 blockade synergized in controlling B16.F10 tumor growth. Our results demonstrate the importance of LRRC33 in tumor biology and highlight the therapeutic potential of dual blockade of the LRRC33/TGFβ1 axis and PD-1/PD-L1 in cancer immunotherapy.
jiang_2022_37950.pdf
Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
Bonazza, K., et al. Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker. Elife 11, (2022).Abstract
Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.
bonazza_2022_38029.pdf
Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities
Anderson, J.M., Li, J. & Springer, T.A. Regulation by metal ions and the ADMIDAS of integrin α5β1 conformational states and intrinsic affinities. Mol Biol Cell mbcE21110536 (2022).Abstract
Activation of integrins by Mn2+ is a benchmark in the integrin field, but how it works and whether it reproduces physiologic activation is unknown. We show that Mn2+ and high Mg2+ concentrations compete with Ca2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg2+ by Mn2+ at the MIDAS increases the intrinsic affinities of both the high affinity open and low affinity closed states of integrins, in agreement with stronger binding of Mn2+ than Mg2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on- and off-states, respectively.
anderson_2022_37926.pdf
Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-β Family
Le, V.Q., et al. Protection of the Prodomain α1-Helix Correlates with Latency in the Transforming Growth Factor-β Family. J Mol Biol 434, 5, 167439 (2022).Abstract
The 33 members of the transforming growth factor beta (TGF-β) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of non-covalently associated prodomains and growth factors (GF). Pro-complexes from a subset of family members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, particularly because of large family diversity. Here, we have examined representative family members in negative stain electron microscopy (nsEM) and hydrogen deuterium exchange (HDX) to identify features that differentiate latent from non-latent members. nsEM showed three overall pro-complex conformations that differed in prodomain arm domain orientation relative to the bound growth factor. Two cross-armed members, TGF-β1 and TGF-β2, were each latent. However, among V-armed members, GDF8 was latent whereas ActA was not. All open-armed members, BMP7, BMP9, and BMP10, were non-latent. Family members exhibited remarkably varying HDX patterns, consistent with large prodomain sequence divergence. A strong correlation emerged between latency and protection of the prodomain α1-helix from exchange. Furthermore, latency and protection from exchange correlated structurally with increased α1-helix buried surface area, hydrogen bonds, and cation-pi bonds. Moreover, a specific pattern of conserved basic and hydrophobic residues in the α1-helix and aromatic residues in the interacting fastener were found only in latent members. Thus, this first comparative survey of TGF-β family members reveals not only diversity in conformation and dynamics but also unique features that distinguish latent members.
le-2022-37756.pdf