Crystal Structure Analysis and Solution Studies of Human Lck-SH3; Zinc-induced Homodimerization Competes with the Binding of Proline-rich Motifs

Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
Journal of Molecular Biology (Impact Factor: 4.33). 03/2007; 365(5):1417-28. DOI: 10.1016/j.jmb.2006.10.058
Source: PubMed


In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.

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    • "This brings the kinase into spatial proximity of its substrates, the immunoreceptor tyrosine-based activation motifs in the TCR's CD3 molecules and the upstream kinase ZAP70 [119] [120]. In addition, homodimerization of Lck is stabilized by another bridging Zn 21 binding to the SH3 domains of two kinase molecules [121]. The latter mechanism is thought to facilitate Lck activation by promoting its intermolecular autophosphorylation . "
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    ABSTRACT: For more than 50 years, it has been known that zinc deficiency compromises immune function. During this time, knowledge about the biochemistry of zinc has continued to grow, but only recent years have provided in-depth molecular insights into the multiple aspects of zinc as a regulator of immunity. A network based on ZnT and ZIP proteins for transport and metallothionein for storage tightly regulates zinc availability, and virtually all aspects of innate and adaptive immunity are affected by zinc. In vivo, zinc deficiency alters the number and function of neutrophil granulocytes, monocytes, natural killer (NK)-, T-, and B-cells. T cell functions and balance between the different subsets are particularly susceptible to changes in zinc status. This article focuses in particular on the main mechanisms by which zinc ions exert essential functions in the immune system. On the one hand, this includes tightly protein bound zinc ions serving catalytic or structural functions in a multitude of different proteins, in particular enzymes and transcription factors. On the other hand, increasing evidence arises for a regulatory role of free zinc ions in signal transduction, especially in cells of the immune system. Identification of several molecular targets, including phosphatases, phosphodiesterases, caspases, and kinases suggest that zinc ions are a second messenger regulating signal transduction in various kinds of immune cells. © 2013 BioFactors, 2013.
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    • "In addition to recruitment to the TCR signaling complex, another zinc dependent interface site mediates dimerization of the SH3 domains of two Lck molecules. The activation of Lck involves trans-autophosphorylation on the activating tyrosine residue 394, and dimerization is likely to facilitate this process [170]. The authors concluded that this kind of zinc-induced activation mechanism is Lck specific, because the residues that form the zinc binding site are not found in other Src kinase family members. "
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    ABSTRACT: The trace element zinc is a crucial cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis. Zinc homeostasis is tightly regulated and disturbance of this homeostasis due to genetic defects, zinc deficiency, or supplementation influences the development and the progression of various infectious and autoimmune diseases. The immune system is strongly impaired during zinc deficiency, predominantly the cell-mediated response by T-lymphocytes. During zinc deprivation T-lymphocyte development, polarization into effector cells, and function are impaired. This leads to reduced T-cell numbers, a decreased ratio of type 1 to type 2 T-helper cells with reduced production of T-helper type 1 cytokines like interferon-gamma, and compromised T-cell mediated immune defense. Accordingly, disturbed zinc homeostasis increases the risk for infections, and zinc supplementation restores normal immune function. Furthermore, several disorders, like mycobacterial infections, asthma, diabetes, and rheumatoid arthritis are accompanied by decreased zinc levels and in some cases disease progression can be affected by zinc supplementation. On the molecular level, apoptosis of T-cell precursors is influenced by zinc via the Bcl-2/Bax ratio, and zinc ions inhibit caspases-3, -6, -7, and -8. In mature T-cells, zinc interacts with kinases involved in T-cell activation, like protein kinase C and the lymphocyte protein tyrosine kinase (Lck), while higher zinc concentrations are inhibitory, reducing the activities of the interleukin-1 receptor-associated kinase (IRAK) and calcineurin. Taken together, zinc homeostasis influences T-lymphocytes via several molecular targets, leading to a modulation of T-cell-dependent immune responses.
    Preview · Article · Jul 2009 · Endocrine Metabolic & Immune Disorders - Drug Targets(Formerly Current Drug Targets - Immune Endocrine & Metabolic Disorders)
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    • "The N-terminal region of Lck is recruited to the intracellular domains of the membrane proteins CD4 or CD8 by a 'zinc clasp' structure [22-24]. At the second zinc-dependent interface site two zinc ions at the dimer interface of the SH3 domains stabilize homodimerization of Lck, which is thought to promote autophosphorylation required for its activation [25]. "
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    ABSTRACT: The trace element zinc is essential for the immune system, and zinc deficiency affects multiple aspects of innate and adaptive immunity. There are remarkable parallels in the immunological changes during aging and zinc deficiency, including a reduction in the activity of the thymus and thymic hormones, a shift of the T helper cell balance toward T helper type 2 cells, decreased response to vaccination, and impaired functions of innate immune cells. Many studies confirm a decline of zinc levels with age. Most of these studies do not classify the majority of elderly as zinc deficient, but even marginal zinc deprivation can affect immune function. Consequently, oral zinc supplementation demonstrates the potential to improve immunity and efficiently downregulates chronic inflammatory responses in the elderly. These data indicate that a wide prevalence of marginal zinc deficiency in elderly people may contribute to immunosenescence.
    Full-text · Article · Jul 2009 · Immunity & Ageing
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