Chafen Lu

Reversibly locking a protein fold in an active conformation with a disulfide bond: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin αLβ2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the αM and α2 subunits has been crystallized in both open and closed conformations; however, the αL I domain has been crystallized in only the closed conformation. We hypothesized that the αL domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the αL I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The kon, koff, and KD values for the locked open I domain were within 1.5-fold of values previously determined for the αLβ2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized αLβ2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.

Sequence and structure relationships within von Willebrand factor

In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)β(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-β and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.

Epitope Mapping of Antibodies to the C-Terminal Region of the Integrin β2 Subunit Reveals Regions that Become Exposed Upon Receptor Activation

The cysteine-rich repeats in the stalk region of integrin β subunits appear to convey signals impinging on the cytoplasmic domains to the ligand-binding headpiece of integrins. We have examined the functional properties of mAbs to the stalk region and mapped their epitopes, providing a structure-function map. Among a panel of 14 mAbs to the β2 subunit, one, KIM127, preferentially bound to αLβ2 that was activated by mutations in the cytoplasmic domains, and by Mn2+. KIM127 also bound preferentially to the free β2 subunit compared with resting αLβ2. Activating β2 mutations also greatly enhanced binding of KIM127 to integrins αMβ2 and αXβ2. Thus, the KIM127 epitope is shielded by the α subunit, and becomes reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2, MEM48, and KIM185, activated αLβ2 and bound equally well to resting and activated αLβ2, differentially recognized resting αMβ2 and αXβ2, and bound fully to activated αMβ2 and αXβ2. The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and MEM48 bind to overlapping epitopes involving residues 534, 536, 541, 543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185 maps near the end of cysteine-rich repeat 4. The nonactivating mAbs, 6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344–432 and 432–487, respectively. We thus define five different β2 stalk subregions, mAb binding to which correlates with effect on activation, and define regions in an interface that becomes exposed upon integrin activation.

Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor

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-Å 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 α4 helix, and a cis-proline. The central β4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the β4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal α6-helix. The α6-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.

An isolated, surface-expressed I domain of the integrin αLβ2 is sufficient for strong adhesive function when locked in the open conformation with a disulfide bond

We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.

Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

ABSTRACT The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two α-helical turns, in contrast to association throughout the membrane over five α-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.

GARP regulates the bioavailability and activation of TGFβ

GARP disulfide links to latent TGFβ on the cell surface, which serves as a reservoir for TGFβ activation by αVβ6 and to a lesser extent αVβ8. Activation requires the RGD motif of latent TGFβ, disulfide linkage between GARP and latent TGFβ, and membrane association of GARP.

Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor

To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.

Folding and Function of I Domain-deleted Mac-1 and Lymphocyte Function-associated Antigen-1

In those integrins that contain it, the I domain is a major ligand recognition site. The I domain is inserted between β-sheets 2 and 3 of the predicted β-propeller domain of the integrin α subunit. We deleted the I domain from the integrin αM and αL subunits to give I-less Mac-1 and lymphocyte function-associated antigen-1 (LFA-1), respectively. The I-less αM and αL subunits were expressed in association with the wild-type β2subunit on the surface of transfected cells and bound to all the monoclonal antibodies mapped to the putative β-propeller and C-terminal regions of the αM and αLsubunits, suggesting that the folding of these domains is independent of the I domain. I-less Mac-1 bound to the ligands iC3b and factor X, but this binding was reduced compared with wild-type Mac-1. In contrast, I-less Mac-1 did not bind to fibrinogen or denatured bovine serum albumin. Binding to iC3b and factor X by I-less Mac-1 was inhibited by the function-blocking antibody CBRM1/32, which binds to the β-propeller domain of the αM subunit. I-less LFA-1 did not bind its ligands intercellular adhesion molecule-1 and -3. Thus, the I domain is not essential for the folding, heterodimer formation, and surface expression of Mac-1 and LFA-1 and is required for binding to some ligands, but not others.

Functional and Structural Stability of the Epidermal Growth Factor Receptor in Detergent Micelles and Phospholipid Nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.