Biology subjects

Springer, T. A.

Publications and source records attributed to Springer, T. A..

14 recordsLinked to original sources

bioRxiv2026

Conformational Preference Classification of Integrin-Binding Ligands Using Free Energy Perturbation

Integrins are crucial cell adhesion receptors and attractive therapeutic targets, but developing safe small-molecule inhibitors has been challenging, at least in part due to inadvertent partial agonism caused by stabilization of the integrins open, high-affinity state. To address this challenge, we present a computational approach using Absolute Binding Free Energy Perturbation (AB-FEP) calculations to predict whether a ligand will stabilize the open or closed integrin states, leveraging the difference between the ligands binding free energy to the respective end states. Despite challenges posed by Ca and Mg ions, metal-coordinating residues in the binding pocket, and the subtlety of structural differences between states, AB-FEP achieved excellent classification performance on a set of known opening and closing ligands, significantly outperforming docking scores and MM-GBSA results. We also showed a good correlation between AB-FEP binding free energy differences and experimental values. Furthermore, AB-FEP provided insights into intermediate integrin states and analysis of simulation trajectories confirmed the formation of a water-mediated hydrogen bond network with an ion in the binding pocket to be characteristic for closing ligands. This work demonstrates AB-FEP as a robust method for classifying integrin ligands by the conformation they stabilize and for understanding their functional mechanisms, offering valuable guidance for designing safe and conformationally selective integrin therapeutics. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/721214v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a0b8eforg.highwire.dtl.DTLVardef@cd6b56org.highwire.dtl.DTLVardef@420cc0org.highwire.dtl.DTLVardef@533963_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

High-Throughput Machine Learning-Aided Antibody Discovery for Cell Surface Antigens

Machine learning (ML) has the potential to revolutionize antibody design and selection, but its success depends on access to extensive, well-curated datasets of antibody-antigen interactions. To address this need, we developed a synthetic Fab yeast display library optimized for seamless ML integration, focusing on sequence diversity within the CDRH3 loop. The library incorporates key sequence features derived from human B cell repertoires essential for efficient antibody generation captured in a compact antigen recognition module (ARM) format. Built using the VH1-69 heavy chain and four light chains, the library was evaluated against ten human and murine cell surface antigens, including PD-L1, TIGIT, and ROBO1. This approach yielded hundreds of antibodies with robust biophysical properties, validated for functional performance in flow cytometry and immunohistochemistry. Furthermore, ML analysis identified additional antibodies for ROBO2 and PD-L2 from the aggregate sequencing data, demonstrating utility for hybrid in silico and experimental workflows. We provide a publicly accessible dataset comprising more than 68,000 Fab sequences and 486 characterized antibodies. This study establishes an ML-compatible framework designed to accelerate and streamline antibody discovery and development.

biophysics↗

bioRxiv2024

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with β subunits

Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins V{beta}3, V{beta}5, V{beta}6, V{beta}8, and 5{beta}1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the V subunit for its 5 {beta}-subunit partners: {beta}6={beta}8>{beta}3>{beta}1={beta}5.

cell biology↗

bioRxiv2023

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

The RGD (Arg-Gly-Asp)-binding integrins v{beta}6 and v{beta}8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of v{beta}6 and v{beta}8 with high selectivity. The v{beta}6 and v{beta}8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7[A] root-mean-square deviation (RMSD) to the computational design models; the designed v{beta}6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-v{beta}6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the v{beta}8 inhibitor maintains the constitutively fixed extended-closed v{beta}8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the v{beta}6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

biochemistry↗

bioRxiv2022

Conformational change of the Plasmodium TRAP I domain is essential for sporozoite migration and transmission of malaria

Eukaryotic cell adhesion and migration relies on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite., while binding ligands on the substrate through its inserted (I)-domain. Crystal structures of TRAP from different Plasmodium species revealed the I-domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I-domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry and transmission. Absence of gliding in sporozoites expressing the open TRAP I-domain could be partly rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility and organ invasion and hence sporozoite transmission from mosquito to mammal.

cell biology↗

bioRxiv2022

Subtype-specific single β1 integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling

Although integrins are known to be mechano-sensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin 4{beta}1 and RGD-binding integrins (V{beta}1 and 5{beta}1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of 4{beta}1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of V{beta}1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.

biophysics↗

bioRxiv2022

Von Willebrand factor A1 domain affinity for GPIbα and stability are differentially regulated by its O-glycosylated N-linker and C-linker

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor to GPIb 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 GPIb, 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 GPIb, which is induced by tensile force physiologically and regulated allosterically by the N-linker. Impact StatementBoth the polypeptide and attached O-glycans N-terminal to the A1 domain in von Willebrand factor lower its affinity for its ligand GPIb on platelets, its stability, and structural dynamics and decrease population of a high-affinity, intermediate state in unfolding. Key pointsO_LILigand-binding affinity of A1 and the stability of its native state is regulated by an N-terminal interdomain, O-glycosylated linker C_LI

biophysics↗

bioRxiv2021

Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2

HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitos. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.

immunology↗

bioRxiv2021

Low affinity integrin states have faster binding kinetics than the high affinity state

Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins 4{beta}1 and 5{beta}1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rate measurements are similar for integrins on cell surfaces and ectodomain fragments. Although the extended-open conformations on-rate is [~]20-fold slower, its off-rate is [~]25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. These kinetic measurements, together with previous equilibrium measurements of integrin conformational state affinity and relative free energy on intact cells, are key to a definitive understanding of the mechanism of integrin activation.

biophysics↗

bioRxiv2021

Protection of the prodomain 1-helix correlates with latency in the transforming growth factor- family

The 33 members of the transforming growth factor beta (TGF-{beta}) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of prodomains that are non-covalently bound to the growth factor (GF). The pro-complexes of some 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, but crystal structures and hydrogen-deuterium exchange (HDX) of four family members have begun to unravel how latency is regulated. Here, we extend this understanding by comparing pro-complex conformation in negative stain EM (nsEM) and HDX of ActA, BMP7, BMP9, BMP10, GDF8, TGF-{beta}1, and TGF-{beta}2. nsEM revealed that family members varied in either adopting cross-armed, open-armed, or V-armed configurations. Latency was achieved in both cross-armed and V-armed but not open-armed conformations. HDX revealed remarkably varying patterns of exchange between family members, consistent with large prodomain sequence divergence. We observed a strong correlation between latency and protection of the prodomain 1-helix from exchange, which in latent members coincided with greater buried surface area of the 1-helix and more hydrogen and cation-pi bonds from the prodomain fastener and GF to the 1-helix. Strong sequence conservation of the 1-helix and fastener only in latent members suggests that similar interactions are conserved and sufficient to confer latency. Moreover, most members exhibited rapid exchange in the unstructured "association region" at the prodomain N-terminus, highlighting their availability for interacting with factors that may regulate latency and extracellular storage.

biophysics↗

bioRxiv2019

Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines

The circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the most advanced CSP-based vaccine RTS,S provides only partial protection, highlighting the need for innovative approaches for vaccine design and development. Here we design and characterize TRAP-CSP fusion antigens, and evaluate their immunogenicity and protection against malaria infection. TRAP N-terminal folded domains were fused to CSP C-terminal fragments consisting of the C-terminal TSR domain with or without the intervening repeat region. Homogenous, monomeric and properly folded fusion proteins were purified from mammalian transfectants. Notably, fusion improved expression of chimeras relative to the TRAP or CSP components alone. Immunization of BALB/c mice with the P. berghei TRAP-CSP fusion antigens formulated in AddaVax adjuvant elicited antigen-specific antibody responses. Remarkably, fusion antigens containing the CSP repeat region conferred complete sterile protection against P. berghei sporozoite challenge, and furthermore, mice that survived the challenge were completely protected from re-challenge 16 weeks after the first challenge. In contrast, fusion antigens lacking the CSP repeat region were less effective, indicating that the CSP repeat region provided enhanced protection, which correlated with higher antibody titers elicited by fusion antigens containing the CSP repeat region. In addition, we demonstrated that N-linked glycans had no significant effect on antibody elicitation or protection. Our results show that TRAP-CSP fusion antigens could be highly effective vaccine candidates. Our approach provides a platform for designing multi-antigen/multi-stage fusion antigens as next generation more effective malaria vaccines.

microbiology↗

bioRxiv2018

Cationic charge and polyspecificity of an integrin domain regulates infectivity of malaria parasites

Cell-cell and cell-substrate adhesion is critical for many functions in life. In eukaryotes, I-domains mediate functions as divergent as tissue traversal by malaria-causing Plasmodium parasites as well as cell adhesion and migration by human leucocytes. The I-domain containing protein TRAP is important for Plasmodium sporozoite motility and invasion. Here we show that the I-domain of TRAP is required to mediate adhesional properties which can be partially preserved when the native I-domain is replaced by I-domains from human integrins or from an apicomplexan parasite that does not infect insects. By putting in vivo data and structural features in perspective we conclude that polyspecificity and positive charge around the ligand binding site of the I-domain are important for TRAP function. Our data suggest a highly preserved functionality of I-domains across eukaryotic evolution that is used by apicomplexan parasites to invade a broad range of tissues in a variety of hosts.

microbiology↗

bioRxiv2017

Prodomain-Growth Factor Swapping in the Structure of pro-TGF-β1

Transforming growth factor (TGF)-{beta} is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-{beta} is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked prior to proprotein convertase (PC) cleavage, and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-{beta}1 with the PC cleavage site mutated, to mimic the structure of the TGF-{beta}1 proprotein. Our structure demonstrates that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure, i.e., the prodomain arm domain and GF domain in each monomer are swapped. Swapping has important implications for the mechanism of biosynthesis in the TGF-{beta} family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-{beta} family. Our structure also provides comparisons between independent TGF-{beta}1 crystal structures and between human and porcine pro-TGF-{beta}1.

biochemistry↗

bioRxiv2017

Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation

Growth differentiation factor 8 (GDF8)/Myostatin is a latent TGF{-}{beta} family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid{-}cleaved GDF8 pro{-}complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro{-}complexes reveals a V{-}shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring{-}like, cross{-}armed conformation of latent TGF{-}{beta}1. Surprisingly, Tolloid{-}cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen-deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain-growth factor dissociation, increased exchange in regions that correspond in pro-TGF-{beta}1 to the 1-helix, latency lasso, and {beta}1 strand in the prodomain and to the {beta}6-7 strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain-growth factor interfaces and primes the growth factor for release from the prodomain.

biochemistry↗