ChemInform Abstract: ADDITIONAL CONFORMATIONAL DATA FOR THE MAPPING OF THE PROGESTIN BINDING SITE: CRYSTAL STRUCTURES OF 21-(PHENYLSELENO)PROGESTERONE AND 17α-(PHENYLSELENO)PROGESTERONE

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

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Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082072 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1793-C1793

Author(s):

Paul Rowland ◽

Onkar SINGH ◽

Leila Ross ◽

Francisco Gamo ◽

Maria Lafuente-Monasterio ◽

...

Keyword(s):

Drug Resistance ◽

Binding Site ◽

Crystal Structures ◽

Point Mutations ◽

Crystal Form ◽

Drug Binding ◽

Binding Modes ◽

Resistance Mutations ◽

Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

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Calixarene-mediated assembly of a small antifungal protein

IUCrJ ◽

10.1107/s2052252519000411 ◽

2019 ◽

Vol 6 (2) ◽

pp. 238-247 ◽

Cited By ~ 23

Author(s):

Jimi M. Alex ◽

Martin L. Rennie ◽

Sylvain Engilberge ◽

Gábor Lehoczki ◽

Hajdu Dorottya ◽

...

Keyword(s):

Crystal Structure ◽

Antifungal Activity ◽

High Resolution ◽

Complex Formation ◽

Binding Site ◽

Crystal Structures ◽

Protein Assembly ◽

Bidentate Ligand ◽

Antifungal Protein ◽

Protein Recognition

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[n]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx4, sclx6 or sclx8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx8, yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.

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Helicase mechanisms and the coupling of helicases within macromolecular machines Part I: Structures and properties of isolated helicases

Quarterly Reviews of Biophysics ◽

10.1017/s0033583502003852 ◽

2002 ◽

Vol 35 (4) ◽

pp. 431-478 ◽

Cited By ~ 111

Author(s):

Emmanuelle Delagoutte ◽

Peter H. von Hippel

Keyword(s):

Crystal Structure ◽

Nucleic Acid ◽

Nucleic Acids ◽

Binding Site ◽

Crystal Structures ◽

Base Pair ◽

Biochemical Properties ◽

Functional Coupling ◽

Hexameric Helicases ◽

Base Pair Opening

1. Mechanisms of nucleic acid (NA) unwinding by helicases 4322. Helicases may take advantage of ‘breathing’ fluctuations in dsNAs 4342.1 Stability and dynamics of dsNAs 4342.2 dsNAs ‘breathe’ in isolation 4352.3 Thermodynamics of terminal base pairs of dsNA 4382.4 Thermal fluctuations may be responsible for sequential base-pair opening at replication forks 4392.5 Helicases may capture single base-pair opening events sequentially 4403. Biochemical properties of helicases 4433.1 Binding of NAs 4433.2 Binding and hydrolysis of NTP 4453.3 Coordination between NA binding and NTP binding and hydrolysis activities 4464. Helicase structures and mechanistic consequences 4474.1 Amino-acid sequence analysis reveals conserved motifs that constitute the NTP-binding pocket and a portion of the NA-binding site 4474.2 Organization of hepatitis virus C NS3 RNA helicase 4494.2.1 Biochemical properties of HCV NS3 4494.2.2 Crystal structures of HCV NS3 helicase 4504.2.2.1 The apoprotein 4504.2.2.2 The protein–dU8 complex 4504.2.3 A possible unwinding mechanism 4524.2.4 What is the functional oligomeric state of HCV NS3? 4524.3 Organization of the PcrA helicase 4534.3.1 The apoenzyme and ADP–PcrA complex 4544.3.2 The protein–DNA–sulfate complex 4564.3.3 The PcrA–DNA–ADPNP complex 4564.3.4 A closer look at the NTP-binding site in the crystal structure of PcrA–ADPNP–DNA 4574.3.5 Communication between domains A and B 4574.3.6 How might ssDNA stimulate the ATPase activity of PcrA? 4574.3.7 A possible helicase translocation mechanism 4584.3.8 A possible unwinding mechanism 4584.4 Organization of the Rep helicase 4594.4.1 Biochemical properties 4594.4.2 Crystal structure of Rep bound to ssDNA 4624.5 Organization of the RecG helicase 4624.6 Hexameric helicases 4664.6.1 Insights from crystal structures of hexameric helicases 4674.6.2 Possible translocation and unwinding mechanisms 4685. Conclusions 4696. Acknowledgments 4727. References 472Helicases are proteins that harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of double-stranded nucleic acids. These enzymes have been much studied in isolation, and here we review what is known about the mechanisms of the unwinding process. We begin by considering the thermally driven ‘breathing’ of double-stranded nucleic acids by themselves, in order to ask whether helicases might take advantage of some of these breathing modes. We next provide a brief summary of helicase mechanisms that have been elucidated by biochemical, thermodynamic, and kinetic studies, and then review in detail recent structural studies of helicases in isolation, in order to correlate structural findings with biophysical and biochemical results. We conclude that there are certainly common mechanistic themes for helicase function, but that different helicases have devised solutions to the nucleic acid unwinding problem that differ in structural detail. In Part II of this review (to be published in the next issue of this journal) we consider how these mechanisms are further modified to reflect the functional coupling of these proteins into macromolecular machines, and discuss the role of helicases in several central biological processes to illustrate how this coupling actually works in the various processes of gene expression.

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Structural studies of ligands for the glycine binding site of the excitatory amino acid receptor complex: 2. 6-bromo-, 6-methyl-, and 6-styryl-1,8-ethano-4-hydro-2,3-quinoxalinedione

Canadian Journal of Chemistry ◽

10.1139/v94-259 ◽

1994 ◽

Vol 72 (10) ◽

pp. 2028-2036 ◽

Cited By ~ 3

Author(s):

Maciej Kubicki ◽

Teresa W. Kindopp ◽

Mario V. Capparelli ◽

Penelope W. Codding

Keyword(s):

Hydrogen Bond ◽

Binding Site ◽

Crystal Structures ◽

Excitatory Amino Acid ◽

Glycine Receptor ◽

Three Dimensional ◽

Unit Cell ◽

Geometric Constraints ◽

Cell Parameters ◽

Π Stacking

The crystal structures of three tricyclic quinoxalinedione derivatives, 6-bromo-1,8-ethano-4-hydro-2,3-quinoxalinedione (1), 6-methyl-1,8-ethano-4-hydro-2,3-quinoxalinedione hydrate (2), and 6-styryl-1,8-ethano-4-hydro-2,3-quinoxalinedione (3), are reported. For 1 and 2, the space groups are P21/n with the unit cell parameters for 1: a = 7.4003(5) Å, b = 8.5799(5) Å, c = 14.3127(9) Å, β = 90.639(6)°, and for 2: a = 7.0590(2) Å, b = 10.7483(3) Å, c = 13.9509(7) Å, β = 103.290(3)°. For 3, the space group is P21/c, with a = 19.3683(10) Å, b = 8.0962(16) Å, c = 19.5801(16) Å, β = 114.028(6)°. Compound 3 crystallizes with two molecules in the asymmetric part of the unit cell; in one of them the styryl group is disordered. The geometries of the 1,8-ethano-4-hydro-2,3-quinoxalinedione fragments are similar in all observations, with the differences mainly caused by the different nature of the substituents in the 6-position. Hydrogen bonds connect the molecules into three-dimensional networks. Head-to-tail π-stacking between molecules connected by a center of symmetry determines the packing modes in 1 and 2 but there is no π-stacking in the crystal structure of 3. The crystal structures of the three quinoxaline derivative ligands for the glycine receptor suggest a mode of recognition that involves an [Formula: see text]receptor hydrogen bond, a three-centre hydrogen bond to the neighboring carbonyl groups on the ligand, and π-stacking between ligand and receptor. This mode is consistent with the geometric constraints of the current binding site model but places greater emphasis on hydrogen-bond interactions.

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Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.186.16.5303-5310.2004 ◽

2004 ◽

Vol 186 (16) ◽

pp. 5303-5310 ◽

Cited By ~ 21

Author(s):

Annette Sauter ◽

Volkmar Braun

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Outer Membrane ◽

Crystal Structures ◽

Transcription Initiation ◽

Ferric Citrate ◽

Globular Domain ◽

Salt Bridges ◽

The Individual ◽

K 12

ABSTRACT The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the β-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the β-barrel and to contribute to the fixation of the globular domain inside the β-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.

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