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It is targeted by the Rap1 effector RIAM to focal adhesion sites and subsequently undergoes force induced conformational opening to recruit the actin interacting protein vinculin. The conformational switch involves the talin R3 domain, which binds RIAM when closed and vinculin when open. Here, we apply pressure to R3 and measure 1 H, 15 N, and 13 C chemical shift changes, which are fitted using a simple model, and indicate that R3 is only 50% closed: the closed form is a four helix bundle, while in the openstate helix 1 is twisted out. Strikingly, a mutant of R3 that binds RIAM with an affinity similar to wild type but more weakly to vinculin is shown to be 0.84 kJ mol 1 more stable when closed. These results demonstrate that R3 is thermodynamically poised to bind either RIAM or vinculin, and thusconstitutes a good mechanosensitive switch. Baxter et al. used high pressure to increase the population of the open state of the R3 domain of talin. They calculated chemical shifts of closed and open states, showing that the open state has helix 1 opened out, representing the start of force dependent opening.

Lilja J, Zacharchenko T, Georgiadou M, Jacquemet G, De Franceschi N, Peuhu E, Hamidi H, Pouwels J, Martens V, Nia FH, Beifuss M, Boeckers T, Kreienkamp HJ, Barsukov IL, Ivaska J SHANK proteins limit integrin activation by directly interacting with Rap1 and R Ras Nature Cell Biology 19 292 305, 2017

2017 Macmillan Publishers Limited, part of Springer Nature. 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 isan 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,
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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.

Zacharchenko T, Barsukov I, Rigden DJ, Bennett D, Mayans O Biophysical Analysis of the N Terminal Domain from the Human Protein Phosphatase 1 Nuclear Targeting Subunit PNUTS Suggests an Extended Transcription Factor TFIIS Like Fold Protein Journal 35 340 345, 2016

Human protein phosphatase 1 nuclear targeting subunit (PNUTS) plays critical roles in DNA repair, cell growth and survival. The N terminal domain of PNUTS mediates interactions with Tox4 and the phosphatase and tensin homolog PTEN, which are essential for the roles of this protein. To study this N terminal domain, we have established its recombinant overproduction in E. coli utilizing NusA fusion. Upon removal of the tag, the remaining PNUTS sample is soluble and highly pure. We have characterized the domain using circular dichroism and nuclear magnetic resonance and analyzed its sequence using bioinformatics. All data agree in suggesting that the PNUTS N terminal segment adopts a compact, globular fold rich in helical content, where the folded fraction is substantially larger than the previously annotated fold. We conclude that this domain adopts a single fold, likely being an extended form of the transcription factor S II leucine/tryptophan conserved motif. Thermal denaturation yielded a melting temperature of 49.5C, confirming the stability of the fold. These findings pave the way for the molecular characterization of functional interactions mediated by the N terminal region of PNUTS.

Zacharchenko T, Qian X, Goult BT, Jethwa D, Almeida TB, Ballestrem C, Critchley DR, Lowy DR, Barsukov IL LD Motif Recognition by Talin: Structure of the Talin DLC1 Complex Structure 24 1130 1141, 2016

Cell migration requires coordination between integrin mediated cell adhesion to the extracellular matrix and force applied to adhesion sites. Talin plays a key role in coupling integrin receptors to the actomyosin contractile machinery, while deleted in liver cancer 1 (DLC1) is a Rho GAP that binds talin and regulates Rho, and therefore actomyosin contractility. We show that the LD motif of DLC1 forms a helix that binds to the four helix bundle of the talin R8 domain in a canonical triple helix arrangement. We demonstrate that the same R8 surface interacts with the paxillin LD1 and LD2 motifs. We identify key charged residues that stabilize the R8 interactions with LD motifs and demonstrate their importance invitro and in cells. Our results suggest a network of competitive interactions in adhesion complexes that involve LD motifs, and identify mutations that can be used to analyze the biological roles of specific protein protein interactions in cell migration. 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,
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the novel concepts derived from these and the emerging translational avenues.