Intermolecular multiple-quantum coherences between bulk water and a glycoprotein fragment at modest concentration (20 mM) have been experimentally produced and detected, although such coherences are inconceivable in the normal theoretical framework of nuclear magnetic resonance. A density matrix treatment explains these results by including the long-range dipolar interaction between spins and by discarding the high-temperature approximation. These results imply that peak intensities (critical for structural determinations) can be distorted in many gradient experiments, and show that magic-angle gradients provide substantial improvements with reduced gradient strengths. They also suggest methods for contrast enhancement in magnetic resonance imaging.

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The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR). The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood. Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine. Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism. The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction. By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly. Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2. We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase of the Tec family that participates in the intracellular signaling events leading to T cell activation. Tec family members contain the conserved SH3, SH2, and catalytic domains common to many kinase families, but they are distinguished by unique sequences outside of this region. The mechanism by which Itk and related Tec kinases are regulated is not well understood. Our studies indicate that Itk catalytic activity is inhibited by the peptidyl prolyl isomerase activity of cyclophilin A (CypA). NMR structural studies combined with mutational analysis show that a prolinedependent conformational switch within the Itk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. CypA and Itk form a stable complex in Jurkat T cells that is disrupted by treatment with cyclosporin A. Moreover, the phosphorylation levels of Itk and a downstream substrate of Itk, PLC␥1, are increased in Jurkat T cells that have been treated with cyclosporin A. These findings support a novel mode of tyrosine kinase regulation for a Tec family member and provide a molecular basis for understanding a cellular function of the ubiquitous peptidyl prolyl isomerase, CypA.N ormal cell growth depends on the precise control of protein tyrosine kinase activity (1). For certain families of kinases, the mechanism of catalytic regulation is well understood. Structures of Src tyrosine kinases (2, 3) reveal intramolecular interactions mediated by the Src homology 2 (SH2) and Src homology 3 (SH3) domains that control catalytic activity of the neighboring kinase domain. Specifically, distortion of the Src kinase active site is achieved in part by SH2 binding to a phosphorylated tyrosine residue in the C-terminal tail of Src (4). For other families of kinases, the mechanistic details of catalytic regulation remain elusive. In particular, the Tec family of nonreceptor tyrosine kinases (5) displays distinguishing characteristics that point to an alternative mode of regulation. The Tec family kinases modulate hematopoietic cellular responses to external stimuli (6). The T cell-specific Tec family member, interleukin-2 tyrosine kinase (Itk) (7,8), plays a role in the maturation of thymocytes, is required for intracellular signaling following T cell receptor (TCR) crosslinking, and is involved in generation of second messengers that mediate cytoskeletal reorganization (9). Itk is homologous to Src in the region spanning the SH3, SH2, and kinase domain but lacks the Src C-terminal tail that contains the regulatory tyrosine. However, activation of Itk depends on SH2-mediated interactions with phosphorylated signaling partners (9) such as Slp-76 (10) and LAT (11), suggesting a regulatory role for the Itk SH2 domain despite the absence of a Src-like C-terminal regulatory tyrosine residue.The Src regulatory mechanism highlights the role of molecular switches in controlling cellular signaling pathways. Well studied covalent modifications such as...