Heart of glass (HEG1) is a transmembrane receptor with a large cytoplasmic domain that plays a crucial role in cardiovascular development. Because almost nothing was known about HEG1, other than its interaction with the FERM (4.1, ezrin, radixin, moesin) domain of KRIT1, I set out to understand the basis of HEG1 function from a structural perspective. Cerebral Cavernous Malformations (CCMs) are a common vascular disease affecting 1 in 200 people. The three genes linked to CCMs, CCM1-3, encode proteins that play an important role in stabilizing the junctions between cells lining the inner surface of blood vessels, but these junctions become leaky in multiple pathological conditions. My studies focused on the protein KRIT1 (CCM1), the protein product of a gene that is linked with CCMs in humans and is also required for cardiovascular development. I established that KRIT1 is an authentic Rap1 effector and that Rap1 acts by displacing KRIT1 from microtubules enabling it to travel to cell-cell junctions. I also showed that the cytoplasmic domain (tail) of HEG1 binds directly to KRIT1. I solved the structure of the complex of KRIT1 FERM domain with HEG1, and used that structure to show that the interaction of HEG1 with KRIT1 recruits KRIT1 to cell-cell junctions thereby supporting cardiovascular development and inhibiting the activation of RhoA/Rho Kinase (ROCK) signaling. I also showed that HEG1 tail, Rap1, and KRIT1 FERM domain form a ternary complex, solved the structure of the complex (see figure), and used the structure to establish the basis of the remarkable Rap1 specificity of KRIT1.
Ras-interacting protein 1 (Rasip1) is an endothelial-specific Rap1 and Ras effector important for vascular development and angiogenesis. Here, we report the crystal structure of the Rasip1 RA domain (RRA) alone revealing the basis of dimerization and in complex with Rap1 at 2.8Å resolution. In contrast to most RA domains, RRA formed a dimer that can bind two Rap1 (KD = 0.9 μM) or Ras (KD = 2.2 μM) molecules. We solved the Rap1-RRA complex and found that Rasip1 binds Rap1 in the Switch I region, and Rap1 binding induces few conformation changes to Rasip1 stabilizing a -strand and an unstructured loop. Our data explain how Rasip1 can act as a Rap1 and Ras effector and show that Rasip1 defines a subgroup of dimeric RA domains that could mediate cooperative binding to membrane-associated Ras superfamily members.
Integrins are important cell adhesion proteins and the cytoskeletal protein talin is a key regulator of their activation that both activates the integrin family of cell adhesion molecules, and couples them to the actin cytoskeleton. I became fascinated by talin because of its critical importance in cell adhesion and its beautiful multi-domain structure that enabled a cut and study strategy that included NMR, crystallography, and peptide arrays. The work provided novel insights into the structure of the large talin rod domain (residues 482-2541) which is made up of a series of amphipathic helical bundles, some of which contain binding sites for the cytoskeletal protein vinculin. We established that the stability of the helical bundles that make up the talin rod is an important factor in determining vinculin binding and that talin contains many cryptic vinculin-binding sites. We also made the novel observation that binding of vinculin induces unfolding of parts of the talin rod domain. In summary, these studies were instrumental in the realisation that talin is a mechanosensitive protein.
I have subsequently continued this research on talin and have played a leading role in the structural biology team. The main focus of my work has been on (i) determining the organization of the 62 α-helices in the talin rod into domains and (ii) in solving the structure of these domains. The paper published in EMBO Journal reports the solution structure of the C-terminal actin-binding domain of talin. Using the structural information, small angle X-ray scattering and cryo-EM (in collaboration with Dorit Hanein, Burnham Institute, La Jolla, CA), I have derived a model showing how this dimeric domain interacts with F-actin. I also have a first author paper on the structure of the integrin binding site in the talin rod. Another manuscript describes the molecular basis of the regulation of talin activity through a head/rod interaction. As part of this work, we defined the structure of all 18 domains in talin, and this has enabled us to develop a structural model of the entire molecule spanning >2,500 amino acids.