Intrinsic differences in the mechanisms of Tie2 binding to angiopoietins exploited by directed evolution to create an Ang2-selective ligand trap

Angiopoietins Ang1 and Ang2 are secreted ligands for the endothelial receptor tyrosine kinase Tie2 essential for vascular development and maintenance. Ang1 acts as an agonist to maintain normal vessel function, whereas Ang2 acts as a Tie2 antagonist. Ang2 is increased in macular edema, sepsis, and other conditions, in which it blocks Ang1-mediated signaling, causing vascular dysfunction and contributing to disease pathology. Therefore, Ang2 is an attractive therapeutic target. Previously, we reported a Tie2 ectodomain variant that selectively binds Ang2 and acts as soluble ligand trap to sequester Ang2; however, the mechanism of Ang2-binding selectivity is unknown. In the present study, we used directed protein evolution to enhance Ang2-binding affinity of this Tie2 ectodomain trap. We examined contributions of individual residues in the ligand-binding interface of Tie2 to Ang1 and Ang2 binding. Surprisingly, different combinations of Tie2 residues were found to bind each ligand, with hydrophobic residues binding both ligands and polar residues contributing selectively to either Ang1 or Ang2 binding. Our analysis also identified a single Tie2 residue, His168, with a pivotal role in both Ang1 and Ang2 binding, enabling competition between binding ligands. In summary, this study reports an enhanced-affinity Ang2-selective ligand trap with potential for therapeutic development and reveals the mechanism behind its selectivity. It also provides the first analysis of contributions of individual Tie2 residues to Ang1 and Ang2 binding and identifies selectivity-determining residues that could be targeted in the future design of small molecule and other inhibitors of Ang2 for the treatment of vascular dysfunction

Sin3A recruits Tet1 to the PAH1 domain via a highly conserved Sin3-Interaction Domain.

The Sin3A complex acts as a transcriptional hub, integrating the function of diverse transcription factors with histone modifying enzymes, notably, histone deacetylases (HDAC) 1 and 2. The Sin3A protein sits at the centre of the complex, mediating multiple simultaneous protein-protein interactions via its four paired-amphipathic helix (PAH) domains (PAH1-4). The PAH domains contain a conserved four helical bundle, generating a hydrophobic cleft into which the single-helix of a Sin3-interaction domain (SID) is able to insert and bind with high affinity. Although they share a similar mode of interaction, the SIDs of different repressor proteins bind to only one of four potential PAH domains, due to the specific combination of hydrophobic residues at the interface. Here we report the identification of a highly conserved SID in the 5-methylcytosine dioxygenase, Tet1 (Tet1-SID), which interacts directly with the PAH1 domain of Sin3A. Using a combination of NMR spectroscopy and homology modelling we present a model of the PAH1/Tet1-SID complex, which binds in a Type-II orientation similar to Sap25. Mutagenesis of key residues show that the 11-amino acid Tet1-SID is necessary and sufficient for the interaction with Sin3A and is absolutely required for Tet1 to repress transcription in cells

A Novel Mechanism for Calmodulin-Dependent Inactivation of Transient Receptor Potential Vanilloid 6

The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+ homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct binding of the universal Ca2+-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular Ca2+ concentrations CaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When the cytosolic Ca2+ concentration increases charging the apo CaM N-lobe with Ca2+, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges two TRPV6 monomers resulting in closure of the channel pore

The Structural Basis of Calcium-Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel

The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca 2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca 2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca 2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca 2+ /CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate, N., et al. (2018) Biochemistry, (57), DOI: 10.1021/acs.biochem.7b01286 ]

Mice carrying a complete deletion of the talin2 coding sequence are viable and fertile.

Mice homozygous for several Tln2 gene targeted alleles are viable and fertile. Here we show that although the expression of talin2 protein is drastically reduced in muscle from these mice, other tissues continue to express talin2 albeit at reduced levels. We therefore generated a Tln2 allele lacking the entire coding sequence (Tln2(cd)). Tln2(cd/cd) mice were viable and fertile, and the genotypes of Tln2(cd/+) intercrosses were at the expected Mendelian ratio. Tln2(cd/cd) mice showed no major difference in body mass or the weight of the major organs compared to wild-type, although they displayed a mildly dystrophic phenotype. Moreover, Tln2(cd/cd) mouse embryo fibroblasts showed no obvious defects in cell adhesion, migration or proliferation. However, the number of Tln2(cd/cd) pups surviving to adulthood was variable suggesting that such mice have an underlying defect

GFP-talin1 calpain2-resistant mutants accumulate in HEK293 cells.

<p>(A) Western blots (anti-GFP) of cells transfected with the following constructs: GFP alone; GFP-talin1; GFP-talin1(L432G); GFP-talin1 (E2492G); a GFP-talin1(L432G,E2492G) double mutant. Anti-GAPDH was used as a loading control. GFP-talin1 head (arrow) was not detected in these cells. (B) Band intensity was quantified using the Odyssey software (LI-COR). Bars indicate SEM of triplicate determinations. Essentially similar results were obtained in three separate experiments.</p

An E2492G mutation in talin1 2300–2541 reduces sensitivity to calpain2 cleavage but does not affect actin binding or dimerisation.

<p>(A) Wild-type talin 2300–2541 (ABS-DD) and an E2492G mutant were incubated with varying dilutions of calpain2, and their sensitivity to cleavage analysed by SDS-PAGE. (B) For comparison, the same experiment was conducted with wild-type talin 309–655 (F3-VBS1) and a L432G mutant in the linker between the head and rod which has previously been shown to partially inhibit calpain2 cleavage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034461#pone.0034461-Franco1" target="_blank">[38]</a>. (C) Actin co-sedimentation assay using wild-type talin 2300–2541 and the E2492G mutant. Comparable amounts of each protein co-sedimented with F-actin (Pellet (P); Supernatant (S). Binding was quantified using imageJ analysis of the relevant bands; wild-type 100±2.3 versus mutant 105±4.3. (D) Gel filtration of wild-type talin1 2300–2541 and the E2492G mutant show that both form dimers. The monomeric R2526G mutant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034461#pone.0034461-Gingras1" target="_blank">[27]</a> is shown for comparison.</p

Both calpain cleavage sites are located in predominantly unstructured regions.

<p>(A) The structures of the C-terminal ABS and the dimerisation domain of talin1 are incorporated into a model of the C-terminal region of the protein. The calpain2 cleavage site is indicated (blue line). (B–D) ¹H,¹⁵N HSQC spectra of the following talin1 polypeptides (150 µM); (B) Residues 2294–2541 spanning the C-terminal ABS and dimerisation domain (DD). The intense poorly dispersed peaks (red asterisks) relate to the 18 amino acid linker between the ABS and the DD (as deduced by comparison of the spectra of residues 2294–2491 and 2300–2482), and show that the linker is predominantly disordered. (C) Residues 400–480 - the linker between the talin1 head and rod that contains the calpain-cleavage site. The intense poorly dispersed signals in the centre of the spectrum indicate that the linker is predominantly unstructured. (D) Residues 309–655 containing the F3 FERM domain, the linker and the VBS1 rod domain. (E) A model of the linker region generated using MODELLER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034461#pone.0034461-Eswar1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034461#pone.0034461-Sali1" target="_blank">[51]</a> showing that it can span ∼200 Å when fully extended. The calpain2 cleavage site (blue line) between Q433–Q434 is shown along with L432, which when mutated to a glycine, reduces calpain2 cleavage. The CDK5 phosphorylation site (S425) that regulates binding of Smurf1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034461#pone.0034461-Huang1" target="_blank">[47]</a> is also shown.</p

The C-terminal calpain2 site in talin1 is less sensitive to cleavage than that in the linker between the head and rod.

<p>(A) A talin1 F3-VBS1 polypeptide (residues 309–655) containing the linker between the head and rod and (B) a talin1 polypeptide containing the C-terminal ABS and DD (residues 2300–2541) were incubated with varying dilutions of calpain2. Cleavage products were analysed by SDS-PAGE and visualised with Coomassie blue. The N-terminal calpain2 cleavage site in talin1 is more sensitive than the C-terminal site. (C, D) Calpain2 cleavage of the equivalent talin2 polypeptides. Both talin2 polypeptides were somewhat more sensitive to calpain2 cleavage than the corresponding talin1 polypeptides.</p

The talin1 C-terminal dimerisation domain is clipped off by calpain2.

<p>(A) Talin consists of an N-terminal head (residues 1–400) containing an atypical extended FERM domain (made up of F0–F3 domains) and a flexible rod containing 61 α-helices (grey elipses) organised into 13 helical bundles and terminating in a single helix responsible for dimerisation (DD). The position of various ligand-binding sites are shown including the C-terminal actin-binding site (ABS). The head and rod are joined by a linker region that is cleaved by calpain2 between Q433 and Q434. (B) Sequence and secondary structure of the last two helices in the talin rod. The two asterisks indicate residues L2515 and R2526, key determinants of the epitope recognised by the TD77 monoclonal antibody. The red arrow indicates the calpain2 cleavage site. (C,D) Purified turkey gizzard talin was incubated with calpain2 to generate a partial digest (PD) and a complete digest (CD). Cleavage products were resolved by SDS-PAGE and stained either with (C) Coomassie blue or (D) the monoclonal antibody TD77 that recognises the DD. (E) A talin polypeptide (residues 2300–2541) containing the C-terminal ABS and DD domain was incubated with calpain2 in the presence (+) or absence (−) of calcium. N-terminal sequencing and mass spectroscopy of the products show that the largest band corresponds to the ABS (2300–2493) and the smallest fragment (M2494) corresponds to the dimerisation domain (2494–2541).</p