Asymmetric framework motion of TCRαß controls load-dependent peptide discrimination.

Mechanical force is critical for the interaction between an αß T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.

Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Asymmetric framework motion of TCRαß controls load-dependent peptide discrimination.

Mechanical force is critical for the interaction between an αßT cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.

Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.

Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.

Harnessing αß T cell receptor mechanobiology to achieve the promise of immuno-oncology.

T cell receptors (TCR) on cytolytic T lymphocytes (CTLs) recognize "foreign" antigens bound in the groove of major histocompatibility complex (MHC) molecules (H-2 in mouse and HLA in human) displayed on altered cells. These antigens are peptide fragments of proteins derived either from infectious pathogens or cellular transformations during cancer evolution. The conjoint ligand formed by the foreign peptide and MHC, termed pMHC, marks an aberrant cell as a target for CTL-mediated destruction. Recent data have provided compelling evidence that adaptive protection is achieved in a facile manner during immune surveillance when mechanical load consequent to cellular motion is applied to the bond formed between an αß TCR and its pMHC ligand arrayed on a disease-altered cell. Mechanobiology maximizes both TCR specificity and sensitivity in comparison to receptor ligation in the absence of force. While the field of immunotherapy has made advances to impact the survival of cancer patients, the latest information relevant to T cell targeting and mechanotransduction has yet to be applied for T cell monitoring and treatment of patients in the clinic. Here we review these data, and challenge scientists and physicians to apply critical biophysical parameters of TCR mechanobiology to the medical oncology field, broadening treatment success within and among various cancer types. We assert that TCRs with digital ligand-sensing performance capability directed at sparsely as well as luminously displayed tumor-specific neoantigens and certain tumor-associated antigens can improve effective cancer vaccine development and immunotherapy paradigms.

Characterizing Biophysical Parameters of Single TCR-pMHC Interactions Using Optical Tweezers.

αß T cells are mechanosensors that leverage bioforces during immune surveillance for highly sensitive and specific antigen discrimination. Single-molecule studies are used to profile the initial TCRαß-pMHC binding event, and various biophysical parameters can be identified. Isolating purified TCRαß and pMHC molecules on a coverslip allows for direct measurements of the kinetics and conformational changes in the system and removes cellular components along the load pathway that may interfere with or mask subtle changes. Optical tweezers provide high resolution position and force information that map the bonding profile, including catch bond, and the ability to measure distinct conformational changes driven by forces. The present method describes the single-molecule optical tweezers assay setup, considerations, and execution. This model can be used for various TCR-pMHC pairs or expanded to measure a wide variety of receptor-ligand interactions operative in multiple biological systems.

Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation.

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific ß chain, is a critical early checkpoint in thymocyte development within the αß T cell lineage5,6. PreTCRs arrayed on CD4-CD8- double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αß T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7-10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αß T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting ß chain repertoire broadening for subsequent αß T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.

Measuring αß T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.

Single-molecule investigations of single-chain cellulose biosynthesis.

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ∼80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

A kinesin-1 variant reveals motor-induced microtubule damage in cells.

Kinesins drive the transport of cellular cargoes as they walk along microtubule tracks; however, recent work has suggested that the physical act of kinesins walking along microtubules can stress the microtubule lattice. Here, we describe a kinesin-1 KIF5C mutant with an increased ability to generate damage sites in the microtubule lattice as compared with the wild-type motor. The expression of the mutant motor in cultured cells resulted in microtubule breakage and fragmentation, suggesting that kinesin-1 variants with increased damage activity would have been selected against during evolution. The increased ability to damage microtubules is not due to the enhanced motility properties of the mutant motor, as the expression of the kinesin-3 motor KIF1A, which has similar single-motor motility properties, also caused increased microtubule pausing, bending, and buckling but not breakage. In cells, motor-induced microtubule breakage could not be prevented by increased α-tubulin K40 acetylation, a post-translational modification known to increase microtubule flexibility. In vitro, lattice damage induced by wild-type KIF5C was repaired by soluble tubulin and resulted in increased rescues and overall microtubule growth, whereas lattice damage induced by the KIF5C mutant resulted in larger repair sites that made the microtubule vulnerable to breakage and fragmentation when under mechanical stress. These results demonstrate that kinesin-1 motility causes defects in and damage to the microtubule lattice in cells. While cells have the capacity to repair lattice damage, conditions that exceed this capacity result in microtubule breakage and fragmentation and may contribute to human disease.

Single Molecule Force Spectroscopy Reveals Distinctions in Key Biophysical Parameters of αß T-Cell Receptors Compared with Chimeric Antigen Receptors Directed at the Same Ligand.

Chimeric antigen receptor (CAR) T-cell therapies exploit facile antibody-mediated targeting to elicit useful immune responses in patients. This work directly compares binding profiles of CAR and αß T-cell receptors (TCR) with single cell and single molecule optical trap measurements against a shared ligand. DNA-tethered measurements of peptide-major histocompatibility complex (pMHC) ligand interaction in both CAR and TCR exhibit catch bonds with specific peptide agonist peaking at 25 and 14 pN, respectively. While a conformational transition is regularly seen in TCR-pMHC systems, that of CAR-pMHC systems is dissimilar, being infrequent, of lower magnitude, and irreversible. Slip bonds are observed with CD19-specific CAR T-cells and with a monoclonal antibody mapping to the MHC α2 helix but indifferent to the bound peptide. Collectively, these findings suggest that the CAR-pMHC interface underpins the CAR catch bond response to pMHC ligands in contradistinction to slip bonds for CARs targeting canonical ligands.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III.

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM-cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1-cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

Pre-T cell receptors topologically sample self-ligands during thymocyte ß-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αßTCRs. Using x-ray crystallography, we show how a preTCR applies the concave ß-sheet surface of its single variable domain (Vß) to "horizontally" grab the protruding MHC α2-helix. By contrast, αßTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVß module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3ß reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αßTCR canonical docking mode. "Horizontal" docking precludes germline CDR1ß- and CDR2ß-MHC binding to broaden ß-chain repertoire diversification before αßTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

The αßTCR mechanosensor exploits dynamic ectodomain allostery to optimize its ligand recognition site.

Each [Formula: see text]T cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCR[Formula: see text] ligand-binding heterodimer and the noncovalently associated CD3 signaling subunits. When bound by ligand, an antigenic peptide arrayed by a major histocompatibility complex molecule (pMHC), the TCR[Formula: see text] has a longer bond lifetime under piconewton-level loads. The atomistic mechanism of this "catch bond" behavior is unknown. Here, we perform molecular dynamics simulation of a TCR[Formula: see text]-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCR[Formula: see text] domain allostery. The TCR[Formula: see text]-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vß variable domains containing the pMHC-binding complementarity-determining region (CDR) loops. Without adequate load, the interfacial contacts are unstable, whereas applying sufficient load suppresses Vα-Vß motion, stabilizing the interface. A second level of control is exerted by Cα and Cß constant domains, especially Cß and its protruding FG-loop, that create mismatching interfaces among the four TCR[Formula: see text] domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCR[Formula: see text] molecule. Thus, the catch bond involves the entire TCR[Formula: see text] conformation, interdomain motion, and interfacial contact dynamics, collectively. This multilayered architecture of the machinery fosters fine-tuning of cellular response to load and pMHC recognition. Since the germline-derived TCR[Formula: see text] ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse [Formula: see text]TCRs constituting the T cell repertoire.

Neck linker docking is critical for Kinesin-1 force generation in cells but at a cost to motor speed and processivity.

Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.

NMR: an essential structural tool for integrative studies of T cell development, pMHC ligand recognition and TCR mechanobiology.

Early studies of T cell structural biology using X-ray crystallography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) focused on a picture of the αßT cell receptor (αßTCR) component domains and their cognate ligands (peptides bound to MHC molecules, i.e. pMHCs) as static interaction partners. Moving forward requires integrating this corpus of data with dynamic technologies such as NMR, molecular dynamics (MD) simulations and real-time single molecule (SM) studies exemplified by optical tweezers (OT). NMR bridges relevant timescales and provides the potential for an all-atom dynamic description of αßTCR components prior to and during interactions with binding partners. SM techniques have opened up vistas in understanding the non-equilibrium nature of T cell signaling through the introduction of force-mediated binding measurements into the paradigm for T cell function. In this regard, bioforces consequent to T-lineage cell motility are now perceived as placing piconewton (pN)-level loads on single receptor-pMHC bonds to impact structural change and αßT-lineage biology, including peptide discrimination, cellular activation, and developmental progression. We discuss herein essential NMR technologies in illuminating the role of ligand binding in the preT cell receptor (preTCR), the αßTCR developmental precursor, and convergence of NMR, SM and MD data in advancing our comprehension of T cell development. More broadly we review the central hypothesis that the αßTCR is a mechanosensor, fostered by breakthrough NMR-based structural insights. Collectively, elucidating dynamic aspects through the integrative use of NMR, SM, and MD shall advance fundamental appreciation of the mechanism of T cell signaling as well as inform translational efforts in αßTCR and chimeric T cell (CAR-T) immunotherapies and T cell vaccinology.

Intracellular cargo transport by single-headed kinesin motors.

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

Simultaneous Imaging and Selective Photothermal Therapy through Aptamer-Driven Au Nanosphere Clustering.

Gold (Au) nanoparticles display enhanced near-infrared (NIR) photothermal effects upon the formation of clusters. We studied the photothermal properties of Au nanosphere clusters on the single-particle level using photothermal heterodyne imaging (PTHI) microscopy to understand the enhancement mechanisms. NIR photothermal responses of Au nanoparticle clusters were found to significantly increase from monomers to trimers. The averaged PTHI signal intensity of Au nanosphere dimers and trimers is ∼10 and ∼25 times that of monomers. The NIR photothermal effect of clustered nanospheres strongly correlates with their longitudinal plasmon mode. Clustered Au nanospheres were demonstrated to exhibit dual-capability NIR photothermal imaging and therapy of human prostate cancer cells with high efficiency and selectivity. This strategy can be potentially utilized for simultaneous cancer imaging and therapy with 3D selectivity.

The T Cell Antigen Receptor α Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations.

Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.