Molecular Details of Itk Activation by Prolyl Isomerization and Phospholigand Binding: The NMR Structure of the Itk SH2 Domain Bound to a Phosphopeptide

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The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings (3hJNC′) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of 3hJNC′ couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the αB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.

Introduction

Interleukin-2 tyrosine kinase (Itk) of the Tec family non-receptor kinases is an important signaling protein that transmits signals downstream of the T-cell antigen receptor and mediates T-cell development.1, 2 More recently, Itk has also been implicated in regulation of actin cytoskeleton reorganization and in chemokine-induced signaling.3, 4, 5 In contrast to the well-understood regulation of the Src kinases, the precise mechanism by which Itk activity is regulated remains largely unknown. Abundant structural data6, 7, 8 have provided key insights into the molecular details of Src kinase regulation. However, no crystallographic data are available to date for any of the full-length Tec family kinases. For Itk, recent crystal structures of the isolated kinase domain in both its phosphorylated (activated) and unphosphorylated (inactivated) forms show little or no discernible conformational differences suggesting that the non-catalytic domains outside of the kinase domain play a significant role in regulating activity.9

As is the case for many other protein kinases, the SH2 domain of Itk plays a critical role in its catalytic regulation. Engagement of the Itk SH2 domain with a phosphotyrosine-containing signaling partner appears to be an important step in Itk activation following TCR/CD3 engagement.10 Deletion of the SH2 domain from Itk and point mutations that disrupt the SH2-phospholigand interaction both lead to severely underphosphorylated (inactivated) Itk.10

The Itk SH2 domain, however, is unusual in that it contains an additional mode of conformational control. Cis/trans isomerization around the Asn286–Pro287 imide bond in the Itk SH2 domain gives rise to two distinct domain conformers in solution.11 The solution structures of the SH2 domain with cis and trans conformations of the Asn286–Pro287 imide bond (referred to as the cis and trans SH2 conformers, respectively) have been solved by nuclear magnetic resonance (NMR) spectroscopy and reveal substantial structural differences that arise from prolyl isomerization.12 Conformational differences between these two conformers extend well beyond Pro287 itself as one-third of the residues in this domain show doubled NMR resonances as a result of the slow conformational exchange between cis and trans conformers. This is in contrast to other protein systems wherein only nearest neighbor residues are influenced by isomerization about a prolyl imide bond.13

The large number of residues strongly affected by prolyl isomerization in the Itk SH2 domain may in fact be a key factor in regulating ligand recognition by the domain. Phospholigand binding by the Itk SH2 domain (the interaction implicated in activation of Itk in T cells) preferentially involves the trans SH2 conformer.14 Interestingly, the cis SH2 conformer also mediates ligand binding; in this case, however, a non-canonical, phosphotyrosine-independent interaction is observed with another domain of Itk.14, 15 Thus, prolyl isomerization within the folded Itk SH2 domain governs ligand recognition and, given the putative regulatory role of phospholigand binding, likely plays a role in regulating Itk activity in T cells.16

To understand the nature of the Itk SH2/phosphopeptide binding event and to assess the structural features responsible for the observed conformer selectivity, we have solved the NMR structure of the trans Itk SH2 domain complexed to a phosphotyrosine-containing peptide (Ac-ADpYEPP-NH2, hereafter referred to as pY) chosen to mimic Slp-76, a phospholigand binding partner of Itk in T cells.17 The structure of the SH2/pY complex has revealed critical contacts between the conformationally heterogeneous BG loop of Itk SH2 and a phosphopeptide. This loop is better pre-organized for pY binding in the ligand-free trans SH2 conformer than the cis SH2 conformer, providing an explanation for the observed ligand discrimination between the cis and trans forms. In addition, changes in the hydrogen bonding network of the SH2 domain upon pY binding suggest a possible mechanism by which this interaction regulates Itk activity.

Section snippets

Chemical shift perturbations in the SH2 domain induced by pY binding

Titration of the pY peptide into a sample containing the uniformly 15N-labeled Itk SH2 domain allowed us to identify protein residues affected by pY binding. Figure 1 shows these residues, both in the context of the primary amino acid sequence (Figure 1(a)) and the three-dimensional structure of the SH2 complex (Figure 1(b)).

There is an overlap between the SH2 residues that are affected by phosphopeptide binding and those that give rise to doubled resonances in the absence of ligand.

Molecular details of Itk activation

The insights gained from these data can be interpreted in terms of Itk regulation. In conjunction with experimental evidence for the importance of Pro287 in primary T cells,16 our emerging model for Itk regulation25 points to a role for the equilibrium between cis and trans prolyl imide bond conformations in the Itk SH2 domain in directing ligand binding and subsequently the activity of the neighboring kinase domain. The trans SH2 conformer preferentially mediates phospholigand binding, an

Preparation of protein samples

The preparation of singly 15N and doubly 15N, 13C-labeled Itk SH2 domain has been described.12, 15 To produce triply 15N, 13C, 2H-labeled protein the cells were gradually adapted to growth in 2H2O using the following protocol.37 A single bacterial colony from an LB-ampicillin plate was used to inoculate 3 ml of LB-ampicillin medium made with 33% 2H2O. This culture was grown for 12 h at 37 °C in a 15 ml Falcon tube. An aliquot (100 μl) was then transferred to 3 ml of 67% 2H2O LB for an additional 12 h.

Acknowledgements

Support for this work was provided by a National Institutes of Health grant (RO1-AI43957) to A.H.A. In addition, this study made use of the National Magnetic Resonance Facility at Madison, which is supported by National Institutes of Health grants P41RR02301 (Biomedical Research Technology Program, National Center for Research Resources) and P41GM66326 (National Institute of General Medical Sciences). Equipment in the facility was purchased with funds from the University of Wisconsin, the

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    †

    E.V.P. and M.S. contributed equally to this work.

    †

    Present addresses: E. V. Pletneva, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA; M. Sundd, RCSB Protein Data Bank, Department of Chemistry and Chemical Biology, Rutgers The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854-8087, USA.

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