Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.
Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demonstrate a detailed understanding of this conformational change by applying computational design techniques to select five amino acid side chains that play an important role in the energetic balance between the open and closed conformations of alphaIIbbeta3. Eight single-point mutants were designed at these sites, of which five bound ligands much better than wild type. Further, these mutants were found to be in a more extended conformation than wild type, suggesting that the conformational change at the ligand binding headpiece was propagated to the legs of the integrin. This detailed understanding of the conformational change will assist in the development of allosteric drugs that either stabilize or destabilize specific integrin conformations without occluding the ligand-binding site.
Selectins are adhesion molecules that resist large tensile forces applied by hydrodynamic forces to leukocytes binding to vessel walls. In crystals, the liganded (high-affinity) and unliganded (low-affinity) conformations differ in orientation between their tandem lectin and EGF domains. I examine how tensile force exerted on a selectin-ligand complex in vivo could favor the more extended, high-affinity conformation. Allostery is transmitted from the EGF-lectin domain interface to the ligand-binding interface on the lectin domain, 30 A away. Trp-1 of the lectin domain and the long axis of the EGF domain form an L-shaped prybar that is welded together by hydrogen bonds to the Trp-1 alpha-amino group. Pivoting of the prybar induced by force demolishes an interface between the Trp-1 side chain and the lectin domain at a switch1 region. These changes are transmitted by rigid body movement of the switch2 region to rearrangements in the switch3 region at the ligand binding site. Another switch region corresponds to a single residue in the EGF domain with large effects on ligand binding and rolling adhesion. Allostery in selectins, and the alignment of tensile force on a selectin-ligand complex with the transition pathway for conformational change, explain much of the structural basis for selectin mechanochemistry.
The lengths of von Willebrand factor (VWF) concatamers correlate with hemostatic potency. After secretion in plasma, length is regulated by hydrodynamic shear force-dependent unfolding of the A2 domain, which is then cleaved by a specific protease. The 1.9-A crystal structure of the A2 domain demonstrates evolutionary adaptations to this shear sensor function. Unique among VWF A (VWA) domains, A2 contains a loop in place of the alpha4 helix, and a cis-proline. The central beta4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the beta4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal alpha6-helix. The alpha6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket. These features may narrow the force range over which unfolding occurs and may also slow refolding. Von Willebrand disease mutations, which presumably lower the force at which A2 unfolds, are illuminated by the structure.
The selectins are cell adhesion proteins that must resist applied forces to mediate leukocyte tethering and rolling along the endothelium and have 2 conformational states. Selectin-ligand bond dissociation increases only modestly with applied force, and exhibits catch bond behavior in a low-force regime where bond lifetimes counterintuitively increase with increasing force. Both allosteric and sliding-rebinding models have emerged to explain catch bonds. Here, we introduce a large residue into a cleft that opens within the lectin domain to stabilize the more extended, high-affinity selectin conformation. This mutation stabilizes the high-affinity state, but surprisingly makes rolling less stable. The position of the mutation in the lectin domain provides evidence for an allosteric pathway through the lectin domain, connecting changes at the lectin-EGF interface to the distal binding interface.
Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alphaIIbbeta3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta3 and the calf-1 domain in alphaIIb and by decreased flexibility between I-EGF2 and I-EGF3.
The activity of integrin LFA-1 (alpha(L)beta(2)) to its ligand ICAM-1 is regulated through the conformational changes of its ligand-binding domain, the I domain of alpha(L) chain, from an inactive, low-affinity closed form (LA), to an intermediate-affinity form (IA), and then finally, to a high-affinity open form (HA). A ligand-mimetic human monoclonal antibody AL-57 (activated LFA-1 clone 57) was identified by phage display to specifically recognize the affinity-upregulated I domain. Here, we describe the crystal structures of the Fab fragment of AL-57 in complex with IA, as well as in its unligated form. We discuss the structural features conferring AL-57's strong selectivity for the high affinity, open conformation of the I domain. The AL-57-binding site overlaps the ICAM-1 binding site on the I domain. Furthermore, an antibody Asp mimics an ICAM Glu by forming a coordination to the metal-ion dependent adhesion site (MIDAS). The structure also reveals better shape complementarity and a more hydrophobic interacting interface in AL-57 binding than in ICAM-1 binding. The results explain AL-57's antagonistic mimicry of LFA-1's natural ligands, the ICAM molecules.
Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.