Timothy Springer

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 αVβ3, αVβ5, αVβ6, αVβ8, and α5β1 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 αV subunit for its 5 β-subunit partners: β6=β8>β3>β1=β5

ZRKFYGHZFMAOKI-QMGMOQQFSA-N Tgfbeta Chemical compound C ([C@ H](NC (= O)[C@ H](C (C) C) NC (= O) CNC (= O)[C@ H](CCC (O)= O) NC (= O)[C@ H](CCCNC (N)= N) NC (= O)[C@ H](CC (N)= O) NC (= O)[C@ H](CC (C) C) NC (= O)[C@ H]([C@@ H](C) O) NC (= O)[C@ H](CCC (O)= O) NC (= O)[C@ H]([C@@ H](C) O) NC (= O)[C@ H](CC (C) C) NC (= O) CNC (= O)[C@ H](C) NC (= O)[C@ H](CO) NC (= O)[C@ H](CCC (N)= O) NC (= O)[C@@ H](NC (= O)[C@ H](C) NC (= O)[C@ H](C) NC (= O)[C@@ H](NC (= O)[C@ H](CC (C) C) NC (= O)[C@@ H](N) CCSC) C (C) C)[C@@ H](C) CC) C (= O) N [C@@ H]([C@@ H](C) O) C (= O) N [C@@ H](C (C) C) C (= O) N [C@@ H](CC= 1C= CC= CC= 1) C (= O) N [C@@ H](C) C (= O) N1 [C@@ H](CCC1) C (= O) N [C@@ H]([C@@ H](C) O) C (= O) N [C@@ H](CC (N)= O) C (= O) N [C@@ H](CCC (O)= O) C (= O) N [C@@ H](C) C (= O) N [C@@ H](CC= 1C …

Activation of latent transforming growth factor (TGF)-β2 is incompletely understood. Unlike TGF-β1 and β3, the TGF-β2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-β2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVβ6 but not αVβ8. Both classes of motifs compete for the same binding site in αVβ6. Multiple changes in the longer motif underlie its specificity. ProTGF-β2 structures define interesting differences from proTGF-β1 and the structural context for activation by αVβ6. Some integrin-independent activation is also seen for proTGF-β2 and even more so for proTGF-β3. Our findings have important implications for therapeutics to αVβ6 in clinical trials for fibrosis, in which inhibition of TGF-β2 activation has not …

The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational …

Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out …

Eukaryotic cell adhesion and migration rely 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 reveal 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 …

Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a …

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 excessive bleeding of type 2A von Willebrand disease (VWD). 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 electron cryomicroscopy (cryo-EM) structures of 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 before and after disulfide bond-mediated concatemerization. The structures and …