Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.
Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the second step in glycolysis, the reversible isomerization of D-glucose 6-phosphate to D-fructose 6-phosphate. The reaction mechanism involves acid-base catalysis with proton transfer and proceeds through a cis-enediol(ate) intermediate. 5-Phospho-D-arabinonohydroxamic acid (5PAH) is a synthetic small molecule that resembles the reaction intermediate, differing only in that it has a nitrogen atom in place of C1. Hence, 5PAH is the best inhibitor of the isomerization reaction reported to date with a Ki of 2 ؋ 10 ؊7 M. Here we report the crystal structure of rabbit phosphoglucose isomerase complexed with 5PAH at 1.9 Å resolution. The interaction of 5PAH with amino acid residues in the enzyme active site supports a model of the catalytic mechanism in which Glu-357 transfers a proton between C1 and C2 and Arg-272 helps stabilize the intermediate. It also suggests a mechanism for proton transfer between O1 and O2. P hosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic glycolytic enzyme that catalyzes the reversible isomerization of D-glucose 6-phosphate (G6P) to D-fructose 6-phosphate (F6P). In addition to isomerase activity, PGI has been shown to display anomerase activity between pyranose anomers of G6P (1), between furanose anomers of F6P (2), and between those of D-mannose 6-phosphate (3), as well as C-2-epimerase activity on G6P (4). It also has roles in protein glycosylation, gluconeogenesis, and the pentose phosphate pathway (5). This central role in the metabolism of phosphorylated sugars explains the strong impact of PGI deficiency in humans (6, 7), as well as the interest as a therapeutic target in parasite metabolism (8). The PGI polypeptide chain, or perhaps a variation thereof, also has extracellular roles as neuroleukin, autocrine motility factor, and differentiation and maturation mediator (9-12). These proteins promote antibody secretion by mononuclear cells, tumor cell motility, and differentiation of leukemia cells and a promyelocytic cell line (HL-60 cells), respectively. More recently, PGI has also been identified as the antigen involved in rheumatoid arthritis of a mouse line (13), as a specific inhibitor toward myofibril-bound serine proteinase (14), and as the surface antigen involved in sperm agglutination (15). Because very little is known about how PGI is precisely involved in these various biological processes, this moonlighting protein (16) has been the subject of intense investigations.Biochemical characterization of PGI began over 50 years ago and includes inhibitor studies (1,(17)(18)(19)(20)(21)(22)(23)(24)(25), labeling studies (26-32), solvent exchange (33, 34), mutagenesis (35,36), and pH profile studies (20,37). A proposed multistep catalytic mechanism includes a ring-opening step followed by an isomerization step. The isomerization step proceeds via general acid-base catalysis with proton transfer. In the G6P to F6P direction, abstraction of the proton from C2 by an active site amino acid residue yields a 1,2-cis-enediol(ate) in...
The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F 1 ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between ␥Leu83 and  DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the ␥-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.The mitochondrial ATP synthase is a molecular motor that couples the transport of protons down a potential gradient with the phosphorylation of ADP. This process can be reversed and the hydrolysis of ATP results in the pumping of protons out of the mitochondrial matrix into the cytoplasm generating a proton gradient. In the synthesis mode, the protomotive force is established by oxidation of NADH or succinate by the electron transport chain. Whereas much is known on the structure/ function of the ATP synthase and the mechanism of ATP hydrolysis, less is known on the molecular details on the coupling of proton transport and ATP synthesis. The structure/ function relationship of the ATP synthase is key to understanding the coupling mechanism; that is, identification of which intra-and intermolecular interactions within the ATP synthase critical for coupling.The ATP synthase is composed of two distinct components, the F 1 and the F o portion and as such is referred to as the F 1 F o ATP synthase. The F 1 portion contains the catalytic sites and is composed of ␣ 3  3 ␥␦⑀ with an overall molecular mass of about 350 kDa (1). The active site is composed of the ␣ pair, and thus there are three catalytic sites per complex. The ␥␦⑀-subunits comprise the central stalk with the ␥-subunit situated in the middle of the ␣ 3  3 subcomplex. The central stalk acts as a rotor and drives conformational changes in the active sites in a sequential manner, thereby effecting ATP synthesis. The ␦-and ⑀-subunits are critical for coupling (2, 3), but their roles are unclear, they may participate directly in the coupling process or needed merely for structural purposes.The F o portion of the ATP synthase acts as a proton turbine whose rotation is physically coupled to the central stalk and drives its rotation. F o is minimally composed of subunits abc 10 with the c 10 arranged as a cylinder within the membrane (4). Subunit a is thought to directly participate in the proton flow while subunit b is minimally involved in the structure of the peripheral stalk. The peripheral stalk is composed of subunits b, d, h, and subunit 5 (5-8) and acts as a stator connecting F 1 with ...
Enzymes of glycolysis in Trypanosoma brucei have been identified as potential drug targets for African sleeping sickness because glycolysis is the only source of ATP for the bloodstream form of this parasite. Several inhibitors were previously reported to bind preferentially to trypanosomal phosphoglucose isomerase (PGI, the second enzyme in glycolysis) than to mammalian PGIs, which suggests that PGI might make a good target for species-specific drug design. Herein, we report recombinant expression, purification, crystallization and X-ray crystal structure determination of T. brucei PGI. One structure solved at 1.6 A resolution contains a substrate, D-glucose-6-phosphate, in an extended conformation in the active site. A second structure solved at 1.9 A resolution contains a citrate molecule in the active site. The structures are compared with the crystal structures of PGI from humans and from Leishmania mexicana. The availability of recombinant tPGI and its first high-resolution crystal structures are initial steps in considering this enzyme as a potential drug target.
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.