Intramolecular Dynamics and Domain Crosstalk in C-terminal Src Kinase (Csk)

Organisms rely on complex forms of chemical, physical and structural sensors that wire their communication networks at the cellular and molecular levels. These signal transduction pathways were classically treated as binary switches that relay information along specific routes from origins to destinations; however, this view is insufficient to describe the scale and diversity of responses offered by a limited number of molecular actors and pathways. Therefore, a more nuanced examination of structural components is needed for deeper understanding of high specificity and diversity of generated signals in the cellular context. The studies presented herein aim to explore the special molecular features of a major signaling component, the C-terminal Src kinase (Csk). It is a master regulator of the Src family of tyrosine kinases (SFKs) whose members participate in almost all aspects of cellular regulation and functions. We first studied the importance of a site-specific divergence within Csk's regulatory Src Homology 2 (SH2) domain and examined its effects on kinase behavior and functions using extended biophysical and activity techniques coupled to molecular dynamics simulations. Induced flexibility in the distal loop position was sensed throughout the protein as it modulated kinase functional motions and activity; revealing intramolecular routes of long-range communications in Csk. Along with insights on intrinsic functions, the results may provide a general framework for identifying potential effector sites for specific targeting by design. And since the distal motif is within a common adaptor domain, we characterized the changes in Csk's interaction with its physiological, membrane- recruiting activator, the Csk Binding Protein (CBP). Our enzymatic activation assays and Nuclear Magnetic Resonance binding analysis indicate that while the magnitude of maximal p-CBP activation of the kinase is largely unaffected, the overall catalytic efficiency is hampered by the non-contacting loop and allows for simultaneous, tunable control of localization and intrinsic activity. Lastly, unlike many modular scaffolds that could routinely be captured in multiple functional states, Csk's intrinsically dynamic character precludes extensive analysis through traditional structural techniques. Therefore, we used computational tools to study full- length Csk aiming to predict molecular transitions and large-scale coupled motions. The theoretical results were corroborated experimentally using our established kinetic, mutational, and structural-dynamic techniques; and construct a clearer model of dynamic intramolecular regulation. The data suggest that previously undetected, directional-global motions of the modular domains about the kinase core are inherently linked to Csk's enzymatic tasks of binding its substrates and catalyzing the phosphotransferase reactio

Intramolecular Dynamics and Domain Crosstalk in C-terminal Src Kinase (Csk)

Organisms rely on complex forms of chemical, physical and structural sensors that wire their communication networks at the cellular and molecular levels. These signal transduction pathways were classically treated as binary switches that relay information along specific routes from origins to destinations; however, this view is insufficient to describe the scale and diversity of responses offered by a limited number of molecular actors and pathways. Therefore, a more nuanced examination of structural components is needed for deeper understanding of high specificity and diversity of generated signals in the cellular context. The studies presented herein aim to explore the special molecular features of a major signaling component, the C-terminal Src kinase (Csk). It is a master regulator of the Src family of tyrosine kinases (SFKs) whose members participate in almost all aspects of cellular regulation and functions. We first studied the importance of a site-specific divergence within Csk's regulatory Src Homology 2 (SH2) domain and examined its effects on kinase behavior and functions using extended biophysical and activity techniques coupled to molecular dynamics simulations. Induced flexibility in the distal loop position was sensed throughout the protein as it modulated kinase functional motions and activity; revealing intramolecular routes of long-range communications in Csk. Along with insights on intrinsic functions, the results may provide a general framework for identifying potential effector sites for specific targeting by design. And since the distal motif is within a common adaptor domain, we characterized the changes in Csk's interaction with its physiological, membrane- recruiting activator, the Csk Binding Protein (CBP). Our enzymatic activation assays and Nuclear Magnetic Resonance binding analysis indicate that while the magnitude of maximal p-CBP activation of the kinase is largely unaffected, the overall catalytic efficiency is hampered by the non-contacting loop and allows for simultaneous, tunable control of localization and intrinsic activity. Lastly, unlike many modular scaffolds that could routinely be captured in multiple functional states, Csk's intrinsically dynamic character precludes extensive analysis through traditional structural techniques. Therefore, we used computational tools to study full- length Csk aiming to predict molecular transitions and large-scale coupled motions. The theoretical results were corroborated experimentally using our established kinetic, mutational, and structural-dynamic techniques; and construct a clearer model of dynamic intramolecular regulation. The data suggest that previously undetected, directional-global motions of the modular domains about the kinase core are inherently linked to Csk's enzymatic tasks of binding its substrates and catalyzing the phosphotransferase reactio

Theoretical Insights Reveal Novel Motions in Csk's SH3 Domain That Control Kinase Activation.

The Src family of tyrosine kinases (SFKs) regulate numerous aspects of cell growth and differentiation and are under the principal control of the C-terminal Src Kinase (Csk). Although Csk and SFKs share conserved kinase, SH2 and SH3 domains, they differ considerably in three-dimensional structure, regulatory mechanism, and the intrinsic kinase activities. Although the SH2 and SH3 domains are known to up- or down-regulate tyrosine kinase function, little is known about the global motions in the full-length kinase that govern these catalytic variations. We use a combination of accelerated Molecular Dynamics (aMD) simulations and experimental methods to provide a new view of functional motions in the Csk scaffold. These computational studies suggest that high frequency vibrations in the SH2 domain are coupled through the N-terminal lobe of the kinase domain to motions in the SH3 domain. The effects of these reflexive movements on the kinase domain can be viewed using both Deuterium Exchange Mass Spectrometry (DXMS) and steady-state kinetic methods. Removal of several contacts, including a crystallographically unobserved N-terminal segment, between the SH3 and kinase domains short-circuit these coupled motions leading to reduced catalytic efficiency and stability of N-lobe motifs within the kinase domain. The data expands the model of Csk's activation whereby separate domains productively interact with two diametrically opposed surfaces of the kinase domain. Such reversible transitions may organize the active structure of the tyrosine kinase domain of Csk

Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity

Botulinum neurotoxins (BoNTs) are among the most potent toxins known and are also used to treat an increasing number of medical disorders. There are seven well-established serotypes (BoNT/A-G), which all act as zinc-dependent endopeptidases targeting specific members of the SNARE proteins required for synaptic vesicle exocytosis in neurons. A new toxin serotype, BoNT/X, was recently identified. It cleaves not only the canonical targets, vesicle associated membrane proteins (VAMP) 1/2/3 at a unique site, but also has the unique ability to cleave VAMP4/5 and Ykt6. Here we report the 1.35 Å X-ray crystal structure of the light chain of BoNT/X (LC/X). LC/X shares the core fold common to all other BoNTs, demonstrating that LC/X is a bona fide member of BoNT-LCs. We found that access to the catalytic pocket of LC/X is more restricted, and the regions lining the catalytic pocket are not conserved compared to other BoNTs. Kinetic studies revealed that LC/X cleaves VAMP1 with a ten times higher efficiency than BoNT/B and the tetanus neurotoxin. The structural information provides a molecular basis to understand the convergence/divergence between BoNT/X and other BoNTs, to develop effective LC inhibitors, and to engineer new scientific tools and therapeutic toxins targeting distinct SNARE proteins in cells

Functional interdomain contacts suggest SH3-mediated activation.

<p>High occupancy polar contacts at the SH3-kinase core interface in Csk show a flexible hydrogen bonding network in the starting, “SH3 up” state, A. The same network of residues is shown from a simulation frame of the “SH3 down” conformation, B.</p

Dynamic interdomain activation in Csk.

<p>In the compact activated form, the functional motions of Csk’s modular domains are correlated for efficient activation of the kinase core to phosphorylate and downregulate SFKs.</p

Effects of SH3 domain truncations on the time-dependent solvent deuterium incorporation into Csk peptide probes.

<p>Left: Deuterium incorporation into several probes in wild type, ΔN8, and ΔSH3 Csk is plotted as a function of time. Data were obtained over time courses of 1000 s at room temperature in deuterated buffer. Peptide identification and analysis were performed on two sets of data for verification. Right: Peptides with significant DXMS changes are plotted on the crystal structure of Csk. Red color gradient indicates level of HDX deprotection for each probe in the truncated constructs (ΔN8, ΔSH3) with respect to that observed for the wild type protein probes.</p

Perturbations to the SH3 domain contacts inhibit kinase activation.

<p>A) Schematic representations of some of the Csk constructs used in this study. B) The Csk kinase activity assay is used to determine the extent of SH3-mediated kinase activation. Kinase activity was monitored in a [γ-³²P]ATP coupled radioactive assay in which a kinase dead substrate (kdSrc) is phosphorylated as a function of time. The reactions typically included 100 nM Csk, 5 μM kdSrc, 10 mM MgCl₂, 1 mM MnCl₂ and were initiated with 100 μM ATP at 23°C.</p

Kinetic parameters for select SH3 domain variants.

<p>Relative Steady-State kinetic values are listed for Csk variants and show differential effects on turnover rates, substrate binding, and catalytic efficiency.</p><p>Kinetic parameters for select SH3 domain variants.</p