The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.
Soluble guanylate cyclases are nitric oxide-responsive signaling proteins in which the nitric oxide sensor is a heme-binding domain of unknown structure that we have termed the heme-NO and oxygen binding (H-NOX) domain. H-NOX domains are also found in bacteria, either as isolated domains, or are fused through a membrane-spanning region to methyl-accepting chemotaxis proteins. We have determined the crystal structure of an oxygen-binding H-NOX domain of one such signaling protein from the obligate anaerobe Thermoanaerobacter tengcongensis at 1.77-Å resolution, revealing a protein fold unrelated to known structures. Particularly striking is the structure of the protoporphyrin IX group, which is distorted from planarity to an extent not seen before in proteinbound heme groups. Comparison of the structure of the H-NOX domain in two different crystal forms suggests a mechanism whereby alteration in the degree of distortion of the heme group is coupled to changes on the molecular surface of the H-NOX domain and potentially to changes in intermolecular interactions.
Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).
The Abl and Src tyrosine kinases are key signaling proteins that are of considerable interest as drug targets in cancer and many other diseases. The regulatory mechanisms that control the activity of these proteins are complex, and involve large-scale conformational changes in response to phosphorylation and other modulatory signals. The success of the Abl inhibitor imatinib in the treatment of chronic myelogenous leukemia has shown the potential of kinase inhibitors, but the rise of drug resistance in patients has also shown that drugs with alternative modes of binding to the kinase are needed. The detailed understanding of mechanisms of protein-drug interaction and drug resistance through biophysical methods demands a method for the production of active protein on the milligram scale. We have developed a bacterial expression system for the kinase domains of c-Abl and c-Src, which allows for the quick expression and purification of active wild-type and mutant kinase domains by coexpression with the YopH tyrosine phosphatase. This method makes practical the use of isotopic labeling of c-Abl and c-Src for NMR studies, and is also applicable for constructs containing the SH2 and SH3 domains of the kinases.
Hemoproteins carry out diverse functions utilizing a wide range of chemical reactivity while employing the same heme prosthetic group. It is clear from high-resolution crystal structures and biochemical studies that protein-bound hemes are not planar and adopt diverse conformations. The crystal structure of an H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) contains the most distorted heme reported to date. In this study, Tt H-NOX was engineered to adopt a flatter heme by mutating proline 115, a conserved residue in the H-NOX family, to alanine. Decreasing heme distortion in Tt H-NOX increases affinity for oxygen and decreases the reduction potential of the heme iron. Additionally, flattening the heme is associated with significant shifts in the N-terminus of the protein. These results show a clear link between the heme conformation and Tt H-NOX structure and demonstrate that heme distortion is an important determinant for maintaining biochemical properties in H-NOX proteins.The very broad range of chemistry carried out by hemoproteins has attracted a vast amount of attention for many years. Representative examples include oxygen-transporting proteins, like the globins, and potent catalysts involving high-valent iron-oxo complexes, such as the cytochrome P-450s. There are a number of factors that direct and control the type of chemistry carried out by this ubiquitous class of proteins. The coordination environment provided by the protein, for example, plays a significant role in dictating both chemistry and function. The globins have evolved to stabilize the unligated Fe(II) oxidation state and the Fe(II)-O 2 complex using a proximal histidine ligand. In contrast, the thiol-ligated cytochrome P450s have a stable Fe(III) oxidation state; however, when reduced to Fe(II), the iron binds O 2 and then generates a high-valent iron-oxo complex (formally the Fe(V) oxidation state). This potent oxidant is competent for hydroxylation chemistry of unactivated C-H bonds.The unique and tunable chemical properties of the heme prosthetic group account for the wide range of functions observed within the hemoprotein family. The tetrapyrrole moiety of the heme is aromatic and as such, hemes in isolation are planar. However, when proteinbound, the heme deviates significantly from planarity (1-5). Although the types of heme * Corresponding author: Michael A. Marletta, QB3 Institute, University of California, Berkeley, 570 Stanley Hall, Berkeley, CA 94720-3220, Telephone: 510-666-2763, Fax: 510-666-2765 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript distortions found in hemoproteins are energetically unfavorable (6), they are conserved in homologous proteins from different organisms (7), suggesting that heme distortion is important for function. Indeed, out-of-plane heme distortions have been shown to influence the biochemical properties of both non-covalent b-type cytochromes, such as the globins, where the heme is bound to the protein through coordination to the iron atom of an am...
The cardiac field has benefited from the availability of several CaMKII inhibitors serving as research tools to test putative CaMKII pathways associated with cardiovascular physiology and pathophysiology. Successful demonstrations of its critical pathophysiological roles have elevated CaMKII as a key target in heart failure, arrhythmia, and other forms of heart disease. This has caught the attention of the pharmaceutical industry, which is now racing to develop CaMKII inhibitors as safe and effective therapeutic agents. While the first generation of CaMKII inhibitor development is focused on blocking its activity based on ATP binding to its catalytic site, future inhibitors can also target sites affecting its regulation by Ca2+/CaM or translocation to some of its protein substrates. The recent availability of crystal structures of the kinase in the autoinhibited and activated state, and of the dodecameric holoenzyme, provides insights into the mechanism of action of existing inhibitors. It is also accelerating the design and development of better pharmacological inhibitors. This review examines the structure of the kinase and suggests possible sites for its inhibition. It also analyzes the uses and limitations of current research tools. Development of new inhibitors will enable preclinical proof of concept tests and clinical development of successful lead compounds, as well as improved research tools to more accurately examine and extend knowledge of the role of CaMKII in cardiac health and disease.
The dodecameric holoenzyme of calcium-calmodulin-dependent protein kinase II (CaMKII) responds to high-frequency Ca(2+) pulses to become Ca(2+) independent. A simple coincidence-detector model for Ca(2+)-frequency dependency assumes noncooperative activation of kinase domains. We show that activation of CaMKII by Ca(2+)-calmodulin is cooperative, with a Hill coefficient of approximately 3.0, implying sequential kinase-domain activation beyond dimeric units. We present data for a model in which cooperative activation includes the intersubunit 'capture' of regulatory segments. Such a capture interaction is seen in a crystal structure that shows extensive contacts between the regulatory segment of one kinase and the catalytic domain of another. These interactions are mimicked by a natural inhibitor of CaMKII. Our results show that a simple coincidence-detection model cannot be operative and point to the importance of kinetic dissection of the frequency-response mechanism in future experiments.
Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C-and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.Phosphorylation of proteins on tyrosine residues is central to a variety of intracellular signal transduction pathways. The enzymes responsible for this modification, tyrosine kinases, occur as transmembrane receptors or nonreceptor cytoplasmic proteins. The uncontrolled signaling resulting from the deregulation of tyrosine kinase activity has been implicated in the onset and progression of a variety of human malignancies (1). Thus, considerable interest exists in the study of tyrosine kinase substrate selection, regulation of activity, and biological function. In vitro studies using peptide libraries have helped to determine the substrate specificities of several tyrosine kinases (2-4). Surprisingly, the catalytic domains of tyrosine kinases possess less rigorous substrate specificity than those of Ser/Thr kinases (2). However, cytoplasmic tyrosine kinases have an array of accessory domains implicated in protein-protein interactions that can further influence substrate selection.Src family tyrosine kinases are organized into a set of modular domains: unique, SH3, 1 SH2, and catalytic. The catalytic domain is responsible for transferring phosphate from ATP onto tyrosine residues of substrate proteins. The noncatalytic SH2, SH3, and unique domains each have multiple roles that are important for the regulation of cellular kinase function. The SH2 and SH3 domains regulate kinase activity by maintaining the catalytic domain in an inact...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.