[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: 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 Leishmania major (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(2+) ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg(2+) ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major 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.
[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.
[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(-).
[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.
[Show abstract][Hide abstract] ABSTRACT: The mechanism by which antibodies elicited against protein-derived peptides achieve cross-reactivity with their cognate proteins remains unknown. To address this question, we have carried out the complete thermodynamic characterization of the association of a monoclonal antibody (260.33.12) raised against a peptide (SNpep) derived from staphylococcal nuclease (SNase) with both eliciting peptide and cognate protein. Although both ligands bind with similar affinity (Kd = 0.42 μM and 0.30 μM for protein and peptide, respectively), protein and peptide binding have highly different thermodynamic signatures: peptide binding is characterized by a large enthalpic contribution (ΔH = − 7.7 kcal/mol) whereas protein binding is dominated by a large entropic contribution (− TΔS = − 7.2 kcal/mol). The structure of the SNpep:Fab complex, determined by X-ray diffraction, reveals that the bound conformation of the peptide differs from the conformation of the corresponding loop region in crystal structures of free SNase. The energy difference, estimated by molecular dynamics simulations between native SNase and a model in which the Ω-loop is built in the conformation of the Fab-bound peptide, shows that the energetic cost of adopting this conformation is compatible with the enthalpic cost of binding the protein vis-à-vis the peptide. These results are compatible with a mechanism by which the anti-peptide antibody recognizes the cognate protein: high affinity is maintained upon binding a non-native conformation by offsetting enthalpic penalties with reduced entropic losses. These findings provide potentially useful guidelines for the identification of linear epitopes within protein sequences that are well suited for the development of synthetic peptide vaccines.
Journal of Molecular Biology 06/2013; 425(11):2027–2038. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene for a Nudix enzyme (SP_1669) was found to code for a UDP-X diphosphatase. The SP_1669 gene is localized among genes encoding proteins that participate in cell division in Streptococcus pneumoniae. One of these genes, MurF, encodes an enzyme that catalyzes the last step of the Mur pathway of peptidoglycan biosynthesis. Mur pathway substrates are all derived from UDP-glucosamine and all are potential Nudix substrates. We showed that UDP-X diphosphatase can hydrolyze the Mur pathway substrates UDP-N-acetylmuramic acid and UDP-N-acetylmuramoyl-L-alanine. The 1.39 Å resolution crystal structure of this enzyme shows that it folds as an asymmetric homodimer with two distinct active sites, each containing elements of the conserved Nudix box sequence. In addition to its Nudix catalytic activity, the enzyme has a 3959 RNA exonuclease activity. We propose that the structural asymmetry in UDP-X diphosphatase facilitates the recognition of these two distinct classes of substrates, Nudix substrates and RNA. UDP-X diphosphatase is a prototype of a new family of Nudix enzymes with unique structural characteristics: two monomers, each consisting of an N-terminal helix bundle domain and a C-terminal Nudix domain, form an asymmetric dimer with two distinct active sites. These enzymes function to hydrolyze bacterial cell wall precursors and degrade RNA. Copyright: ß 2013 Duong-Ly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PLoS ONE 05/2013; 8(5):e64241. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein that mediates active I(-) transport in the thyroid, the first step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report a thorough characterization of the ITD-causing NIS mutation in which the sixth intracellular loop residues 439-443 are missing. This mutant protein was intracellularly retained, incompletely glycosylated, and intrinsically inactive. Engineering 5 Ala at positions 439-443 partially recovered cell surface targeting and activity (∼15%). Strikingly, NIS with the sequence 439-AANAA-443, in which Asn was restored at position 441, was targeted to the plasma membrane and exhibited ∼95% the transport activity of WT NIS. Based on our NIS homology model, we propose that the side chain of N441, a residue conserved throughout most of the SLC5 family, interacts with the main chain amino group of G444, capping the α-helix of transmembrane segment XII and thus stabilizing the structure of the molecule. Our data provide insight into a critical interhelical interaction required for NIS folding and activity.-Li, W., Nicola, J. P., Amzel, L. M., Carrasco, N. Asn441 plays a key role in folding and function of the Na(+)/I(-) symporter (NIS).
[Show abstract][Hide abstract] ABSTRACT: [*F.Z. and S.B. are joint first authors] Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms. While MetAP2 has been demonstrated to be a therapeutic target for inhibiting angiogenesis in mammals, MetAP1 seems to be vital for cell proliferation. Our earlier efforts identified two structural classes of human MetAP1 (HsMetAP1)-selective inhibitors (1-4), but all of them failed to inhibit cellular HsMetAP1. Using Mn(II) or Zn(II) to activate HsMetAP1, we found that 1-4 could only effectively inhibit purified HsMetAP1 in the presence of physiologically unachievable concentrations of Co(II). In an effort to seek Co(II)-independent inhibitors, a novel structural class containing a 2-(pyridin-2-yl)quinazoline core has been discovered. Many compounds in this class potently and selectively inhibited HsMetAP1 without Co(II). Subsequently, we demonstrated that 11j, an auxiliary metal-dependent inhibitor, effectively inhibited HsMetAP1 in primary cells. This is the first report that an HsMetAP1-selective inhibitor is effective against its target in cells.
Journal of Medicinal Chemistry 05/2013; 56(10):3996-4016. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure-activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5'-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a-30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.
[Show abstract][Hide abstract] ABSTRACT: The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380-385) and these phosphorylations are proposed to induce a reduction in PTEN's plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN's regulation and suggest pharmacologic approaches for direct PTEN activation. DOI:http://dx.doi.org/10.7554/eLife.00691.001.
[Show abstract][Hide abstract] ABSTRACT: Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O(2)-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O(2) is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (Cu(H) and Cu(M)). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-Å-resolution structure (hydro)peroxide binds solely to Cu(M) in a slightly asymmetric side-on mode. The O-O interatomic distance of the copper-bound ligand is 1.5 Å, characteristic of peroxide/hydroperoxide species, and the Cu-O distances are 2.0 and 2.1 Å. Density functional theory calculations using the first coordination sphere of the Cu(M) active site as a model system show that the computed energies of the side-on L(3)Cu(M)(II)-O(2) (2-) species and its isomeric, end-on structure L(3)Cu(M)(I)-O(2) (·-) are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L(3)Cu(M)(II)O(2) (2-) is in good agreement with the results of a hybrid quantum mechanical-molecular mechanical optimization of this species.
European Journal of Biochemistry 12/2012; · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: the cell wall of Mycobacterium tuberculosis: Structure and Mechanism of L,D-transpeptidase 2, Structure, doi:10.1016/j.str.2012.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
[Show abstract][Hide abstract] ABSTRACT: Using the "pull and wait" (PNW) simulation protocol at 300 K, we studied the unfolding of a ubiquitin molecule by force. PNW was implemented in the CHARMM program using an integration time step of 1 fs and a uniform dielectric constant of 1. The ubiquitin molecule, initially solvated, was put under mechanical stress, exerting forces from different directions. The rupture of five hydrogen bonds between parallel strands β1 and β5 takes place during the extension from 13 to 15 Å, defines a mechanical barrier for unfolding and dominates the point of maximum unfolding force. The simulations described here show that given adequate time, a small applied force can destabilize those five H-bonds relative to the bonds that can be created to water molecules; allowing the formation of stable H-bonds between a single water molecule and the donor and acceptor groups of the interstrand H-bonds. Thus, simulations run with PNW show that the force is not responsible for "ripping apart" the backbone H-bonds; it merely destabilizes them making them less stable than the H-bonds they can make with water. Additional simulations show that the force necessary to destabilize the H-bonds and allow them to be replaced by H-bonds to water molecules depends strongly on the pulling direction. By using a simulation protocol that allows equilibration at each extension we have been able to observe the details of the events leading to the unfolding of ubiquitin by mechanical force.
[Show abstract][Hide abstract] ABSTRACT: With multidrug-resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug targets are becoming an urgent need. Traditional β-lactam antibiotics that inhibit D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase Ldt(Mt2) containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for Ldt(Mt2) in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights to facilitate development of drugs targeting this validated yet unexploited enzyme.
[Show abstract][Hide abstract] ABSTRACT: Equilibrium free-energy differences can be computed from nonequilibrium molecular dynamics (MD) simulations using Jarzynski's equality (Jarzynski, C. Phys. Rev. Lett.1997, 78, 2690) by combining a large set of independent trajectories (path ensemble). Here we present the multistep trajectory combination (MSTC) method to compute free-energy differences, which by combining trajectories significantly reduces the number of trajectories necessary to generate a representative path ensemble. This method generates well-sampled work distributions, even for large systems, by combining parts of a relatively small number of trajectories carried out in steps. To assess the efficiency of the MSTC method, we derived analytical expressions and used them to compute the bias and the variance of the free-energy estimates along with numerically calculated values. We show that the MSTC method significantly reduces both the bias and variance of the free-energy estimates compared to the estimates obtained using single-step trajectories. In addition, because in the MSTC method the process is divided into steps, it is feasible to compute the reverse transition. By combining the forward and reverse processes, the free-energy difference can be computed using the Crooks' fluctuation theorem (Crooks, G. E. J. Stat. Phys.1998, 90, 1481 and Crooks, G. E. Phys. Rev. E2000, 61, 2361) or Bennett's acceptance ratio (Bennett, C. H. J. Comput. Phys. 1976, 22, 245), which further reduces the bias and variance of the estimates.
The Journal of Physical Chemistry B 07/2012; 116(36):10986-95. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Linear 2-alkylaminoethyl-1,1-bisphosphonates are effective agents against proliferation of Trypanosoma cruzi , the etiologic agent of American trypanosomiasis (Chagas disease), exhibiting IC(50) values in the nanomolar range against the parasites. This activity is associated with inhibition at the low nanomolar level of the T. cruzi farnesyl diphosphate synthase (TcFPPS). X-ray structures and thermodynamic data of the complexes TcFPPS with five compounds of this family show that the inhibitors bind to the allylic site of the enzyme, with their alkyl chain occupying the cavity that binds the isoprenoid chain of the substrate. The compounds bind to TcFPPS with unfavorable enthalpy compensated by a favorable entropy that results from a delicate balance between two opposing effects: the loss of conformational entropy due to freezing of single bond rotations and the favorable burial of the hydrophobic alkyl chains. The data suggest that introduction of strategically placed double bonds and methyl branches should increase affinity substantially.
Journal of Medicinal Chemistry 06/2012; 55(14):6445-54. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although lectins are "hard-wired" in the germline, the presence of tandemly arrayed carbohydrate recognition domains (CRDs), of chimeric structures displaying distinct CRDs, of polymorphic genes resulting in multiple isoforms, and in some cases, of a considerable recognition plasticity of their carbohydrate binding sites, significantly expand the lectin ligand-recognition spectrum and lectin functional diversification. Analysis of structural/functional aspects of galectins and F-lectins-the most recently identified lectin family characterized by a unique CRD sequence motif (a distinctive structural fold) and nominal specificity for l-Fuc-has led to a greater understanding of self/nonself recognition by proteins with tandemly arrayed CRDs. For lectins with a single CRD, however, recognition of self and nonself glycans can only be rationalized in terms of protein oligomerization and ligand clustering and presentation. Spatial and temporal changes in lectin expression, secretion, and local concentrations in extracellular microenvironments, as well as structural diversity and spatial display of their carbohydrate ligands on the host or microbial cell surface, are suggestive of a dynamic interplay of their recognition and effector functions in development and immunity.
Annals of the New York Academy of Sciences 04/2012; 1253(1):E14-26. · 4.38 Impact Factor