Crystal Structure Of Mutant Research Articles

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Rep protein accommodates together dsDNA and ssDNA which enables a loop-back mechanism to plasmid DNA replication initiation.

For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the replication origin. The structural-functional relationship of the nucleoprotein complex involving initiator proteins is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), the analysis of mutant proteins and crystal structures, we defined amino acid residues essential for the interaction between plasmid Rep proteins, TrfA and RepE, and ssDNA. This interaction and Rep binding to dsDNA could not be provided in trans, and both are important for dsDNA melting at DNA unwinding element (DUE). We solved two crystal structures of RepE: one in a complex with ssDNA DUE, and another with both ssDNA DUE and dsDNA containing RepE-specific binding sites (iterons). The amino acid residues involved in interaction with ssDNA are located in the WH1 domain in stand β1, helices α1 and α2 and in the WH2 domain in loops preceding strands β1' and β2' and in these strands. It is on the opposite side compared to RepE dsDNA-recognition interface. Our data provide evidence for a loop-back mechanism through which the plasmid replication initiator molecule accommodates together dsDNA and ssDNA.

Crystal structure of the Thr316Ala mutant of a yeast JAMM deubiquitinase: implication of active-site loop dynamics in catalysis.

AMSH, an endosome-associated deubiquitinase (DUB) with a high specificity for Lys63-linked polyubiquitin chains, plays an important role in endosomal-lysosomal sorting and down-regulation of cell-surface receptors. AMSH belongs to the JAMM family of DUBs that contain two insertion segments, Ins-1 and Ins-2, in the catalytic domain relative to the JAMM core found in the archaebacterial AfJAMM. Structural analyses of the AMSH homologs human AMSH-LP and fission yeast Sst2 reveal a flap-like structure formed by Ins-2 near the active site that appears to open and close during its catalytic cycle. A conserved phenylalanine residue of the flap interacts with a conserved aspartate residue of the Ins-1 β-turn to form a closed `lid' over the active site in the substrate-bound state. Analyses of these two residues (Phe403 and Asp315) in Sst2 showed that their interaction plays an important role in controlling the flexibility of Ins-2. The Lys63-linked diubiquitin substrate-bound form of Sst2 showed that the conserved phenylalanine also interacts with Thr316 of Ins-1, which is substituted by tyrosine in other AMSH orthologs. Although Thr316 makes no direct interaction with the substrate, its mutation to alanine resulted in a significant loss of activity. In order to understand the contribution of Thr316 to catalysis, the crystal structure of this mutant was determined. In spite of the effect of the mutation on catalytic activity, the structure of the Sst2 Thr316Ala mutant did not reveal significant changes in either the overall structure or the active-site arrangement relative to the wild type. The Phe403-Thr316 van der Waals interaction is impaired by the Thr316Ala mutation, abrogating the adoption of the closed active-site conformation required for catalysis. Since van der Waals interactions with phenylalanine are conserved across substrate-bound forms of AMSH-LP and Sst2, these interactions may be critical for loop immobilization and the positioning of the isopeptide bond of Lys63-linked polyubiquitin-chain substrates.

Nano Schiff base and its metal complexes: Synthesis, Characterization tools, biological applications and molecular docking studies Walaa H. Mahmouda,b*, Ahmed M. Refaata, Gehad G. Mohameda, b a. Chemistry Department, Faculty of Science, Cairo University, Giza, 126103, Egypt b. Egypt Nanotechnology Center, Cairo University, 6th October City, Giza, El-Sheikh Zayed, 12588, Egypt

A total of eight new metal complex derivatives of 4,4'-((1Z,1'Z)-(naphthalene-1,8-diylbis(azanylylidene))bis(methanylylidene))dibenzaldehyde, H2L with the metal ions Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) have been successfully prepared in alcoholic medium. The complexes obtained are characterized quantitatively and qualitatively by using micro elemental analysis, conductivity measurements, FT-IR spectroscopy, UV–Vis spectroscopy, mass spectroscopy, 1HNMR, magnetic susceptibility, thermal (TG/DTG) and SEM data to illuminate their structures. The data showed that the complexes had composition of MH2L type. From the spectral study, all the complexes obtained as monomeric structure and the metals center moieties are six-coordinated with an octahedral geometry. 1H NMR spectral data of the ligand (H2L) and its Zn(II) and Cd(II) complexes agreed well with the proposed structures. Thermogravimetric data (TG and DTG) were also studied. SEM analysis confirm the nano-structures of the ligand and some of its complexes. The preliminary in vitro antibacterial and antifungal screening activity revealed that complexes showed moderate activity against tested bacterial strains and slightly higher compared to the ligand (H2L). Anticancer activity of the ligand and its metal complexes were evaluated in human cancer (MCF-7 cells viability) and human normal melanocytes (HFB-4) cell lines. The binding between H2L with receptors of crystal structure of S. aureus (PDB ID: 3Q8U), crystal structure of sialidase NanC of Streptococcus pneumoniae (4YW4), receptors of breast cancer mutant oxidoreductase (PDB ID: 3HB5) and crystal structure of Escherichia coli (PDB ID: 3T88) were expected and set in details using molecular docking.

Molecular and structural analysis of a mechanical transition of helices in the L. donovani coronin coiled-coil domain

Protein-protein interactions of cellular importance are mediated by coiled coils (CCs), the ubiquitous structural motif formed by the association of two or more α-helices in a knobs into holes manner. Coronins, actin-associated multi-functional proteins that possess distinct cytoskeleton-dependent and independent functions, oligomerize through their C-terminal CC domain. The structure of the L. donovani coronin CC domain (LdCoroCC; PDB ID 5CX2) revealed, in addition to a novel topology and architecture, an inherent asymmetry, with one of the helices of the 4-helix bundle axially shifted (~2 turns). The structural analysis identified that steric hindrance by Ile 486, Leu 493 and Met 500 as the cause for this asymmetry. To experimentally validate this hypothesis and to better understand the sequence-structure relationship in CCs, these amino acids have been mutated (I486A, L493A, M500V and the double mutant I486A-L493A) and characterized. Thermal CD studies suggest that the I486A and M500V mutants have comparable Tm values to LdCoroCC, while the other mutants have lower melting temperatures. The mutant crystal structures (I486A, M500V and the double mutant) retain the ‘ade’ core packing as LdcoroCC. While the M500V structure is similar to LdCoroCC, the I486A and the I486A-L493A structures show an asymmetry to symmetry transition. This study reveals crucial role of residues at position ‘a’ in coiled-coil domain play an important role in stabilizing the asymmetry in LdCoroCC, which might be necessary pursue specific biological function(s) inside the Leishmania.

Structural studies of antiviral inhibitor with HIV-1 protease bearing drug resistant substitutions of V32I, I47V and V82I

HIV-1 protease inhibitors are effective in HIV/AIDS therapy, although drug resistance is a severe problem. This study examines the effects of four investigational inhibitors against HIV-1 protease with drug resistant mutations of V32I, I47V and V82I (PRTri) that model the inhibitor-binding site of HIV-2 protease. These inhibitors contain diverse chemical modifications on the darunavir scaffold and form new interactions with wild type protease, however, the measured inhibition constants for PRTri mutant range from 17 to 40 nM or significantly worse than picomolar values reported for wild type enzyme. The X-ray crystal structure of PRTri mutant in complex with inhibitor 1 at 1.5 Å resolution shows minor changes in interactions with inhibitor compared with the corresponding wild type PR complex. Instead, the basic amine at P2 of inhibitor together with mutation V82I induces two alternate conformations for the side chain of Arg8 with new interactions with inhibitor and Leu10. Hence, inhibition is influenced by small coordinated changes in hydrophobic interactions.

Physical Origin of Thermostabilization by a Quadruple Mutation for the Adenosine A2a Receptor in the Active State.

The G protein-coupled receptors (GPCRs) form a large, physiologically important family of membrane proteins and are currently the most attractive targets for drug discovery. We investigate the physical origin of thermostabilization of the adenosine A2a receptor (A2aR) in the active state, which was experimentally achieved by another research group using the four point mutations: L48A, A54L, T65A, and Q89A. The investigation is performed on the basis of our recently developed physics-based free-energy function (FEF), which has been quite successful for the thermodynamics of GPCRs in the inactive state. The experimental condition for solving the wild-type and mutant crystal structures was substantially different from that for comparing their thermostabilities. Therefore, all-atom molecular dynamics simulations are necessitated, which also allows us to account for the structural fluctuations of the membrane protein. We show that the quadruple mutation leads to the enlargement of the solvent-entropy gain upon protein folding. The solvent is formed by hydrocarbon groups constituting nonpolar chains within the lipid bilayer, and the entropy is relevant to the thermal motion of the hydrocarbon groups. From an energetic point of view (e.g., in terms of protein intramolecular hydrogen bonds), the mutation confers no improvement upon the structural stability of A2aR. The reliability of our FEF and the crucial importance of the solvent-entropy effect have thus been demonstrated for a GPCR in the active state. We are now ready to identify thermostabilizing mutations of GPCRs not only in the inactive state but also in the active one.

Predicting allosteric mutants that increase activity of a major antibiotic resistance enzyme.

The CTX-M family of beta lactamases mediate broad-spectrum antibiotic resistance and are present in the majority of drug-resistant Gram-negative bacterial infections worldwide. Allosteric mutations that increase catalytic rates of these drug resistance enzymes have been identified in clinical isolates but are challenging to predict prospectively. We have used molecular dynamics simulations to predict allosteric mutants increasing CTX-M9 drug resistance, experimentally testing top mutants using multiple antibiotics. Purified enzymes show an increase in catalytic rate and efficiency, while mutant crystal structures show no detectable changes from wild-type CTX-M9. We hypothesize that increased drug resistance results from changes in the conformational ensemble of an acyl intermediate in hydrolysis. Machine-learning analyses on the three top mutants identify changes to the binding-pocket conformational ensemble by which these allosteric mutations transmit their effect. These findings show how molecular simulation can predict how allosteric mutations alter active-site conformational equilibria to increase catalytic rates and thus resistance against common clinically used antibiotics.

Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase.

Proton transfer reactions are ubiquitous in enzymes and utilize active site residues as general acids and bases. Crystal structures and site-directed mutagenesis are routinely used to identify these residues, but assessment of their catalytic contribution remains a major challenge. In principle, effective molarity measurements, in which exogenous acids/bases rescue the reaction in mutants lacking these residues, can estimate these catalytic contributions. However, these exogenous moieties can be restricted in reactivity by steric hindrance or enhanced by binding interactions with nearby residues, thereby resulting in over- or underestimation of the catalytic contribution, respectively. With these challenges in mind, we investigated the catalytic contribution of an aspartate general base in ketosteroid isomerase (KSI) by exogenous rescue. In addition to removing the general base, we systematically mutated nearby residues and probed each mutant with a series of carboxylate bases of similar pKa but varying size. Our results underscore the need for extensive and multifaceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ∼5 × 10(4) M for KSI from Comamonas testosteroni (tKSI) and ∼10(3) M for KSI from Pseudomonas putida (pKSI). An X-ray crystal structure of a tKSI general base mutant showed no additional structural rearrangements, and double mutant cycles revealed similar contributions from an oxyanion hole mutation in the wild-type and base-rescued reactions, providing no indication of mutational effects extending beyond the general base site. Thus, the high effective molarities suggest a large catalytic contribution associated with the general base. A significant portion of this effect presumably arises from positioning of the base, but its large magnitude suggests the involvement of additional catalytic mechanisms as well.

Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase.

Aristolochene synthase (ATAS) is a high-fidelity terpenoid cyclase that converts farnesyl diphosphate exclusively into the bicyclic hydrocarbon aristolochene. Previously determined crystal structures of ATAS complexes revealed trapped active site water molecules that could potentially interact with catalytic intermediates: water "w" hydrogen bonds with S303 and N299, water molecules "w1" and "w2" hydrogen bond with Q151, and a fourth water molecule coordinates to the Mg(2+)C ion. There is no obvious role for water in the ATAS mechanism because the enzyme exclusively generates a hydrocarbon product. Thus, these water molecules are tightly controlled so that they cannot react with carbocation intermediates. Steady-state kinetics and product distribution analyses of eight ATAS mutants designed to perturb interactions with active site water molecules (S303A, S303H, S303D, N299A, N299L, N299A/S303A, Q151H, and Q151E) indicate relatively modest effects on catalysis but significant effects on sesquiterpene product distributions. X-ray crystal structures of S303A, N299A, N299A/S303A, and Q151H mutants reveal minimal perturbation of active site solvent structure. Seven of the eight mutants generate farnesol and nerolidol, possibly resulting from addition of the Mg(2+)C-bound water molecule to the initially formed farnesyl cation, but no products are generated that would suggest enhanced reactivity of other active site water molecules. However, intermediate germacrene A tends to accumulate in these mutants. Thus, apart from the possible reactivity of Mg(2+)C-bound water, active site water molecules in ATAS are not directly involved in the chemistry of catalysis but instead contribute to the template that governs the conformation of the flexible substrate and carbocation intermediates.

Key Players in I-DmoI Endonuclease Catalysis Revealed from Structure and Dynamics.

Homing endonucleases, such as I-DmoI, specifically recognize and cleave long DNA target sequences (∼20 bp) and are potentially powerful tools for genome manipulation. However, inefficient and off-target DNA cleavage seriously limits specific editing in complex genomes. One approach to overcome these limitations is to unambiguously identify the key structural players involved in catalysis. Here, we report the E117A I-DmoI mutant crystal structure at 2.2 Å resolution that, together with the wt and Q42A/K120M constructs, is combined with computational approaches to shed light on protein cleavage activity. The cleavage mechanism was related both to key structural effects, such as the position of water molecules and ions participating in the cleavage reaction, and to dynamical effects related to protein behavior. In particular, we found that the protein perturbation pattern significantly changes between cleaved and noncleaved DNA strands when the ions and water molecules are correctly positioned for the nucleophilic attack that initiates the cleavage reaction, in line with experimental enzymatic activity. The proposed approach paves the way for an effective, general, and reliable procedure to analyze the enzymatic activity of endonucleases from a very limited data set, i.e., structure and dynamics.

Creation of a S1P Lyase bacterial surrogate for structure-based drug design

S1P Lyase (SPL) has been described as a drug target in the treatment of autoimmune diseases. It plays an important role in maintaining intracellular levels of S1P thereby affecting T cell egress from lymphoid tissues. Several groups have already published approaches to inhibit S1P Lyase with small molecules, which in turn increase endogenous S1P concentrations resulting in immunosuppression. The use of structural biology has previously aided SPL inhibitor design. Novel construct design is at times necessary to provide a reagent for protein crystallography. Here we present a chimeric bacterial protein scaffold used for protein X-ray structures in the presence of early small molecule inhibitors. Mutations were introduced to the bacterial SPL from Symbiobacterium thermophilum which mimic the human enzyme. As a result, two mutant StSPL crystal structures resolved to 2.8Å and 2.2Å resolutions were solved and provide initial structural hypotheses for an isoxazole chemical series, whose optimization is discussed in the accompanying paper.

Crystal Structure Analysis of Wild Type and Fast Hydrolyzing Mutant of EhRabX3, a Tandem Ras Superfamily GTPase from Entamoeba histolytica

The enteric protozoan parasite, Entamoeba histolytica, is the causative agent of amoebic dysentery, liver abscess and colitis in human. Vesicular trafficking plays a key role in the survival and virulence of the protozoan and is regulated by various Rab GTPases. EhRabX3 is a catalytically inefficient amoebic Rab protein, which is unique among the eukaryotic Ras superfamily by virtue of its tandem domain organization. Here, we report the crystal structures of GDP-bound fast hydrolyzing mutant (V71A/K73Q) and GTP-bound wild type EhRabX3 at 3.1 and 2.8Å resolutions, respectively. Though both G-domains possess “phosphate binding loop containing nucleoside triphosphate hydrolases fold”, only the N-terminal domain binds to guanine nucleotide. The relative orientation of the N-terminal domain and C-terminal domain is stabilized by numerous inter-domain interactions. Compared to other Ras superfamily members, both the GTPase domains displayed large deviation in switch II perhaps due to non-conservative substitutions in this region. As a result, entire switch II is restructured and moved away from the nucleotide binding pocket, providing a rationale for the diminished GTPase activity of EhRabX3. The N-terminal GTPase domain possesses unusually large number of cysteine residues. X-ray crystal structure of the fast hydrolyzing mutant of EhRabX3 revealed that C39 and C163 formed an intra-molecular disulfide bond. Subsequent mutational and biochemical studies suggest that C39 and C163 are critical for maintaining the structural integrity and function of EhRabX3. Structure-guided functional investigation of cysteine mutants could provide the physiological implications of the disulfide bond and could allow us to design potential inhibitors for the better treatment of intestinal amebiasis.

Abstract P3-05-07: Determining the role of somatic ERα mutations in acquired hormone (or SERM) resistance

Abstract The estrogen receptor alpha (ERα) is a member of the nuclear hormone receptor (NHR) family and is critical for the etiology and treatment of breast cancer. Approximately 70% of breast cancers express ERα and many of these are sensitive to anti-estrogen therapies. Selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, are approved to treat or reduce the risk of ER-dependent breast cancers. SERMs act by competitively binding to the ERα ligand-binding domain (LBD). Unfortunately, metastatic breast tumors recur in approximately half of patients and become SERM resistant while remaining ER-positive in many cases. Recently, conserved somatic mutations in the ERα LBD were identified in patients who received SERM/aromatase inhibitor (AI)/selective estrogen receptor disruptor (SERD) therapy for an average of five years. Because these mutations were observed in approximately 25% of tumors and the most frequent mutations (Y537S or D538G) were located in or just prior to helix 12 (H12), the molecular switch that controls AF-2 activity, they represent a possible mechanism for acquired SERM insensitivity for a significant population of patients. Further studies revealed that these mutations conferred hormone-independent ERα activity and that the inhibitory efficacy of currently approved SERMs was reduced. Our goal is to understand the effects of these mutations on the structure and function of ERα in these tumors to guide the generation of novel compounds which bypass the effects of these somatic mutations. Here, we employ x-ray crystallography, molecular dynamics simulations, biochemical assays in addition to breast cancer cell proliferation assays to dissect the role of somatic mutation in acquired hormone/SERM resistance. X-ray crystal structures of the ERα LBD D538G mutant in the unliganded (apo), agonist and SERM-bound states, combined with molecular dynamics simulations, reveal a stabilized loop between H11 and H12 that allows the receptor to preferentially adopt an agonist conformation versus an antagonist conformation. The biochemical and breast cancer cell proliferation assays reveal structural insights that may explain mutant ERα function within the tumor. Further, we use these methods to explore the utility of next generation SERMs and SERDs to inhibit these mutant ERs as well as to guide the synthesis of additional novel compounds. Importantly, our work is expected to yield more potent and effective SERMs/SERDs that overcome the impact of acquired activating mutations and result in improved patient survival. Citation Format: Sean W Fanning, Christopher Mayne, Weiyi Toy, Yang Shen, Abhishek Sharma, Srinivas Panchamukhi, Jason Nowak, Kendall W Nettles, Sarat Chandarlapaty, John A Katzenellenbogen, Geoffrey L Greene. Determining the role of somatic ERα mutations in acquired hormone (or SERM) resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-07.

Molecular basis of Streptococcus mutans sortase A inhibition by the flavonoid natural product trans-chalcone.

Sortase A (SrtA) from Gram positive pathogens is an attractive target for inhibitors due to its role in the attachment of surface proteins to the cell wall. We found that the plant natural product trans-chalcone inhibits Streptococcus mutans SrtA in vitro and also inhibited S. mutans biofilm formation. Mass spectrometry revealed that the trans-chalcone forms a Michael addition adduct with the active site cysteine. The X-ray crystal structure of the SrtA H139A mutant provided new insights into substrate recognition by the sortase family. Our study suggests that chalcone flavonoids have potential as sortase-specific oral biofilm inhibitors.

Potential activity of some natural products compounds as Neuraminidase inhibitors based on molecular docking simulation and in vitro test

Neuraminidase (NA) plays an important role in replication and the release of a new avian influenza virion. In consequence, NA has been considered as a valid target in drug design against influenza virus. The aim of this study was to identify the new neuraminidase inhibitors using molecular docking simulation based on virtual screening from natural products compounds. The X-ray crystal structure of neuraminidase type N1 (PDB id: 3B7E) and N1 mutant (PDB id: 3NNS) using Autodock 4.2 program. Zanamivir was used as the control ligand and docked against neuraminidase type N1, further plotted between log IC50 value experiments of sialic acid derivatives compound versus log of Ki value of molecular docking. Molecular docking simulation was performed on 113 herb compounds along with zanamivir and oseltamivir as the control ligands. The result showed that the best interaction against of neuraminidase N1 and N1 mutant from herbs compound is katsumadain-A withfree energy value -7,54 kcal/mol and -7,46 kkal/mol, respectively. Katsumadain-Aformed hydrogen bond with amino acid residue Arg118 and Arg371 on neuraminidase and neuraminidase N1 mutant Katsumadain-A was also connected with Arg118 through hydrogen binding interaction. This in silico results also was proved by in vitro MUNANA assay.

Tyrosine 110 plays a critical role in regulating the allosteric inhibition of Campylobacter jejuni dihydrodipicolinate synthase by lysine.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-β-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

Substrate-induced DNA Polymerase β Activation

DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.

Structural and mutation studies of two DNA demethylation related glycosylases: MBD4 and TDG.

Two mammalian DNA glycosylases, methyl-CpG binding domain protein 4 (MBD4) and thymine DNA glycosylase (TDG), are involved in active DNA demethylation via the base excision repair pathway. Both MBD4 and TDG excise the mismatch base from G:X, where X is uracil, thymine, and 5-hydroxymethyluracil (5hmU). In addition, TDG excises 5mC oxidized bases i.e. when X is 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) not 5-hydroxymethylcytosine (5hmC). A MBD4 inactive mutant and substrate crystal structure clearly explains how MBD4 glycosylase discriminates substrates: 5mC are not able to be directly excised, but a deamination process from 5mC to thymine is required. On the other hand, TDG is much more complicated; in this instance, crystal structures show that TDG recognizes G:X mismatch DNA containing DNA and G:5caC containing DNA from the minor groove of DNA, which suggested that TDG might recognize 5mC oxidized product 5caC like mismatch DNA. In mutation studies, a N157D mutation results in a more 5caC specific glycosylase, and a N191A mutation inhibits 5caC activity while that when X=5fC or T remains. Here I revisit the recent MBD4 glycos ylase domain co-crystal structures with DNA, as well as TDG glycosylase domain co-crystal structures with DNA in conjunction with its mutation studies.

In silico Analysis of 2, 4-Substituted Heterocycles and Glutamic Acid Containing Antifolates as Inhibitors of Malarial (Plasmodium falciparum) Protein, Dihydrofolate Reductase-Thymidylate Synthase

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) function is effectively inhibited by antifolates. The binding affinity of antifolates to PfDHFR-TS is reduced due to mutations in its active site. In the present study, 33 analogues of Methotrexate (MTX), Trimetrexate (TMX), Raltitrexed (RTX) and Pemetrexed (PTX) were designed and evaluated for interaction with PfDHFR-TS by in silico methods. Analyses of drug candidates were performed by generating their docking complexes with quadruple mutant crystal structure of PfDHFR-TS using Molecular Operating Environment (MOE). Initially eight top scoring complexes and then finally two (MTX04 and PTX03) were found suitable for further optimization based on interaction pattern with active site amino acids. Analyses of structural characteristics, binding energy calculations and interaction patterns of MTX04 and PTX03 with DHFR and TS domains respectively as best drug candidates. The comparative docking studies of these two compounds with human proteins provided a strong evidence of selectivity for MTX04 as effective antimalarial drug candidate. It is considered that the drug will inhibit the activity of folate pathway and it will be effective source to control malaria.