Making the Right Moves

The structure of the nucleotide-free F1-ATPase from a thermoalkaliphilic bacterium presented in this issue of Structure (Stocker et al., 2007) reveals the structural interactions that prevent the enzyme from operating naturally in the hydrolytic direction. The data provide new insights into the mechanism of the FoF1-ATP synthase

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): Structural and computational study

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine a-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Ca position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests thatO2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). 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-A° -resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O-O interatomic distance of the copperbound ligand is 1.5 A ° , characteristic of peroxide/hydroperoxide species, and the Cu-O distances are 2.0 and 2.1 A ° . Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)-O2 2- species and its isomeric, end-on structure L3CuM(I)-O2 - 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 L3CuM(II)O2 2- is in good agreement with the results of a hybrid quantum mechanical-molecular mechanical optimization of this species.Fil: Rudzka, Katarzyna. University Johns Hopkins; Estados UnidosFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina. Universidad de Buenos Aires; ArgentinaFil: Eipper, Betty. University Of Connecticut; Estados UnidosFil: Mains, Richard. University Of Connecticut; Estados UnidosFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; ArgentinaFil: Amzel, L. Mario. University Johns Hopkins; Estados Unido

Bisphosphonate-based molecules as potential new antiparasitic drugs

Neglected tropical diseases such as Chagas disease and leishmaniasis affect millions of people around the world. Both diseases affect various parts of the globe and drugs traditionally used in therapy against these diseases have limitations, especially with regard to low efficacy and high toxicity. In this context, the class of bisphosphonate-based compounds has made significant advances regarding the chemical synthesis process as well as the pharmacological properties attributed to these compounds. Among this spectrum of pharmacological activity, bisphosphonate compounds with antiparasitic activity stand out, especially in the treatment of Chagas disease and leishmaniasis caused by Trypanosoma cruzi and Leishmania spp., respectively. Some bisphosphonate compounds can inhibit the mevalonate pathway, an essential metabolic pathway, by interfering with the synthesis of ergosterol, a sterol responsible for the growth and viability of these parasites. Therefore, this review aims to present the information about the importance of these compounds as antiparasitic agents and as potential new drugs to treat Chagas disease and leishmaniasis.publishersversionpublishe

Structural basis of nSH2 regulation and lipid binding in PI3Kα

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

Mimicking damaged DNA with a small molecule inhibitor of human UNG2

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases

Estimation of Free-Energy Differences from Computed Work Distributions: An Application of Jarzynski’s Equality

Equilibrium free-energy differences can be computed from nonequilibrium molecular dynamics (MD) simulations using Jarzynski’s equality (Jarzynski, C. <i>Phys. Rev. Lett.</i> <b>1997</b>,<i> 78</i>, 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. <i>J. Stat. Phys.</i> <b>1998</b>,<i> 90</i>, 1481 and Crooks, G. E. <i>Phys. Rev. E</i> <b>2000</b>,<i> 61</i>, 2361) or Bennett’s acceptance ratio (Bennett, C. H. <i>J. Comput. Phys</i>. <b>1976</b>,<i> 22</i>, 245), which further reduces the bias and variance of the estimates

Asn441 plays a key role in folding and function of the Na+/I− symporter (NIS)

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).Fil: Li, Wenjing. Albert Einstein College of Medicine; Estados UnidosFil: Nicola, Juan Pablo. University of Yale; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amzel, L. Mario. University Johns Hopkins; Estados UnidosFil: Carrasco, Nancy. University of Yale; Estados Unido