Linker Interaction Research Articles

Structural basis of human Nav1.5 gating mechanisms.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

Stability of Metal Hydroxide Organic Frameworks for Oxygen Evolution

The oxygen evolution reaction (OER) is central to storing electrical energy via chemical bonds in energy carriers and fuels through reactions such as electrochemical water splitting to produce hydrogen, CO2 reduction for CO and liquid hydrocarbons, and nitrogen to ammonia. While there has been considerable work towards the engineering of inexpensive yet highly active OER catalysts, current state-of-the-art materials are still at least an order of magnitude less active than oxygen evolving complexes found in biological systems that intricately combine inorganic metal-oxo clusters with organic ligands.1 As a result, metal organic frameworks (MOFs) have drawn considerable attention as hybrid organic-inorganic systems that can potentially mimic the unique structure of biological oxygen evolving complexes. Recently, metal hydroxide organic frameworks (MHOFs),2 a new class of MOFs that combine layered hydroxides with aromatic carboxylate linkers that stabilize the structure via π-π interactions, have been shown to display three times the tunability of layered hydroxides, offering extensive opportunities for further engineering of its electrochemical properties for numerous applications. However, the long-term stability of these material during OER is still unclear, which would be paramount to understand for further rational design of this class of material.In this study, we investigated Ni-based MHOFs with carboxylate linkers of varying π-π interaction strengths to understand how these differences affect the electrochemical stability of these materials during OER. We observed that the MHOFs all undergo activation during OER leading to two orders of magnitude increase in OER activity, where the MHOFs with weaker π-π interaction strengths tend to transform at a faster rate than the MHOFs with stronger π-π interaction strengths. We further characterized the MHOFs using a wide range of analytical techniques, including scanning transmission electron microscopy, Raman spectroscopy, x-ray photoelectron spectroscopy, and hard x-ray absorption spectroscopy, before and after extended OER cycling and galvanostatic tests to understand the transformed phase, which suggested that while the bulk structure largely remains unchanged, the surface undergoes significant restructuring into a Ni(OH)2-like phase during OER. Using operando UV-vis and Raman spectroscopy measurements on the MHOFs during OER to understand the factors that induce the transformation process, we found that there was a clear link between the Ni2+/3+, 4+ redox couple observed around 1.4 VRHE and a loss in the carboxylate organic linkers for the linkers with weak π-π interactions. However, for the MHOFs synthesized with linkers exhibiting strong π-π interaction strengths, there were smaller changes to the overall material during OER, suggesting that the bulk stability of these materials is largely dictated by the linker interaction strength and activation are primarily only surface transformations. These results directly demonstrate that linker selection also plays a key role in the stability MOFs under electrochemical conditions and are pertinent for rational design and understanding of the stability and activity of hybrid organic-inorganic materials as electrocatalysts.

Abstract B132: Discovery of small molecule allosteric modulators of Abl kinase activity and function

Abstract The Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and forms oncogenic fusion proteins that drive several cancer types. The kinase activity of Abl is repressed by intramolecular interactions involving its regulatory SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the back of the C-lobe of the kinase domain. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate Abl kinase activity through its non-catalytic domains may represent selective probes of Abl function. In this study, we developed a high-throughput screening (HTS) assay to identify chemical modulators of Abl kinase activity that target the regulatory SH3:linker interaction. The fluorescence polarization (FP) assay employs a purified recombinant Abl protein comprised of the regulatory N-cap, SH3 and SH2 domains plus the SH2-kinase linker (Abl N32L protein) and a short fluorescein-labeled peptide probe that selectively binds to the Abl SH3 domain. During assay development, we found that the peptide probe binds to the recombinant Abl N32L protein in vitro, producing a robust FP signal that can be competitively displaced by excess unlabeled peptide. The FP signal is not observed in control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved compounds in the FP assay identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays demonstrated that one of the hits, the antithrombotic drug dipyridamole, enhances Abl kinase activity in vitro to a greater extent than the previously described Abl agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates Abl by disrupting this regulatory interaction. Interestingly, this compound does not significantly affect the kinase activity of Hck, a Src family member that is regulated by similar SH3:linker interactions. Treatment with dipyrimadole in combination with DPH led to strong enhancement of Abl kinase activity in transfected cells. The Abl N32L FP assay has been implemented in a fully automated format and used to screen diversity libraries totaling 60,000 compounds. The Z-factor coefficients from this HTS campaign indicated that the FP assay performed well and several additional scaffolds were identified for future development. These results demonstrate that screening assays that incorporate the non-catalytic domains of Abl can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. Citation Format: Prerna Grover, Haibin Shi, Matthew Baumgartner, David Close, Paul A. Johnston, Carlos J. Camacho, Thomas E. Smithgall. Discovery of small molecule allosteric modulators of Abl kinase activity and function. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B132.

Abstract B190: Allosteric modulation of Src family kinases via SH3 domain displacement

Abstract The Src family of non-receptor protein-tyrosine kinases (SFKs) plays a crucial role in cancer progression by affecting multiple signaling pathways linked to tumor cell adhesion, invasion and survival. Recent work from our group and others has shown that individual Src-family members may have unique and in some cases opposing roles in the regulation of cell growth and differentiation. Discovery and development of SFK isoform-selective inhibitors and agonists is therefore essential for the most effective therapeutic targeting of this ubiquitous kinase family. Current SFK inhibitors target the ATP-binding site of the kinase domain or an allosteric pocket nearby. However, the highly conserved nature of the active site across the Src kinase family limits identification of isoform-selective compounds. One possible solution to this problem involves small molecule modulation of SFK activity via the regulatory SH3 and SH2 domains. SFK activity is tightly regulated by intramolecular interactions of the SH3 domain with the SH2:kinase linker and the SH2 domain with the phosphorylated C-terminal tail. Small molecules that either enhance or disrupt these interactions are anticipated to result in kinase inhibition or activation, respectively. Recently, we reported that binding of a short proline-rich peptide to the SH3 domains of several Src-family members is sufficient to induce kinase activation in vitro. This peptide, known as VSL12 (VSLARRPLPLP), was postulated to disrupt the regulatory SH3:linker interaction in the downregulated kinase. In the present study, we used VSL12 as probe to better understand the structural basis of kinase activation by SH3 displacement. We first compared the affinity and kinetics of the VSL12 peptide interaction with SFK SH3 domains vs. the near full-length kinase proteins using surface plasmon resonance. We found that VSL12 bound to the isolated SH3 domains of Src, Hck, Lyn and Fyn with similar micromolar potencies. Surprisingly, the KD values for VSL12 binding to the corresponding near-full-length kinases were in the low nanomolar range, suggesting that the peptide makes additional contacts with residues outside of the SH3 domain that result in the stable, active complex. This idea was tested further using differential scanning fluorimetry to explore the thermal stability of the protein:peptide complexes. Addition of VSL12 enhanced the thermal stability of a recombinant Src SH3-SH2-linker protein, consistent with increased molecular order. However, VSL12 decreased the thermal stability of near full-length c-Src, which may reflect conformational changes in the kinase domain that accompany kinase activation. Addition of the kinase domain inhibitor dasatinib restored thermal stability of the Src:VSL12 complex. Complexes of the Src SH3-SH2-linker protein with the VSL12 peptide readily formed crystals that diffract X-rays to 2.6 Å resolution, providing further support for the stabilization of the complex by VSL12 binding. Determining the structures of the Src SH3-SH2-linker protein and the near-full-length kinase in complex with VSL12 will provide fresh insight as to the mechanism of c-Src activation by SH3 domain displacement and aid in the design of potent and selective non-peptide allosteric modulators. Citation Format: Heather L. Rust, Jamie A. Moroco, John J. Alvarado, John J. Engen, Thomas E. Smithgall. Allosteric modulation of Src family kinases via SH3 domain displacement. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B190.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Abstract 3728: A discovery platform for allosteric modulators of c-Abl kinase function

Abstract The c-Abl protein-tyrosine kinase regulates intracellular signaling pathways controlling cell growth, adhesion, and responses to genotoxic stress. The kinase activity of c-Abl is repressed by intramolecular interactions involving its non-catalytic SH3 and SH2 domains. The SH3 domain interacts with a polyproline type II helix formed by the SH2-kinase linker, while the SH2 domain interacts with the C-lobe of kinase domain away from the active site. In addition, the myristoylated N-terminal cap region (Ncap) binds a hydrophobic pocket in the C-lobe of the kinase domain, clamping the SH3 and SH2 domains against the back of the kinase domain. Small molecules that allosterically regulate c-Abl kinase activity through its non-catalytic domains may represent selective probes of c-Abl function in diverse tumor types. In this study, we developed a chemical library screening assay designed to identify chemical modulators of c-Abl kinase activity that either disrupt or stabilize the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a recombinant purified protein consisting of the c-Abl Ncap, SH3 and SH2 domains plus the linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant c-Abl N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. The assay was optimized with respect to probe peptide sequence, probe and N32L protein concentrations, DMSO tolerance, and FP signal stability over time. Pilot library screens were then performed with the NCI Diversity Set III and an FDA-approved drug library (2800 compounds total). Seven compounds were identified that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds enhances c-Abl kinase activity in vitro, suggesting that it disrupts regulation of kinase activity through the non-catalytic region. These results show that screening assays based on the non-catalytic domains of c-Abl can identify small molecule regulators of kinase function. Citation Format: Prerna Grover, Thomas E. Smithgall. A discovery platform for allosteric modulators of c-Abl kinase function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3728. doi:10.1158/1538-7445.AM2014-3728

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement.

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo.

Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink–turn motif formed through a leader–linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader–linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink–turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.

Thermally Active Hybridization Drives the Crystallization of DNA-Functionalized Nanoparticles

The selectivity of DNA recognition inspires an elegant protocol for designing versatile nanoparticle (NP) assemblies. We use molecular dynamics simulations to analyze dynamic aspects of the assembly process and identify ingredients that are key to a successful assembly of NP superlattices through DNA hybridization. A scale-accurate coarse-grained model faithfully captures the relevant contributions to the kinetics of the DNA hybridization process and is able to recover all experimentally reported to date binary superlattices (BCC, CsCl, AlB2, Cr3Si, and Cs6C60). We study the assembly mechanism in systems with up to 10(6) degrees of freedom and find that the crystallization process is accompanied with a slight decrease of enthalpy. Furthermore, we find that the optimal range of the DNA linker interaction strengths for a successful assembly is 12-16kBT, and the optimal mean lifetime of a DNA hybridization event is 10(-4)-10(-3) of the total time it takes to form a crystal. We also obtain the optimal percentage of hybridized DNA pairs for different binary systems. On the basis of these results, we propose suitable linker sequences for future nanomaterials design.

Abstract 2221: Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors.

Abstract Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Imatinib selectivity is due in part to its ability to recognize and stabilize the inactive conformation of the kinase domain. This conformation is stabilized by intramolecular binding of the SH3 domain to the SH2-kinase linker, which in turn packs against the back of the kinase domain. Imatinib resistance can be caused by point mutations that either affect drug binding directly (e.g., through mutation of the gatekeeper position, Thr315) or that allosterically disturb the downregulated kinase domain conformation (e.g., through mutation or phosphorylation of the SH3 domain). These observations suggest that strengthening the regulatory SH3:linker interaction present in Bcr-Abl may stabilize a downregulated kinase domain conformation, thus sensitizing the kinase domain to inhibitor action. To explore this possibility, we created a series of modified c-Abl and Bcr-Abl proteins with enhanced SH3-linker interactions. By systematically increasing the proline content of the linker, we identified high-affinity linkers that stabilized intramolecular SH3 binding without disturbing the overall regulation of the kinase core. Enhanced SH3:linker interaction completely reversed c-Abl core activation by a C-lobe myristate binding pocket mutation, and substantially reduced activation by mutations in the ATP binding site (T315I) as well as the SH2:C-lobe interface (Y158D). Remarkably, enhanced SH3:linker interaction dramatically sensitized Bcr-Abl not only to imatinib but also to the myristic acid binding pocket inhibitor, GNF-2. These effects were observed in the context of both wild-type and imatinib-resistant forms of Bcr-Abl. Hydrogen exchange mass spectrometry of recombinant Abl core proteins with high-affinity linkers revealed dynamic coupling between the SH3:linker interface, the GNF-2 binding site in the C-lobe myristate binding pocket, and the active site. Taken together, these studies provide strong evidence that regulatory SH3:linker interaction is retained in the context of Bcr-Abl, and supports the idea that small molecules enhancing natural regulatory interaction at the SH3:linker interface may have utility as chemical sensitizers of Bcr-Abl drug action in the kinase domain. Citation Format: Shoghag B. Panjarian, Roxana Iacob, Chen Shugui, Thomas Wales, John R. Engen, Thomas E. Smithgall. Enhanced SH3:linker interaction allosterically sensitizes Abl kinases to small molecule inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2013-2221

Abstract 5599: Allosteric regulation of Abl and Bcr-Abl kinases by enhanced SH3:linker interaction

Abstract Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Despite the clinical success of imatinib in the chronic stage of CML, it is less effective in advanced disease due to the emergence of drug resistance often caused by point mutations in the Bcr-Abl kinase domain. Recently, we demonstrated that introduction of the “gatekeeper” resistance mutation T315I into a recombinant c-Abl “core” protein enhanced basal kinase activity. Imatinib selectivity is achieved in part by binding and stabilizing the unique inactive conformation of the Abl kinase domain. This conformation is stabilized allosterically by intramolecular binding of the SH3 domain to the SH2-kinase linker, which in turn docks against the back of the kinase domain. The goal of this project, therefore, was to determine whether kinase activation induced by T315I, as well as a mutation in a recently described allosteric inhibitor binding site in the kinase domain C-lobe (A356N mutant), could be overcome by enhancing the SH3:linker interaction. For these experiments, we engineered nine High Affinity Linker (HAL) variants of Abl in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction was confirmed by analysis of SH3 dynamics using hydrogen-deuterium exchange mass spectrometry. Using cell based assays, we identified Abl-HAL core proteins that remain locked in an inactive conformation. To determine the impact of the HAL sequences on activating mutations in the kinase domain, the strongest HAL sequence was combined with the T315I and A356N mutations. Remarkably, the HAL substitution completely reversed Abl core activation by the A356N mutation, and partially reversed the effect of T315I. We next introduced the HAL sequence into p210 Bcr-Abl coding region (wild-type and T315I), and expressed these forms of Bcr-Abl along with matched controls in the human myeloid cell line, TF-1. The Bcr-Abl HAL mutants transformed TF-1 cells to a cytokine-independent phenotype as effectively as wild-type Bcr-Abl but were much more sensitive to induction of apoptosis by the allosteric inhibitor, GNF-2. Furthermore, TF-1 cells transformed with Bcr-Abl-HAL bearing a wild-type kinase domain also showed greater sensitivity to imatinib and the related inhibitor nilotinib, suggesting that enhanced SH3:linker interaction structures the kinase domain in a favorable conformation for drug action. Our results provide new evidence that SH3:linker interaction not only has a strong allosteric effect on the regulation of the c-Abl kinase core, but also on Bcr-Abl sensitivity to both ATP-competitive and allosteric inhibitors as well. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5599. doi:1538-7445.AM2012-5599

An energetically beneficial leader–linker interaction abolishes ligand-binding cooperativity in glycine riboswitches

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences. In >50% of the glycine riboswitches, the leader-linker interaction forms a kink-turn motif. Characterization of three glycine ribsowitches showed that the leader-linker interaction improved the glycine-binding affinities by 4.5- to 86-fold. In-line probing and native gel assays with two aptamers in trans suggested synergistic action between glycine-binding and interaptamer interaction during global folding of the glycine riboswitch. Mutational analysis showed that there appeared to be no ligand-binding cooperativity in the glycine riboswitch when the leader-linker interaction is present, and the previously measured cooperativity is simply an artifact of a truncated construct missing the leader sequence.

Abstract B75: Allosteric regulation of Abl and Bcr-Abl kinases by enhanced SH3:linker interaction.

Abstract Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Despite the clinical success of imatinib in the chronic stage of CML, it is less effective in advanced disease due to the emergence of drug resistance often caused by point mutations in the Bcr-Abl kinase domain. Recently, we demonstrated that introduction of the “gatekeeper” resistance mutation T315I into a recombinant c-Abl “core” protein (consisting of the myristoylated N-Cap, SH3, SH2 and kinase domains) enhanced basal kinase activity. Imatinib selectivity is achieved in part by binding and stabilizing the unique inactive conformation of the Abl kinase domain. This conformation is stabilized allosterically by intramolecular binding of the SH3 domain to the SH2-kinase linker, which in turn docks against the back of the kinase domain. The goal of this project, therefore, was to determine whether kinase activation induced by T315I, as well as a mutation in a recently described allosteric inhibitor binding site in the kinase domain C-lobe (A356N mutant), could be overcome by enhancing the SH3:linker interaction. For these experiments, we engineered ten recombinant High Affinity Linker (HAL) proteins based on the c-Abl regulatory region (SH3-SH2-Linker) in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction was confirmed by analysis of SH3 dynamics using hydrogen-deuterium exchange mass spectrometry. We then incorporated the HAL coding sequences into the c-Abl core, and assessed kinase activation following transfection of 293T cells. Immunoblot analysis with autophosphorylation site-specific antibodies showed that five of the Abl-HAL core proteins remained inactive, suggesting that linker proline substitution did not alter the downregulated conformation. To determine the impact of the HAL sequences on activating mutations in the kinase domain, the strongest HAL sequence was combined with the T315I and A356N mutations. Remarkably, the HAL substitution completely reversed Abl core activation by the A356N mutation, and partially reversed the effect of T315I. We next introduced the HAL sequence into p210 Bcr-Abl coding region (wild-type and T315I), and expressed these forms of Bcr-Abl along with matched controls in the human myeloid leukemia cell line, TF-1. The Bcr-Abl HAL mutants transformed TF-1 cells to a cytokine-independent phenotype as effectively as wild-type Bcr-Abl but were much more sensitive to induction of apoptosis by the allosteric inhibitor, GNF-2. Furthermore, TF-1 cells transformed with Bcr-Abl-HAL bearing a wild-type kinase domain also showed greater sensitivity to imatinib and the related inhibitor nilotinib, suggesting that enhanced SH3:linker interaction structures the kinase domain in a favorable conformation for drug action. Our results provide new evidence that SH3:linker interaction not only has a strong allosteric effect on the regulation of the c-Abl kinase core, but also on Bcr-Abl sensitivity to both ATP-competitive and allosteric inhibitors as well. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B75.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

AbstractAbstract 1208Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures:No relevant conflicts of interest to declare.

Structural basis of the auto-inhibition mechanism of nonreceptor tyrosine kinase PTK6

Protein tyrosine kinase 6 (PTK6) is composed of SH3, SH2, and Kinase domains, with a linker region (Linker) between the SH2 and Kinase domains. Here, we report the structural basis of the SH3–Linker interaction that results in auto-inhibition of PTK6. The solution structures of the SH3 domain and SH3/Linker complex were determined by NMR spectroscopy. The structure of the SH3 domain forms a conventional β-barrel with two β-sheets comprised of five β-strands. However, the molecular topology and charge distribution of PTK6-SH3 slightly differs from that of the other SH3 domains. The structure of the N-terminal Linker within the complex showed that the proline-rich region (P175–P187) of the Linker forms a compact hairpin structure through hydrophobic interactions. The structure of the SH3/Linker complex revealed intra-molecular interaction between the amino acid pairs R22/E190, W44/W184, N65/P177, and Y66/P179. Mutations in PTK6 at R22, W44, N65, and Y66 residues in the SH3 domain increased catalytic activity compared with wild-type protein, implying that specific interactions between hydrophobic residues in the proline-rich linker region and hydrophobic residues in the SH3 domain are mainly responsible for down-regulating the catalytic activity of PTK6.

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3–Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3–Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

The Abl SH2‐kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.