Native State Proline Isomerization: An Intrinsic Molecular Switch

Exquisite control of biological function is achieved via tight regulation of the catalytic and binding activities of cellular proteins. The mechanistic details of protein regulation vary from targeted chemical modification of amino acid side chains (1) to the quite drastic global unfolding of an entire polypeptide chain (2). Peptidyl prolyl cis/trans isomerization is emerging as a potentially general mechanism for the control of protein function (3). While most structures of native, folded proteins reveal peptidyl-prolyl imide bonds that adopt either the cis or trans conformation, there are a growing number of folded proteins that exhibit conformational heterogeneity about one or more peptidyl-prolyl bonds. Unlike covalent modification or global unfolding, proline isomerization is an intrinsic conformational exchange process that has the potential to direct ligand recognition and to control protein activity within the confines of the native state

Controlling the Activity of the Tec Kinase Itk by Mutation of the Phenylalanine Gatekeeper Residue

The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2−kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or by removal of the amino-terminal SH2−kinase linker region or by mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2−kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases

Bacterial expression and purification of Interleukin-2 Tyrosine kinase: Single step separation of the chaperonin impurity

Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl2/ATP/KCl incubation procedure to remove the copurifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets

The Linker between SH2 and Kinase Domains Positively Regulates Catalysis of the Tec Family Kinases

Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead

Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the β-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively

Identification of an Allosteric Signaling Network within Tec Family Kinases

The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the ‘regulatory spine’, has been identified that assembles and disassembles as a kinase switches between its active and inactive states. Here we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the SH2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Btk R544 on the activation loop and Btk E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases

Mechanism and Functional Significance of Itk Autophosphorylation

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full length Itk mutant containing a phenylalanine in place of the autophosphorylated tyrosine has been previously studied in Itk deficient primary T cells. These studies revealed that the nonphosphorylated enzyme only partially restores Itk mediated signaling. In spite of these insights, the precise role of the Tec kinase autophosphorylation site remains unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay we also show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand binding capability of the Itk SH3 domain in a ligand dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation

Solution and Micelle-Bound Structures of Tachyplesin I and Its Active Aromatic Linear Derivatives

Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachyplesus tridentatus. It has high antimicrobial activity and adopts a β-hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar β-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the β-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the β-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity:  a β-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association

Ligand Specificity Modulated by Prolyl Imide Bond Cis/Trans Isomerization in the Itk SH2 Domain: A Quantitative NMR Study

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) binds two separate ligands:  a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain. Binding specificity for these ligands is regulated via cis/trans isomerization of the Asn 286−Pro 287 imide bond in the Itk SH2 domain. In this study, we develop a novel method of analyzing chemical shift perturbation and cross-peak volumes to measure the affinities of both ligands for each SH2 conformer. We find that the cis imide bond containing SH2 conformer exhibits a 3.5-fold higher affinity for the Itk SH3 domain compared with binding of the trans conformer to the same ligand, while the trans conformer binds phosphopeptide with a 4-fold greater affinity than the cis-containing SH2 conformer. In addition to furthering the understanding of this system, the method presented here will be of general application in quantitatively determining the specificities of conformationally heterogeneous systems that use a molecular switch to regulate binding between multiple distinct ligands

Murine Itk SH3 domain

Interleukin-2 tyrosine kinase (Itk) is a non-receptor tyrosine kinase of the Tec family that is activated upon antigen binding to the T cell receptor (Schwartzberg et al. 2005; Berg 2007). Itk is comprised of four regulatory domains: PH (Pleckstrin homology), TH (Tec homology), SH3, SH2 and the catalytic kinase domain. SH3 domains share a common fold consisting of five anti-parallel β strands that form a β barrel and bind canonical proline rich ligands as well as a variety of noncanonical ligands (Agrawal and Kishan 2002)