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

Conformational snapshots of Tec kinases during signaling

The control of cellular signaling cascades is of utmost importance in regulating the immune response. Exquisitely precise protein-protein interactions and chemical modification of substrates by enzymatic catalysis are the fundamental components of the signals that alert immune cells to the presence of a foreign antigen. In particular, the phosphorylation events induced by protein kinase activity must be spatially and temporally regulated by specific interactions to maintain a normal and effective immune response. High resolution structures of many protein kinases along with supporting biochemical data are providing significant insight into the intricate regulatory mechanisms responsible for controlling cellular signaling. The Tec family kinases are immunologically important kinases for which regulatory details are beginning to emerge. This review focuses on bringing together structural insights gained over the years to develop an understanding of how domain interactions both within the Tec kinases and between the Tec kinases and other signaling molecules control immune cell function

Cyclophilin A Restricts Influenza A Virus Replication through Degradation of the M1 Protein

Cyclophilin A (CypA) is a typical member of the cyclophilin family of peptidyl-prolyl isomerases and is involved in the replication of several viruses. Previous studies indicate that CypA interacts with influenza virus M1 protein and impairs the early stage of the viral replication. To further understand the molecular mechanism by which CypA impairs influenza virus replication, a 293T cell line depleted for endogenous CypA was established. The results indicated that CypA inhibited the initiation of virus replication. In addition, the infectivity of influenza virus increased in the absence of CypA. Further studies indicated that CypA had no effect on the stages of virus genome replication or transcription and also did not impair the nuclear export of the viral mRNA. However, CypA decreased the viral protein level. Additional studies indicated that CypA enhanced the degradation of M1 through the ubiquitin/proteasome-dependent pathway. Our results suggest that CypA restricts influenza virus replication through accelerating degradation of the M1 protein

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members

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