Thymus-derived lymphocytes protect mammalian hosts against virus-or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric ␣␤, CD3⑀␥, CD3⑀␦, and CD3 subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3⑀ lobe that they approach diagonally, adjacent to the lever-like C␤ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3⑀ and CD3␥ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (ϳ50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands. The T cell receptor (TCR)3 is a multimeric transmembrane complex composed of a disulfide-linked antigen binding clonotypic heterodimer (␣␤ or ␥␦) in non-covalent association with the signal-transducing CD3 subunits (CD3⑀␥, CD3⑀␦, and CD3) (reviewed in Ref. 1). TCR signaling via CD3 dimers evokes T cell lineage commitment and repertoire selection during development, maintains the peripheral T cell pool, and further differentiates naïve T cells into effector or memory cell populations upon immune stimulation (2-5). The interaction between an Fab-like ␣␤ TCR heterodimer and an antigenic peptide bound to a major histocompatibility complex molecule (pMHC) initiates a cascade of downstream signaling events via the immunoreceptor tyrosine-based activation motif elements in the cytoplasmic tails of the associated CD3 subunits (6 -9). The length of these CD3 cytoplasmic tails is substantial, relative to those of the TCR ␣ and ␤ chains (6, 7).How recognition of pMHC by a weakly interacting (ϳ1-100 M K d ) clonotypic heterodimer on the T cell surface evokes intracellular signaling via the adjacent CD3 components remains undefined (1). Solution structures of CD3⑀␥ and CD3⑀␦ heterodimers reveal a unique side-to-side hydrophobic interface with conjoined ␤-sheets involving the G-strands of the two Ig-like ectodomains of the pair (10, 11). The squat and rigid CD3 connecting segments contrast sharply with the long and flexible TCR ␣ and ␤ connecting peptides linking their respective constant domains to the transmembrane segments.To investigate the basis of signal transduction involving the ectodomain components within the TCR membrane complex,...

The Fourier transform has been the gold standard to transform data from time to frequency domain in many spectroscopic methods, including NMR. While reliable, it has as a drawback that it requires a grid of uniformely sampled data points, which is not efficient for decaying signals, while it also suffers from artifacts when dealing with non-decaying signals. Over several decades many alternative sampling and transformation schemes have been proposed. Their common problem is that relative signal amplitudes are not well preserved. Here we demonstrate superior performance of a sineweighted Poisson gap distribution sparse sampling scheme, combined with FM reconstruction. While the relative signal amplitudes are well preserved, we also find that the signal-to-noise ratio is enhanced up to four fold per unit of data acquisition time as compared to the traditional linear sampling.The capabilities of modern NMR spectrometers have recently improved dramatically due to higher magnetic field instruments. To utilize the potential resolution of these spectrometers in multi-dimensional experiments various forms of sparse or non-uniform sampling (NUS) were proposed1 , 2. For optimal processing of such spectra we have recently developed the Forward Maximum entropy (FM) reconstruction method3, which was improved and combined with a distillation procedure4.The quality of spectra obtained form NUS depends crucially on the sampling schedules. In the past we have examined various forms of random sampling and realized that the quality of data retrieval depended significantly on the choice of the seed number when using standard Unix random number generators (e.g. drand48). We realized that (1) big gaps in the sampling Gerhard_Wagner@hms.harvard.edu. Supporting Information Available: Four figures comparing signal to peak-noise ratio, effect of order parameters in linear prediction, resolution between linear prediction with Poisson-gap sampling and a graphical representation of the used SPS schedule as well as a source code for C-program generating SPS schedule are available at http://pubs.acs.org/paragonplus/submission/jacsat/. schedule are generally unfavorable, and (2) gaps at the beginning or end of the sampling are worse than in the middle. A third, crucial criterion is: (3) the sampling requires suitably random variation to prevent violation of the Nyquist theorem. NIH Public AccessTo cope with this we have evaluated a sinusoidal weighted Poisson distribution of the gap lengths between sampling points followed by FM reconstruction. To achieve this distribution, we assume an average gap length of λ in the common Fourier grid and a specific gap size k between two acquired data points. Thus, a λ of 0.0 yields uniform sampling, a λ of 1.0, for example, creates a non-uniform schedule of 50% overall sampling density.The overall probability f for a specific gap size k ≥ 0 is assumed by the Poisson distribution:. This would satisfy the criteria (1) and (3). Obviously, no integer values k less than zero is allowed as no negative ga...

GTP-loaded Ras induces multiple signaling pathways by binding to its numerous effectors such as Raf and PI3K. Ras activity can be influenced by activation of Ras-GEFs that stimulate GDP release and GTP loading or by inhibition of Ras-GAPs that stimulate GTP hydrolysis. Here, we report that monoubiquitination of Lys147 within the G domain of wild-type K-Ras, the Ras gene most frequently mutated in cancer, leads to enhanced GTP loading. Furthermore, ubiquitination increases the ability of the oncogenic Gly-12-Val mutant of K-Ras to bind the downstream effectors PI3K and Raf. These results indicate that monoubiquitination both enhances GTP loading on K-Ras and increases its affinity for specific downstream effectors, providing a previously unidentified mechanism for Ras activation.

Obtaining atomic level information about the structure and dynamics of biomolecules is critical to understand their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR to large biological systems. Here, we take advantage of the high sensitivity and the broad chemical-shift range of 19 F nuclei, and leverage the remarkable relaxation properties of the aromatic 19 F- 13 C spin pair to disperse 19 F resonances in a 2-dimensional transverse relaxation optimized TROSY spectrum. We demonstrate the application of the 19 F- 13 C TROSY to investigate proteins and nucleic acids. This experiment expands the scope of 19 F NMR in the study of structure, dynamics and function of large and complex biological systems and provides a powerful background-free NMR probe.