[Show abstract][Hide abstract] ABSTRACT: Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositide-3-kinase-α (PI3Kα) is a lipid kinase that catalyzes the phosphorylation of PIP2 to produce PIP3 in response to phosphorylated receptor tyrosine kinases (RTK) or their substrates. The increased levels of PIP3 initiate a number of signaling pathways by recruiting other kinases, such as Akt, to the plasma membrane. The enzyme is composed of two subunits, p110 and p85, each comprising five domains. PI3Kα is frequently mutated in many cancer types and the mutations increase PI3K kinase activity leading to increased tumor cell survival, cell motility, cell metabolism, and cell cycle progression. Several atomic resolution structures of the enzyme reveal that the enzyme has a complex architecture in which each domain interacts with several domains of the same or the other subunit. Structural and biochemical data show that physiological activation, as well as activation by some oncogenic mutations, involves relief of autoinhibition by dislodging the inhibitory nSH2 domain of the regulatory subunit p85 from its inhibitory position. Computational studies show that most of these effects involve, in addition to structural changes, modifications of the dynamics of the protein that alter the relative stabilities of the different states accessible to the enzyme.
Recent progress toward determining the mechanism of activation benefited from two developments: the determination of the structure PI3K bound to short chain phosphoinositides, and the characterization of the conformations accessible to the activation loop in molecular dynamics simulations.
[Show abstract][Hide abstract] ABSTRACT: The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1. 4, shows significant calcium dependent inactivation, while its homologue Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these three Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.
[Show abstract][Hide abstract] ABSTRACT: Iodide (I(-)), an essential constituent of the thyroid hormones, is actively accumulated in the thyroid by the Na(+)/I(-) symporter (NIS), a key plasma membrane protein encoded by the slc5a5 gene. Mutations in slc5a5 cause I(-) transport defects (ITDs), autosomal recessive disorders in which I(-) accumulation is totally or partially impaired, leading to congenital hypothyroidism. The characterization of NIS mutants has yielded significant insights into the molecular mechanism of NIS.
To determine the basis of a patient's clinical ITD phenotype, we sequenced her slc5a5 gene. As we identified a new mutation in NIS (V270E), we extensively characterized it to determine the molecular requirements of NIS at position 270.
Genomic DNA was purified and the slc5a5 sequence determined. Functional in vitro studies were performed to characterize the V270E NIS mutant.
The index patient was diagnosed with hypothyroidism with minimal radioiodide uptake in a normally located, although enlarged, thyroid gland.
We identified a new NIS mutation: V270E. The patient had the compound heterozygous NIS mutation R124H/V270E. R124H NIS has been characterized previously. We show that V270E markedly reduces I(-) uptake via a pronounced (but not total) impairment of the protein's plasma membrane targeting. Remarkably, V270E is intrinsically active. Therefore, a negative charge at position 270 interferes with NIS cell surface trafficking. The patient's minimal I(-) uptake enabled sufficient thyroid hormone biosynthesis to prevent cognitive impairment.
A non-polar residue at position 270-which all members of the SLC5A family have-is required for NIS plasma membrane targeting.
No preview · Article · Jul 2015 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule.
Full-text · Article · Jul 2015 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 3-kinase α (PI3Kα) is a heterodimeric lipid kinase that catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2 ) to phosphoinositide-3,4,5-trisphosphate (PIP3 ). The PI3Kα signaling pathway plays an important role in cell growth, proliferation and survival. This pathway is activated in numerous cancers, where the PI3KCA gene, which encodes for the p110α PI3Kα subunit, is mutated. Its mutation often results in gain of enzymatic activity; however, the mechanism of activation by oncogenic mutations remains unknown. Here, using computational methods, we show that oncogenic mutations that are far from the catalytic site and increase the enzymatic affinity, destabilize the p110α/p85α dimer. By affecting the dynamics of the protein, these mutations favor the conformations that reduce the autoinhibitory effect of the p85α nSH2 domain. For example, we determined that in all the mutants, the nSH2 domain exhibits increased positional heterogeneity compared to the wild type (WT), as evidenced by changes in the fluctuation profiles computed by normal mode analysis (NMA) of coarse-grained elastic network models (ENM). Analysis of the inter-domain interactions of the WT and mutants at the p110α/p85α interface obtained using molecular dynamics (MD) simulations suggest that all the tumor-associated mutations effectively weaken the interactions between the p110α and the p85α subunits by disrupting key stabilizing interactions. These findings have important implications for understanding how oncogenic mutations change the conformational multiplicity of PI3Kα and lead to increased enzymatic activity. This mechanism may apply to other enzymes and/or macromolecular complexes that play a key role in cell signaling. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Nearly one-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB. A key factor that contributes to the widespread infection of Mtb is its capacity to survive inside the host macrophage. Understanding how Mtb withstands the hostile intracellular environment of this phagocytic cell may reveal targets for development of therapeutics that enhance the innate anti-Mtb activities of the macrophage. We discovered a novel signaling pathway in mycobacteria which regulates cellular redox homeostasis through NADH and FAD, regulators of metabolism and redox balance. NADH induces the expression of a protein kinase, PknG, which then phosphorylates the ribosomal protein L13 and promotes its presence in the cytoplasm. L13 therein forms a complex with RenU, a Nudix (Nucleoside diphosphate linked moiety X) hydrolase that degrades NADH and FAD. Genetic disruption of this signaling cascade leads to cellular accumulation of these molecules, increased mycobacterial sensitivity to oxidative stress, impaired surface biofilm growth, and most importantly, reduced survival of Mtb in macrophages.
[Show abstract][Hide abstract] ABSTRACT: Despite a constant decrease in tuberculosis (TB) incidence rates, nearly two billion people worldwide are estimated to have latent TB. Five to ten percent of people with latent TB will develop the active form of the disease. Of the estimated 8.6 million new cases of active TB in 2012, 1.3 million people died of the disease and 450,000 developed multidrug-resistant TB. Resistance to oxidative stress is essential for Mycobacterium tuberculosis (Mtb) survival in host macrophages and the onset of latent TB infection. We have identified a Nudix (nucleoside diphosphate-linked moiety X) hydrolase, RenU, necessary for Mtb survival in oxidative stress environments. We show that RenU preferentially degrades adenosine derivatives over other nucleoside derivatives. Through a novel fluorescence based assay, we also determined that RenU prefers NADH as a substrate over NAD. Furthermore, we show that RenU is required for Mtb survival within macrophages. The link between RenU, NADH, and Mtb survival warrants further investigation as it could be the basis for novel therapeutic approaches to prevent and combat latent TB.
No preview · Article · Jan 2015 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: TRPV1 has been shown to alter its ionic selectivity profile in a time- and agonistdependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-offunction substitutions within the TRPV1 pore turret (N628P, S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in
wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large
cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered
capsaicin-induced changes in NMDG permeability, the loss of function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.
No preview · Article · Jan 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Voltage-gated sodium channels (Nav) underlie the rapid upstroke of action potentials in excitable tissues. Binding of channel-interactive proteins is essential for controlling fast and long-term inactivation. In the structure of the complex of the carboxy-terminal portion of Nav1.5 (CTNav1.5) with calmodulin (CaM)-Mg(2+) reported here, both CaM lobes interact with the CTNav1.5. On the basis of the differences between this structure and that of an inactivated complex, we propose that the structure reported here represents a non-inactivated state of the CTNav, that is, the state that is poised for activation. Electrophysiological characterization of mutants further supports the importance of the interactions identified in the structure. Isothermal titration calorimetry experiments show that CaM binds to CTNav1.5 with high affinity. The results of this study provide unique insights into the physiological activation and the pathophysiology of Nav channels.
Full-text · Article · Nov 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
[Show abstract][Hide abstract] ABSTRACT: The Na(+)/I(-) symporter (NIS) mediates active I(-) transport-the first step in thyroid hormonogenesis-with a 2Na(+):1I(-) stoichiometry. NIS-mediated (131)I(-) treatment of thyroid cancer post-thyroidectomy is the most effective targeted internal radiation cancer treatment available. Here to uncover mechanistic information on NIS, we use statistical thermodynamics to obtain Kds and estimate the relative populations of the different NIS species during Na(+)/anion binding and transport. We show that, although the affinity of NIS for I(-) is low (Kd=224 μM), it increases when Na(+) is bound (Kd=22.4 μM). However, this Kd is still much higher than the submicromolar physiological I(-) concentration. To overcome this, NIS takes advantage of the extracellular Na(+) concentration and the pronounced increase in its own affinity for I(-) and for the second Na(+) elicited by binding of the first. Thus, at physiological Na(+) concentrations, ~79% of NIS molecules are occupied by two Na(+) ions and ready to bind and transport I(-).
Preview · Article · Jun 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2) to phosphoinositide-3,4,5-trisphosphate (PIP3), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP2, a membrane lipid.
Full-text · Article · Mar 2014 · Biophysical Reviews
[Show abstract][Hide abstract] ABSTRACT: Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from
(the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca
ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg
ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of
FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.
Full-text · Article · Mar 2014 · Acta Crystallographica Section D Biological Crystallography