Proline cis/trans isomerization regulates a T cell specific tyrosine kinase

This dissertation examines the role of protein-protein interactions that regulate the conformation and function of the Tec family kinase, interleukin-2 tyrosine kinase (Itk). Tec family kinases are expressed in hematopoetic cells and modulate intracellular signaling cascades in response to external stimuli. The domain structure of Tec family members contains the conserved SH3, SH2, and catalytic domains common to many kinase families, yet they are distinguishable by the presence of a unique N-terminal sequence. The mechanism by which Itk is regulated is not well understood. Both nuclear magnetic resonance spectroscopy and functional assays were used to elucidate a novel regulatory mechanism for Itk in the work presented in this dissertation. These studies demonstrate that the Itk SH2 domain adopts two distinct conformations in solution that are in slow exchange. The observed conformational heterogeneity is due to proline cis/trans isomerization around a single prolyl imide bond, generating a cis proline-containing conformer and a trans proline-containing conformer. Each conformer displays unique ligand binding properties. The trans conformer preferentially binds phosphotyrosine-containing ligands, whereas the cis conformer is required for a novel intermolecular interaction with the Itk SH3 domain. This SH3-SH2 self-association interaction is mediated by the conserved aromatic ligand binding pocket on the Itk SH3 domain and a newly defined surface on the Itk SH2 domain.;Proline cis/trans isomerization is not only an important switch in regulating ligand binding, we also observe that the conformationally heterogenous proline residue is required for recognition of Itk as a substrate for the peptidyl-prolyl isomerase, cyclophilin A. Cyclophilin A accelerates the interconversion between the cis and trans Itk SH2 domain conformers. Furthermore, both in vitro and in vivo cellular experiments reveal that cyclophilin A inhibits Itk kinase activity. This observation allows us to propose a mechanistic model for a completely new mode of kinase regulation and reveals a cellular role for cyclophilin A in T cell signaling. In sum, this dissertation provides molecular details about the structure of the Tec family kinase, Itk, and the functional implications of these results

TCR Mechanobiology: Torques and Tunable Structures Linked to Early T Cell Signaling

Mechanotransduction is a basis for receptor signaling in many biological systems. Recent data based upon optical tweezer experiments suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into biochemical signals upon specific peptide-MHC complex (pMHC) ligation. Tangential force applied along the pseudo-twofold symmetry axis of the TCR complex post-ligation results in the αβ heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell–APC interface. Accompanying TCR quaternary change likely fosters signaling via the lipid bilayer predicated on the magnitude and direction of the TCR–pMHC force. TCR glycans may modulate quaternary change, thereby altering signaling outcome as might the redox state of the CxxC motifs located proximal to the TM segments in the heterodimeric CD3 subunits. Predicted alterations in TCR TM segments and surrounding lipid will convert ectodomain ligation into the earliest intracellular signaling events

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

High-acuity αβT 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 αβTCR–pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αβTCRs and pre-TCRs within the αβT 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 αβ. The chimeric γδ–αβTCR 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 αβT 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 αβT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection

Proline cis/trans isomerization regulates a T cell specific tyrosine kinase

This dissertation examines the role of protein-protein interactions that regulate the conformation and function of the Tec family kinase, interleukin-2 tyrosine kinase (Itk). Tec family kinases are expressed in hematopoetic cells and modulate intracellular signaling cascades in response to external stimuli. The domain structure of Tec family members contains the conserved SH3, SH2, and catalytic domains common to many kinase families, yet they are distinguishable by the presence of a unique N-terminal sequence. The mechanism by which Itk is regulated is not well understood. Both nuclear magnetic resonance spectroscopy and functional assays were used to elucidate a novel regulatory mechanism for Itk in the work presented in this dissertation. These studies demonstrate that the Itk SH2 domain adopts two distinct conformations in solution that are in slow exchange. The observed conformational heterogeneity is due to proline cis/trans isomerization around a single prolyl imide bond, generating a cis proline-containing conformer and a trans proline-containing conformer. Each conformer displays unique ligand binding properties. The trans conformer preferentially binds phosphotyrosine-containing ligands, whereas the cis conformer is required for a novel intermolecular interaction with the Itk SH3 domain. This SH3-SH2 self-association interaction is mediated by the conserved aromatic ligand binding pocket on the Itk SH3 domain and a newly defined surface on the Itk SH2 domain.;Proline cis/trans isomerization is not only an important switch in regulating ligand binding, we also observe that the conformationally heterogenous proline residue is required for recognition of Itk as a substrate for the peptidyl-prolyl isomerase, cyclophilin A. Cyclophilin A accelerates the interconversion between the cis and trans Itk SH2 domain conformers. Furthermore, both in vitro and in vivo cellular experiments reveal that cyclophilin A inhibits Itk kinase activity. This observation allows us to propose a mechanistic model for a completely new mode of kinase regulation and reveals a cellular role for cyclophilin A in T cell signaling. In sum, this dissertation provides molecular details about the structure of the Tec family kinase, Itk, and the functional implications of these results.</p

Structural features of the αβTCR mechanotransduction apparatus that promote pMHC discrimination

The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide-MHC complex (pMHC). Here we review the structural features that optimize this transmembrane receptor for mechanotransduction. Specialized adaptations include: 1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic TCR-pMHC bond formation and lifetime; 2) the rigid super β-sheet amalgams of heterodimeric CD3εγ as well as CD3εδ ectodomain components of the αβTCR complex; 3) the αβTCR subunit connecting peptides (CP) linking the extracellular and transmembrane (TM) segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and 4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single molecule FRET, optical tweezers and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the preTCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4+CD8+ double positive thymocyte stage of development