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Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross-linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (RcopLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In RcopLS20, this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that RcopLS20 can form octamers. This domain was named TetDloop and its occurrence was identified in other Bacillus species. The TetDloop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetDloop fold has evolved through divergent evolution and that the TetDloop originates from a common ancestor predating the occurrence of multicellular life.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix–turn–helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU), to D.R.B., BFU2017-86378-P to F.dlC.] and by the Spanish Ministryof Science (MCI/AEI/FEDER,UE) [PGC2018-093885-BI00 and PID2021-122164NB-I00 to G.M., PID2020- 117028GB-I00 to D.R.B. and PID2019-110216GB-I00 to R. F-L.].

15 p.-6 fig.1 graph. abst., DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., Ministry of Science and Innovation of Spain [BFU2017-83720-P, PID2020-120141GB-100 to M.C., 2022AEP028 (RT12018-097114-B-I00), BFU2010-19597 to G.dS.]; RYC fellowship [RYC-2011-09071 to C.M.]; CSIC [2020AEP116 to M.C.]. Funding for open access charge: CSIC.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1–3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems. The minimal regulatory logic and the evolutionary history of the auxin signalling pathway are explored by studying the much simpler auxin network in Marchantia with a combination of genetic, domain shuffling and biochemical strategies.

Significance The plant hormone auxin controls many aspects of growth and development. It does so by changing the activity of AUXIN RESPONSE FACTOR (ARF) proteins that recognize specific DNA elements in plant genes and switch gene expression on or off. A major question in plant biology is how these ARF proteins bind unique DNA sequences and thus select which genes are regulated by auxin. Here, the authors systematically study the DNA architecture required for high-affinity binding of ARF proteins, and for auxin-dependent gene regulation in the plant Arabidopsis thaliana. This work shows that repeats of ARF recognition sites are critical for function, and reveals the molecular basis for the distinct activities of different repeat structures., The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum- signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed., Ministry of Science and Innovation of the Spanish Government (PID2019 108778GB C21 (AEI/FEDER, EU)) to WM, (BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU)), to DB, (PID2019-104544GBI00 (AEI/FEDER/EU)) to CA, (PID2019-109320GB-100/AEI/10.13039/501100011033) to SA, (PGC2018-094143-B-I00, (AEI/FEDER, EU)) to FR, and (FIS2016-78313-P) to SA. The Ministry of Science and Innovation of the Spanish Government also funded the Centro Nacional de Biotecnología (CNB) by means of the “Severo Ochoa” Center of Excellence grant SEV 2017-0712. A Wellcome Investigator grant (209500) to Jeff Errington supported LW. Institutional grants from the ‘Fundación Ramón Areces’ and ‘Banco de Santander’ supported

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1–3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development2–4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. Expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned, or prevented. This fundamental design likely represents the ancestral system, and formed the basis for inflated, complex systems.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria., The Institute for Research in Biomedicine Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish Ministry of Economy and Competitiveness (MINECO). The Molecular Biology Institute, Barcelona Spanish Research Council’s Structural Biology Unit is the recipient of a Maria de Maeztu Award of Excellence from MINECO. This work was supported by grants from MINECO (BFU2008-02372/ BMC, CSD-2006-23, BFU2011-22588, CSD-2008/00013, BIO2013-49148-C2-2-R, BFU2014-53550-P, BIO2015-69085-REDC, and BIO2015-64802), the Generalitat de Catalunya (2014-SGR134 and 2014-SGR1530), and the European Commission [project no. 260644, ERC_SimDNA, and the H2020 program (MuG and BioExcel Projects)]. R.P. and H.G. received a La Caixa/Institute for Research in Biomedicine Barcelona International PhD and Juan de La Cierva fellowship, respectively. M.O. is a Catalan Institution for Research and Advanced Studies Academia researcher.

The crystal structure of the Δ,Δ enantiomer of the binuclear >light-switch> ruthenium complex [μ-(11,11′-bidppz)(1,10- phenanthroline)4 Ru2]4+ bound to the oligonucleotide d(CGTACG) shows that one dppz moiety of the dumbbell-like compound inserts into the DNA stack through the extrusion of an AT base pair. The second dppz moiety recruits a neighboring DNA molecule, and the complex thus cross-links two adjacent duplexes by bridging their major grooves. Pinned together by molecular rivets: The crystal structure of the complex between double-stranded DNA and the binuclear complex [μ-(11,11′-bidppz)(1,10- phenanthroline)4 Ru2]4+ (1, shown in blue; bidppz=bis(dipyridophenazine)), showed that one half of the bidppz ligand inserts into a DNA duplex, and the other half stacks onto the blunt end of a nearby DNA duplex. Two molecules of 1 with the same binding mode effectively cross-link two DNA duplexes. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This research was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, and BFU2011-22588), the Generalitat de Catalunya (grant SGR2009-1309), and the European Commission (FP7 Cooperation Project SILVER, GA No. 260644)

Boer, Roeland et al., Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response. © 2014 Elsevier Inc., This work was supported by the Netherlands Organization for Scientific Research (NWO; ECHO grant 711.011.002 to D.W.), the Spanish Ministry of Science and Innovation (grant BFU2011-22588 to M.C.), the Generalitat de Catalunya (grant SGR2009-1309 to M.C.), and the European Commission (FP7 Cooperation Project SILVER - GA 260644 to M.C.)

SuperStar is an empirical method for identifying interaction sites in proteins, based entirely on the experimental information about non-bonded interactions, present in the IsoStar database. The interaction information in IsoStar is contained in scatterplots, which show the distribution of a chosen probe around structure fragments. SuperStar breaks a template molecule (e.g. a protein binding site) into structural fragments which correspond to those in the scatterplots. The scatterplots are then superimposed on the corresponding parts of the template and converted into a composite propensity map. The original version of SuperStar was based entirely on scatterplots from the CSD. Here, scatterplots based on protein-ligand interactions are implemented in SuperStar, and validated on a test set of 122 X-ray structures of protein-ligand complexes. In this validation, propensity maps are compared with the experimentally observed positions of ligand atoms of comparable types. Although non-bonded interaction geometries in small molecule structures are similar to those found in protein-ligand complexes, their relative frequencies of occurrence are different. Polar interactions are more common in the first class of structures, while interactions between hydrophobic groups are more common in protein crystals. In general, PDB and CSD-based SuperStar maps appear equally successful in the prediction of protein-ligand interactions. PDB-based maps are more suitable to identify hydrophobic pockets, and inherently take into account the experimental uncertainties of protein atomic positions. If the protonation state of a histidine, aspartate or glutamate protein side-chain is known, specific CSD-based maps for that protonation state are preferred over PDB-based maps which represent an ensemble of protonation states.

Heats of formation of five series of diene sterol isomers were calculated and compared with synthetic yields of acid-catalysed isomerisation reactions starting from Δ5,7 isomers. Calculations were based on molecular mechanics, using the MM3 program package. For each of the five Δ5,7 starting compounds, three possible reaction paths were considered, in which heats of formation were calculated for theoretically possible intermediate double bond isomers. Similar results are found for all five series. The starting compounds are found to have the unfavourable heats of formation compared to all other isomers considered within one series. In general, isomerisation reactions of diene sterols ultimately yield spiro compounds when allowed to proceed for a sufficient amount of time. These compounds are found to have the lowest heats of formation in each series. However, they were not formed in the reactions considered in this paper, because the reactions were stopped after the desired isomer was formed in excess, before the spiro compounds could occur. Most compounds identified as products in the syntheses have a favourable heat of formation compared to the isomers preceding the (most stable) spiro compounds. However, when the reactions were carried out at low temperature, isomers with less favourable heats of formation could be trapped. We show that calculated heats of formation correspond well with synthetic yields and we suggest they can be a useful tool in planning syntheses.

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N-and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself. © 2013, American Society for Microbiology. [Journal], This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, and BFU2011-22588 to M.C. and grants CSD2008-00013, INTERMODS, and BFU2010-19597 to M.E.), the Generalitat de Catalunya (grant SGR2009-1309 to M.C.), the “La Caixa”/IRB Barcelona International PhD Programme Fellowship (IRB Barcelona call 01/09/FLC to R.P.), and the European Commission (FP7 Cooperation Project SILVER-GA no. 260644 to M.C.)

The crystal structures of five follicular fluid meiosis-activating sterol-related Δ8,14-sterol compounds are presented. These are 4,4-di­methyl-23-phenyl-24-nor-5α-chola-8,14-dien-3β-ol, C31H44O, 4,4-di­methyl-22-phenyl-23,24-dinor-5α-chola-8,14-dien-3β-ol, C30H42O, (20R)-4,4-di­methyl-22-oxa-5α,20-chol­es­ta-8,14,24-trien-3β-ol, C28H44O2, 4,4-di­methyl-23-phenyl-22-oxa-24-nor-5α-chola-8,14-dien-3β-ol–water (4/1), 4C30H42O2·H2O, and 4,4-di­methyl-5α-cholesta-8,14-dien-3-one, C29H46O. Two of the derivatives are inactive and three are active as agonists. Preliminary structure–activity relationship studies showed that the positions of the double bonds in the skeleton and the structures of the side chains are important determinants for activity. The conformations of the skeletons were compared with double-bond isomers retrieved from the Cambridge Structural Database [Allen & Kennard (1993). Chem. Des. Autom. News, 8, 1, 31–37]; no significant differences were found. Thus, conformational changes induced by the double bonds are not discriminative with respect to the activity of the compounds. Comparisons of the side-chain conformations of active and inactive structures revealed that the crystal structures were not conclusive as far as correlation of conformation and activity of the side chains were concerned.

This research was supported by the EU Marie Curie Research Training Network (HPRN-CT-2002-00175) and by grants from the Spanish Ministerio de Ciencia y Innovación (BFU2008-02372, BFU2005-24122-E, BFU2005-24123-E, and CSD2006-00023 to M.C., BIO2006-14139 to I.U.), the Generalitat de Catalunya (grant 2009-SGR-1309 to M.C.), the Fundació La Marató de TV3 (grant 052810 to M.C.), and the EU projects SPINE2-Complexes and 3D-Repertoire. The research was conducted in the context of the EU COST Action D39. Synchrotron data collection was supported by the ESRF (ID29 and ID14-4) and the EU.

In this paper, we review recent DNA-binding agents that are expected to influence the field of DNA-targeting. We restrict ourselves to binders for which the three-dimensional structure in complex with DNA or RNA has been determined by X-ray crystallography or NMR. Furthermore, we primarily focus on unprecedented ways of targeting peculiar DNA structures, such as junctions, quadruplexes, and duplex DNAs different from the B-form. Classical binding modes of small molecular weight compounds to DNA, i.e. groove binding, intercalation and covalent addition are discussed in those cases where the structures represent a novelty. In addition, we review 3D structures of triple-stranded DNA, of the so-called Peptide Nucleic Acids (PNAs), which are oligonucleotide bases linked by a polypeptide backbone, and of aptamers, which are DNA or RNA receptors that are designed combinatorially. A discussion on perspectives in the field of DNA-targeting and on sequence recognition is also provided. © 2009 The Royal Society of Chemistry.

Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map. publishersversion published

The crystal structures of five follicular fluid meiosis-activating sterol-related Delta(8,14)-sterol compounds are presented. These are 4,4-dimethyl-23-phenyl-24-nor-5alpha-chola-8,14-dien-3beta-ol, C(31)H(44)O, 4,4-dimethyl-22-phenyl-23,24-dinor-5alpha-chola-8,14-dien-3beta-ol, C(30)H(42)O, (20R)-4,4-dimethyl-22-oxa-5alpha,20-cholesta-8,14,24-trien-3beta-ol, C(28)H(44)O(2), 4,4-dimethyl-23-phenyl-22-oxa-24-nor-5alpha-chola-8,14-dien-3beta-ol-water (4/1), 4C(30)H(42)O(2).H(2)O, and 4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one, C(29)H(46)O. Two of the derivatives are inactive and three are active as agonists. Preliminary structure-activity relationship studies showed that the positions of the double bonds in the skeleton and the structures of the side chains are important determinants for activity. The conformations of the skeletons were compared with double-bond isomers retrieved from the Cambridge Structural Database [AllenKennard (1993). Chem. Des. Autom. News, 8, 1, 31-37]; no significant differences were found. Thus, conformational changes induced by the double bonds are not discriminative with respect to the activity of the compounds. Comparisons of the side-chain conformations of active and inactive structures revealed that the crystal structures were not conclusive as far as correlation of conformation and activity of the side chains were concerned.