Natalia Beglova

Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation

Cysteine-rich repeats in the integrin β subunit stalk region relay activation signals to the ligand-binding headpiece. The NMR solution structure and disulfide bond connectivity of Cys-rich module-3 of the integrin β2 subunit reveal a nosecone-shaped variant of the EGF fold, termed an integrin-EGF (I-EGF) domain. Interdomain contacts between I-EGF domains 2 and 3 observed by NMR support a model in which the modules are related by an approximate two-fold screw axis in an extended arrangement. Our findings complement a 3.1 Å crystal structure of the extracellular portion of integrin αVβ3, which lacks an atomic model for I-EGF2 and a portion of I-EGF3. The disulfide connectivity of I-EGF3 chemically assigned here differs from the pairings suggested in the αVβ3 structure. Epitopes that become exposed upon integrin activation and residues that restrain activation are defined in β2 I-EGF domains 2 and 3. Superposition on the αVβ3 structure reveals that they are buried. This observation suggests that the highly bent αVβ3 structure represents the inactive conformation and that release of contacts with I-EGF modules 2 and 3 triggers a switchblade-like opening motion extending the integrin into its active conformation.

Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin β subunits

Integrin β subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169–178]. N-terminally truncated β2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a β2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

Structural features of the low-density lipoprotein receptor facilitating ligand binding and release

The LDLR (low-density lipoprotein receptor) is a modular protein built from several distinct structural units: LA (LDLR type-A), epidermal growth factor-like and beta-propeller modules. The low pH X-ray structure of the LDLR revealed long-range intramolecular contacts between the propeller domain and the central LA repeats of the ligand-binding domain, suggesting that the receptor changes its overall shape from extended to closed, in response to pH. Here we discuss how the LDLR uses flexibility and rigidity of linkers between modules to facilitate ligand binding and low-pH ligand release.

A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

Conformational activation of 2 integrins, their I domains, and regulation by small molecule antagonists

The inserted or I domain of the integrin LFA-1 ( L 2) mediates binding to ligands such as the IgSF member ICAM-1. Mutation has been used to introduce disulfide bonds that lock the L I domain in two distinct conformations termed open and closed. Wild type, resting L 2 requires activation for binding to ICAM-1. Locked closed L 2 is inactive and resistant to activation whereas locked open L 2 is constitutively active. I domains can be expressed in the absence of other integrin domains with artificial membrane anchors. In static adhesion assays, the locked open I domain is adhesive and the locked closed and wild type I domains are not. However, in flow chamber assays, the wild type I domain mediates rolling, whereas the locked open I domain mediates firm adhesion, reflecting how adhesive function in the vasculature is poised to mediate rolling under basal conditions and firm adhesion after activation. Real time ligand binding assays using surface plasmon resonance show that locking open the I domain increases its affinity for ICAM-1 by 9,000 fold, and that its kinetics and KD are comparable to measurements on active, intact L 2. Two classes of small molecule antagonists can be discriminated.