Structural aspects of talin regulation through Rap1A signalling

At the nascent focal adhesion, GTPase Rap1A recruits the adaptor protein RIAM (Rap1 Interacting Adaptor Molecule), and RIAM recruits the scaffold protein talin facilitating integrin activation (Lee et al., 2009). As the nascent adhesion matures, actin linkages are strengthened via recruitment of the talin-actin bridging protein vinculin. In vivo data shows that RIAM is not found in mature focal adhesions; furthermore RIAM and vinculin occupy the same binding sites within talin (Goult et al., 2013). This thesis shows that RIAM-talin interaction is synergistic, with multiple domains simultaneously contributing to the binding. Vinculin and RIAM compete for talin, providing a mechanism of RIAM displacement by vinculin as adhesions mature. The interactions are thus characterised by a range biophysical techniques. SHARPIN (Shank associated RH-domain interacting Protein) potentially competes with talin for integrin binding (Rantala et al., 2011). SHARPIN interaction with the synaptic protein SHANK3 (SH3 and multiple ankyrin repeat domains protein 3) may provide a mechanism for SHANK3 recruitment to the adhesion (Lim et al., 2001). This thesis solves the structure of the SHANK N-terminal domain that contains the SHARPIN binding region. The structure shows the presence of a previously uncharacterised RA (Ras-association) domain that packs against the following ankyrin repeat domain. We show the RA domain binds with high affinity to the GTPase-Rap1A. Rap1A-SHANK could provide a negative feedback pathway for integrin regulation in adhesion. In the course of adhesion maturation multiple proteins are recruited to the adhesion complex; these proteins facilitate linkages to the actomyosin contractile machinery. One such protein is DLC1 (Deleted in Liver Cancer 1); DLC1 contains an LD-like motif that is homologues to the LD domains of the adhesion protein paxillin (Brown et al., 1996). This LD-like motif is responsible for tumour suppressor function and binding to talin (Li et al., 2011; Cao et al., 2012). In this thesis we characterise the talin-DLC1 interaction using X-ray crystallography, and suggest mutations to disrupt the interaction in cell experiments

The structural basis of the Talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions

Adhesion between cells and the extracellular matrix (ECM) is mediated by heterodimeric (alphabeta) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organises cytosolic signalling proteins into discrete complexes on beta-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we developed a novel crystallographic method to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN motif of KANK1 has a novel fold, where a beta-turn stabilises the alpha-helical region, explaining its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localises throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery

Scalable, Non-denaturing Purification of Phosphoproteins Using Ga³⁺-IMAC: N2A and M1M2 Titin Components as Study case

The purification of phosphorylated proteins in a folded state and in large enough quantity for biochemical or biophysical analysis remains a challenging task. Here, we develop a new implementation of the method of gallium immobilized metal chromatography (Ga3+-IMAC) as to permit the selective enrichment of phosphoproteins in the milligram scale and under native conditions using automated FPLC instrumentation. We apply this method to the purification of the UN2A and M1M2 components of the muscle protein titin upon being monophosphorylated in vitro by cAMP-dependent protein kinase (PKA). We found that UN2A is phosphorylated by PKA at its C-terminus in residue S9578 and M1M2 is phosphorylated in its interdomain linker sequence at position T32607. We demonstrate that the Ga3+-IMAC method is efficient, economical and suitable for implementation in automated purification pipelines for recombinant proteins. The procedure can be applied both to the selective enrichment and to the removal of phosphoproteins from biochemical samples

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes

Molecular insights into titin’s A-band

The thick filament-associated A-band region of titin is a highly repetitive component of the titin chain with important scaffolding properties that support thick filament assembly. It also has a demonstrated link to human disease. Despite its functional significance, it remains a largely uncharacterized part of the titin protein. Here, we have performed an analysis of sequence and structure conservation of A-band titin, with emphasis on poly-FnIII tandem components. Specifically, we have applied multi-dimensional sequence pairwise similarity analysis to FnIII domains and complemented this with the crystallographic elucidation of the 3D-structure of the FnIII-triplet A84-A86 from the fourth long super-repeat in the C-zone (C4). Structural models serve here as templates to map sequence conservation onto super-repeat C4, which we show is a prototypical representative of titin’s C-zone. This templating identifies positionally conserved residue clusters in C super-repeats with the potential of mediating interactions to thick-filament components. Conservation localizes to two super-repeat positions: Ig domains in position 1 and FnIII domains in position 7. The analysis also allows conclusions to be drawn on the conserved architecture of titin’s A-band, as well as revisiting and expanding the evolutionary model of titin’s A-band

Structural advances on titin : towards an atomic understanding of multi-domain functions in myofilament mechanics and scaffolding

Titin is a gigantic filamentous protein of the muscle sarcomere that plays roles in myofibril mechanics and homoeostasis. 3D-structures of multi-domain fragments of titin are now available that start revealing the molecular mechanisms governing its mechanical and scaffolding functions. This knowledge is now being translated into the fabrication of self-assembling biopolymers. Here we review the structural advances on titin, the novel concepts derived from these and the emerging translational avenues.publishe