- Email: [email protected]
Structural biology
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Structural biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology. Current Opinion in Structural Biology 2000, 10:129–134 Contents (chosen by) 129 Protein–nucleic acid interactions (Convery et al.) 129 Folding and binding (Kuhlman et al.) 130 Macromolecular assemblages (Engel and Heymann) 131 Theory and simulation (Melo) 131 Nucleic acids (Parkinson) 132 Sequences and topology (Russell) 132 Lipids (Newman) 132 Carbohydrates and glycoconjugates (Flitsch and Lowden) 133 Biophysical methods (Matthews) 133 Proteins (Kleywegt) 133 Catalysis and regulation (Stewart)
• ••
of special interest of outstanding interest
Protein–nucleic acid interactions Selected by Máire Convery, Caitríona Dennis and Siân Rowsell* University of Leeds, Leeds, UK *AstraZeneca, Macclesfield, UK
Structural basis of preinitiation complex assembly on human Pol II promoters. Tsai FTF, Sigler PB: EMBO J 2000, 19:25-36. •• Significance: The first structural evidence that the Pol II preinitiation complex has directionality conferred by the binding of transcription initiation factor TFIIB to a sequence flanking the TATA-box. Findings: The structure described consists of a ternary complex between the cores of the human TATA-box-binding protein and TFIIB, and a DNA sequence. The structure is unique because of the length of the DNA promoter sequence, which allows the TFIIB core to make more extensive interactions with the DNA. TFIIB differentiates between the major groove upstream and the minor grove immediately downstream of the bound TATA-box sequence, thus conferring polarity on the complex. DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination. Mol CD, Izumi T, Mitra S, Tainer JA: Nature 2000, 403:451-456. • Significance: Three structures that reveal a structure-based catalytic mechanism for phosphate bond cleavage. Findings: Upon binding, the protein engulfs the DNA with a preformed, positively rigid surface. The DNA is strongly kinked and has the abasic site flipped out. There is an unusual interaction between an arginine and the phosphate 3′ to the abasic site. When this arginine is mutated to alanine, the catalytic rate of the enzyme is increased, suggesting that the protein is optimised to retain the cleaved product, possibly in order to coordinate DNA repair.
Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 Å resolution. Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK: Nat Struct Biol 2000, 7:134-140. • Significance: The highest resolution protein–DNA complex solved to date yields insight into the specificity of restriction endonuclease target recognition. Findings: The crystal structure of the restriction endonuclease BglII in complex with its target DNA has been determined to 1.5 Å resolution. A comparison of the structure with that of endonuclease BamHI, which recognises a closely related DNA sequence, reveals the different strategies for site-specific recognition employed by the restriction endonuclease family. Base pairs in common in the two structures are recognised in rather different ways as a result of different distortions of the DNA. The new structure suggests a novel mechanism for DNA hydrolysis. Structure of a transcribing T7 RNA polymerase initiation complex. Cheetham GMT, Steitz TA: Science 1999, 286:2305-2309. •• Significance: The crystal structure of a T7 RNA polymerase initiation complex transcribing a trinucleotide of RNA from a DNA promoter has been determined for the first time. Findings: The structure of a transcribing DNA-dependent RNA polymerase initiation complex reveals that, during synthesis, the template strand scrunches into the active site pocket of the enzyme in order to place the +4 base at the catalytic site. Only the observed three base pairs of DNA–RNA heteroduplex are likely to form before the RNA peels off the template. Numerous hydrogen bonds between the 2′-OH of the incoming nucleotide and the transcript account for the specificity of the polymerase for RNA, rather than DNA, synthesis.
Folding and binding Selected by Brian Kuhlman, Tanja Kortemme, Viara Grantcharova and Alex Watters University of Washington, Seattle, Washington, USA
Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Merkel R, Nassoy P, Leung A, Ritchie K, Evans E: Nature 2000, 397:50-53. •• Significance: Atomic force microscopy can be used to map the energy landscape of a receptor–ligand dissociation pathway. Findings: By varying the rate at which biotin is pulled from streptavidin (or avidin), it is possible to probe activation barriers at different distances along the reaction coordinate. Two prominent barriers were detected for streptavidin–biotin dissociation, whereas three barriers were detected for avidin–biotin dissociation. The location of these barriers agrees approximately with the location of transition states identified in molecular dynamics simulations. Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex. Lee AL, Kinnear SA, Wand AJ: Nat Struct Biol 2000, 7:72-77. AND
Increased protein backbone conformational entropy upon hydrophobic ligand binding. Zídek L, Novotny MV, Stone MJ: Nat Struct Biol 1999, 6:1118-1121.
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• Significance: NMR relaxation methods can yield information on changes in protein sidechain and backbone dynamics upon ligand binding and provide a high-resolution picture of entropic effects occurring during molecular recognition. Findings: Zídek et al. observed an increase in backbone flexibility when a small hydrophobic ligand binds mouse major urinary protein and propose that the resulting increase in conformational entropy could be a general mechanism favouring complex formation. Lee at al. found that peptide binding to calmodulin is accompanied by both increases and decreases in sidechain methyl group dynamics, with largely unchanged backbone mobility. This suggests site-specific enthalpy/entropy compensation at the interface. Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol. Hamada D, Chiti F, Guijarro JI, Kataoka M, Taddei N, Dobson CM: Nat Struct Biol 2000, 7:58-61. • Significance: The effect of the co-solvent trifluoroethanol (TFE) on protein folding was previously found to be very proteindependent. In this paper, by examining the folding kinetics of 13 proteins (both two-state and multistate folding proteins), the authors have been able to correlate the effect of different concentrations of TFE to structural properties of the transition state. Findings: The ability of low concentrations of TFE to increase the rate of folding has been quantitatively correlated to the number of local hydrogen bonds in the native protein, whereas the decrease in folding rate with high concentrations of TFE was found to be proportional to the change in the solvent-accessible surface area of polar residues. The effect of TFE on folding kinetics can, therefore, serve as a probe of the rate-limiting step in folding. The introduction of strain and its effects on the structure and stability of T4 lysozyme. Lio R, Walter AB, Matthews BW: J Mol Biol 2000, 295:127-145. • Significance: The mutation of core residues to have larger sidechains has been observed to cause varying amounts of strain on the structure and stability of a protein. Understanding how a protein accommodates such strain may prove to be important in understanding structural differences between related proteins. Findings: Lio et al. show that structural strain due to mutation is usually accommodated by many small changes in the torsion angles of neighbouring sidechains or backbone atoms and, very rarely, by relaxation as a result of a small number of large changes in torsion angles. In addition, the results suggest that the elasticity of the protein to such substitutions, although correlated to increases in sidechain size at an individual site, can differ significantly throughout the core.
Macromolecular assemblages Selected by Andreas Engel and J Bernard Heymann Universitat Basel, Basel, Switzerland
Maturation dynamics of a viral capsid: visualization of transitional intermediate states. Lata R, Conway JF, Cheng N, Duda RL, Hendrix RW, Wikoff WR, Johnson JE, Tsuruta H, Steven AC: Cell 2000, 100:253-263. •• Significance: The maturation of eicosahedral viruses involves large conformational changes in their capsid proteins. Bacteriophage HK97, studied here, is a useful model system to understand the expansion from the relatively unstable prohead formed from soluble capsid proteins to the much more stable head. This expansion occurs concurrent with or just before
packaging of the DNA into the head. This has implications for protein folding and pathways of viral proliferation, allowing the development of agents against viral infection. Findings: The expansion of the prohead II stage is induced at a pH of around 4. Low-angle X-ray scattering revealed an endstate at low pH with a size that is intermediate between the prohead II and head particles. Cryo-electron microscopy further showed three distinct expansion intermediates (E-I, E-II and EIII), where E-III corresponds to the end-state at low pH. Prohead II has a very short lifetime at pH 4.18 (> 30 s), whereas E-I and E-II have lifetimes of about 100 min. E-III is stable at low pH, but converts rapidly to head particles at neutral pH. During the E-II to E-III to head transition, the particle expands by about 25%, increasing the available space for DNA packaging. The result is that the capsid wall becomes much thinner and surface features become less pronounced. 15 Å resolution model of the monomeric kinesin motor, KIF1A. Kikkawa M, Okada Y, Hirokawa N: Cell 2000, 100:241-252. • Significance: Kinesins are (mostly) dimeric motor proteins that move that along microtubules. It is commonly thought that both heads of a kinesin dimer are required for processive movement. However, the authors describe a monomeric kinesin with a higher affinity for microtubules and much better processive movement than normal dimeric kinesins. This intriguing situation is elegantly explained by the presence of an additional loop region in monomeric kinesin that has a high affinity for a region of the microtubule. Findings: The increased affinity and processivity of monomeric kinesin KIF1A compared with the dimeric kinesins is due to the presence of a 12 amino acid insert in the L12 loop region (the ‘K loop’). Deletion of the K loop in C351, the catalytic core of KIF1A, abolishes processivity and decreases its affinity for microtubules. The K loop cross-links with both α- and β-tubulin in the microtubule. Cryo-electron microscopy of the C351–microtubule complex clearly shows an additional protrusion on the kinesin compared with normal human dimeric kinesin. Docking the atomic structures of human kinesin and tubulin into the cryo-electron microscopy 3D reconstructions placed the K loop in this protrusion. Gold cluster labelling further established the correct placement of the K loop. The K loop appears to interact with the very flexible C terminus of tubulin, which would allow it to move a significant distance along the microtubule surface. Therefore, the K loop is proposed to play the role of the dimeric second head in the monomeric kinesins. The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity. Chen L, Sigler PB: Cell 1999, 99:757-768. • Significance: GroEL binds a large variety of unfolded cytosolic proteins in E. coli to accelerate their folding. This X-ray analysis suggests that various modes of molecular plasticity warrant promiscuous binding of non-native substrates. Findings: GroEL is a cylindrical complex comprising two heptameric rings, each one forming a cavity where nascent or non-native proteins fold. These are trapped by interactions with the apical domains (AD) at the cavity entrance and are pushed into the cavity by the co-chaperonin GroES, which binds even more strongly to the AD. To assess the nature of this process, the binding of a phage peptide display library to the AD was tested and a strongly binding peptide (SBP) was selected.
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SBP–AD complexes were crystallised and their structure solved to 2 Å. The AD consists of three helices (H, I and J) and seven β strands. The binding site for SBP consists of parallel helices H and I, which form a hydrophobic cleft accommodating bulky hydrophobic sidechains of SBP. GroES also binds to this site, but more strongly as result of the perfect steric match of heptameric GroES to the heptameric GroEL half-cylinder. A comparison of unliganded AD, the SBP–AD complex and GroES–AD reveals structural plasticity mainly in helices H and I, but also in two other regions. These four regions have also been found to differ in different X-ray structures of AD.
Theory and simulation Selected by Francisco Melo Rockefeller University, New York, New York, USA
Increased protein backbone conformational entropy upon hydrophobic ligand binding. Zídek L, Novotny MV, Stone MJ: Nat Struct Biol 1999, 6:1118-1121. •• Significance: This paper provides evidence that the motion of a protein can increase upon ligand binding. Moreover, these results may provide a general mechanism for the binding of small hydrophobic ligands. Findings: The results presented in this paper indicate that the conventional assumption, that ligand binding reduces motion, may be incorrect. The authors show that the backbone flexibility of mouse major urinary protein increases upon binding a small hydrophobic ligand. The associated entropy appears to make a substantial contribution towards the stabilisation of the protein–ligand complex. kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction. Pilpel Y, Ben-Tal N, Lancet D: J Mol Biol 1999, 294:921-935. • Significance: The authors have generated a new and more accurate method for modelling and predicting the orientation of transmembrane segments in integral membrane proteins. Findings: The authors have developed a new scale for the propensity of residue orientation in transmembrane segments. They used this scale to predict angular helical orientation in a benchmark test of several multispan α-helical transmembrane proteins, showing that their method yielded an average angular error that is significantly lower than the error observed with alternative propensity scales. Filtered neighbors threading. Bienkowska JR, Rogers RG Jr, Smith TF: Proteins 1999, 37:346-359. • Significance: The authors developed a function that gives a significant improvement in the accuracy of sequence-to-structure alignment in fold recognition prediction. Findings: The authors developed a new statistical scoring function and have used it to filter the sequence-to-structure alignment scores by eliminating stereochemically improbable physical contacts for each possible alignment. They show that the accuracy of the sequence-to-structure alignments generated by this method is approximately twofold better than those obtained with unfiltered threading alignments. Analysis and prediction of inter-strand packing distances between β-sheets of globular proteins. Nagarajaram HA, Reddy BVB, Blundell TL: Protein Eng 1999, 12:1055-1062. •• Significance: The authors show how to improve the prediction of rigid-body shifts in the modelling of homologous proteins.
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Findings: The authors found a correlation between the packing of β strands and the weighted sum of the volumes of interacting residues at the packing interface. Using this correlation, the authors show that they can often improve predictions of the distances between β strands, thus making their protein models closer to the target structure. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. Jones DT: J Mol Biol 1999, 287:797-815. • Significance: The author has developed a fast and reliable method for fold recognition that can be applied to complete genomes. Findings: This method has been applied to the whole genome of Mycoplasma genitalium. This algorithm correlated 46% of the genes with a known protein fold. In several instances, however, only one domain of a protein can be assigned, thus giving a total fold assignment coverage of about 30% of the number of residues in the whole proteome.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Sutton, UK
Structure of a triple helical DNA with a triplex-duplex junction. Rhee S, Han Z, Liu K, Miles TH, Davies DR: Biochemistry 1999, 38:16810-16815. • Significance: Using the design of novel DNA sequences, the authors have determined the first single crystal structure of a DNA triplex and its junction with a duplex. Triplex DNA structures have been proposed to play a role in gene expression and recombination. Recent interest has focused on antisense inhibitors that block transcription and, thus, control gene expression. This 1.7 Å resolution crystal structure complements several recent NMR solution structures of intermolecular DNA triplexes. Findings: The structure was determined using multiple-wavelength anomalous diffraction with bromine–uracil bases. The asymmetric unit consists of two 1:1 complexes of a 12-mer and a 9-mer related by a noncrystallographic twofold axis. This arrangement generates a palindromic sequence with two independent triple and double helical regions in the asymmetric unit. The two triple helix segments consist of three base triplets that have canonical dimensions between A- and B-form DNA. The triplex to duplex step shows only modest changes in helical conformation. The Hoogsteen strand is positioned within the major groove of the B-DNA structure, with the Hoogsteen pairs between cytosine and guanine protonated on the N3 atom of cytosine. A typical spine of hydration is seen in the minor groove of the Watson–Crick duplex. This spine of hydration is not observed in the Crick–Hoogsteen region because of a reduced groove width that results from electrostatic interactions between cytosine+ and the phosphate group of the Crick strand. The hydration structure and groove width may have implications for the recognition of triplex DNA by protein molecules. Structure and recognition of sheared tandem G·A base pairs associated with human centromere DNA sequence atomic resolution. Gao Y-G, Robinson H, Sanishvili R, Joachimiak A, Wang AH-J: Biochemistry 1999, 38:16452-16460. • Significance: The structure of a DNA sequence [d(CCGAATGAGG)] in which the centromere core sequence motif GAATG is embedded has been determined. Near atomic resolution reveals the detailed conformation of tandem sheared G·A mismatched base pairs. These mismatches, frequently found in
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nucleic acids, help the purine-rich strand of the centromere to form duplex and hairpin structures with unusual stability. These results are important for further understanding centromere structure and the role played by mismatches in ribozymes and other nucleic acids. Findings: The structures of three crystal forms were determined using the multiple-wavelength anomalous diffraction method and refined to near atomic resolution. The incorporation of two tandem GpA steps causes the decamer duplex to bend towards the major groove and produces unusual interstrand base stacking, which contributes to the overall stability. Examination of the six independent CGAA/TGAG tetranucleotide motifs obtained reveals consistent conformations, with large propeller twist and buckle for the sheared G·A base pairs. This produces uneven helical twists, resulting in an irregular double helix. Water, spermine, magnesium ion and cobalt (III) hexaammine ion interactions with the tandem G·A motif are presented. The observation of tertiary base pair interactions is suggested to have implications for the biological activity of ribozymes.
Sequences and topology Selected by Rob Russell SmithKline Beecham Pharmaceuticals, Harlow, UK
A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Uetz P, Giot L, Cagne G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P et al.: Nature 2000, 403:623-627. •• Significance: The first large-scale experimental analysis of protein–protein interactions within yeast. Findings: Despite the current availability of complete genomic sequence data for several organisms, many genes are still of unknown function. Other experimental and/or theoretical techniques must thus be employed to elucidate gene function. These authors have performed yeast two-hybrid experiments on a large scale to determine the interactions between the 6000 proteins in Saccharomyces cerevisiae. The idea behind the two-hybrid experiments is simple: proteins are fused to either the activation or the DNA-binding domain of Gal4, which is essential for galactose metabolism. The yeast are denied galactose and only those cells containing interacting proteins (fused to either of the two domains of Gal4) survive, thus providing experimental evidence that the two proteins interact. Using two screens, the authors identified a total of 957 putative protein–protein interactions in yeast. Moreover, they have developed a new bioinformatics platform for interrogating the data (http://portal.curagen.com). Several interesting results are discussed, ranging from interactions between proteins of unknown function and those of known function, to insights into the complex interactions between proteins known to be involved in common pathways (e.g. autophagy, cyclin-mediated signalling, translation, cross-overs during meiosis etc). The data provide an incredibly useful resource; the results described in this paper are likely to be just the tip of the iceberg.
Lipids Selected by Richard Newman Imperial Cancer Research Fund, London, UK
Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-bisphosphate-specific PH domain. Hodgkin MN, Masson MR, Powner D, Saqib KM, Ponting CR, Wakelam JO: Curr Biol 2000, 10:43-46. • Significance: The signalling pathway leading to actin cytoskeletal reorganisation, secretion or superoxide generation
involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. The localisation and regulation of PLD is shown to be dependent upon its phosphatidylinositol 4,5-bisphosphate-specific pleckstrin homology (PH) domain. Findings: Sequence analysis indicating the presence of a PH domain was used to instigate studies, using surface plasmon resonance on supported lipid monolayers, of the binding of PLD1b to phosphatidylethanolamine, phosphatidylcholine and an activating lipid, phosphatidylinositol 4,5-bisphosphate. Results suggesting a key role for the PH domain in PLD function were obtained. Confocal microscopy results confirmed that human PLD1b containing a functional PH domain is critical in regulating its subcellular localisation. Its interaction with polyphosphoinositide-containing membranes is mediated by inducing a conformational change in the enzyme, thereby regulating catalytic activity. Trienoic fatty acids and plant tolerance of high temperatures. Murakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K: Science 2000, 287:476-479. •• Significance: Photosynthesis is inhibited by moderate to high temperatures, but the causes of this inhibition are not clear. This study shows that the number of unsaturated lipids in the thylakoid membrane of chloroplasts, which contain the lightabsorbing system, electron import chain and ATP synthase, is important in determining a plant’s ability for growth and photosynthesis at temperatures of 35°C or more. This report may provide valuable information about the best approach to engineering plants that can carry out photosynthesis in the face of heat stress. Findings: Transgenic tobacco plants in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesises trienoic fatty acids, was silenced contained a lower level of trienoic fatty acids than wild-type plants and grew much better than controls at higher temperatures.
Carbohydrates and glycoconjugates Selected by Sabine L Flitsch and Philip AS Lowden* Edinburgh University, Edinburgh, UK *University of Exeter, Exeter, UK
Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes. Shpak E, Leykam JF, Kiesliszewski MJ: Proc Natl Acad Sci USA 1999, 96:14736-14741. • Significance: Hydroxyproline-O-glycosylation is an important feature of plant cell wall structural glycoproteins. The authors provide the first documentation that the extent and type of this glycosylation is determined by codes of specific polypeptide sequences. Findings: Analysis of hydroxyproline-O-glycosylated glycoproteins in the cell walls of plants had previously suggested that glycosylation is dependent on the polypeptide sequence. To test this hypothesis, synthetic genes that encode proposed glycosylation sites were constructed fused to green fluorescent protein. As predicted, a repetitive (serine–hydroxyproline) polypeptide sequence was highly decorated with arabinogalactan polysaccharides, whereas a polypeptide sequence containing contiguous hydroxyproline blocks contained arabinoside sidechains. These experiments not only confirm the existence of a glycosylation polypeptide code, but also open the way to the design and synthesis of unnatural glycoproteins with defined hydroxyproline glycosylation patterns.
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On the meaning of affinity: cluster glycoside effects and concanavalin A. Dimmick SM, Powell SC, McMahon SA, Moothoo DN, Naismith JH, Toone EJ: J Am Chem Soc 1999, 121:10286-10296. •• Significance: One of the first thorough structural investigations of multivalency effects in protein–carbohydrate interactions, this paper suggests that the behaviour of polyvalent ligands is more complicated than previously assumed. This should have important implications for the design of therapeutically useful polyvalent ligands. Findings: The interaction of the plant lectin concanavalin A with synthetic multivalent dendritic saccharides was investigated by agglutination assays, calorimetric titration and low-angle dynamic light scattering, and by the determination of crystal structures of multivalent ligands cross-linking concanavalin A dimers. Although a 30-fold enhancement in the affinity of polyvalent ligands was observed from the agglutination assays, the results obtained from the other measurements suggest that, in this case, this is not due to increased protein–ligand binding (which is unchanged), but is a result of the ability of the ligand to drive aggregation processes. Statements concerning increased binding that are based on agglutination assays must therefore be treated with caution.
Biophysical methods Selected by Steve Matthews Imperial College of Science, Technology and Medicine, London, UK
Characterisation of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid. Chalk SG, MacMillan AM: Biochemistry 2000, 39:2-8. • Significance: A description of the folding pathways by which three-dimensional RNA structures are formed is critical for an indepth understanding of their biological activities. This study demonstrates the use of a readily available reagent, peroxynitrous acid, to kinetically footprint the Tetrahymena ribozyme during folding. Findings: Freshly prepared potassium peroxynitrite was added at various time points to a solution of folding 32P-labelled RNA. In agreement with results from other studies, this work suggested an ordered hierarchical folding pathway for the ribozyme. A ratedetermining step for folding was not observed, which suggests that the formation of the global, protected structure is followed by slow local arrangements to produce the final active species.
Proteins Selected by Gerard J Kleywegt Uppsala University, Uppsala, Sweden
βDirected evolution of new catalytic activity using the α/β barrel scaffold. Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Nature 2000, 403:617-622. •• Significance: A new catalytic function was successfully evolved on the scaffold of an α/β-barrel protein using a combination of rational design, in vitro mutation, recombination and in vivo selection. Findings: The α/β barrel has its substrate-binding residues predominantly within the barrel itself and its catalytic residues mainly in the loop regions. This structural separation suggests that evolution might be mimicked in vitro by grafting a new catalytic function onto an existing binding site (or the other way around). This principle is demonstrated using indole-3-glycerolphosphate synthase (IGPS) as a scaffold and switching its activity to that of phosphoribosyl-anthranilate isomerase (PRAI).
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In E. coli, IGPS and PRAI form two covalently linked domains of a bifunctional enzyme and they share a ligand (the product of PRAI is the substrate of IGPS). The engineered enzyme shares only 28% sequence identity with PRAI (and 90% with IGPS), but has catalytic properties similar to those of PRAI, with an even higher specificity constant. Specific chemical and structural damage to proteins produced by synchrotron radiation. Weik M, Ravelli RBG, Kryger G, McSweeney S, Raves ML, Harel M, Gros P, Silman I, Kroon J, Sussman J: Proc Natl Acad Sci USA 2000, 97:623-628. • Significance: The experiments described in this paper show how intense synchrotron radiation can cause specific structural damage to proteins in frozen crystals. Findings: Radiation damage, a problem inherent to X-ray crystallography, is usually presumed to be a nonspecific phenomenon, manifested as a gradual decay of the quality of the diffraction data. In this work, rapid collection of a time series of datasets at cryogenic temperatures at a third-generation synchrotron facility was applied to two different enzymes (acetylcholinesterase and lysozyme). Analysis of the experimental data reveals that synchrotron radiation can cause highly specific damage, in addition to the loss of diffractive power of the crystal and an overall increase in the atomic B factors. In particular, breakage of certain disulfide bonds is observed, as well as the loss of definition (in the electron density) of the carboxyl groups of acidic residues.
Catalysis and regulation Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA
The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA. Gu X, Liu Y, Santi DV: Proc Natl Acad Sci USA 1999, 96:14270-14275. • Significance: The mechanistic course of an important tRNA modification enzyme has been established, along with its relationship to other enzyme-catalysed reactions involving the pyrimidine ring of uracil. Findings: Incubation of pseudouridine synthase I with yeast tRNAPhe containing 5-fluorouracil at the site modified by this enzyme (position 39) led to the formation of a covalent intermediate that was stable to detergent and urea, but heat labile. By incorporating radioactive isotopes at various positions in the 5-fluorouracil-modified tRNA, it was established that the covalent linkage involved the pyrimidine of 5-fluorouracil. All of the data were consistent with an intermediate formed by conjugate addition of the sidechain carboxylate of Asp60 to the 6-position of the fluorouracil ring, with protonation occurring on the opposite face to yield the cis-5S,6R stereochemistry. This intermediate is believed to be analogous to that formed during the normal catalytic cycle, in which the pyrimidine of U39 is isomerised to form ψ39, and it provides a logical chemical mechanism for this conversion. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Jaeger L, Wright MC, Joyce GF: Proc Natl Acad Sci USA 1999, 96:14712-14717. •• Significance: This paper represents a strategy for more completely exploring the sequence space associated with the randomised portion of a ribozyme, while still affording a high probability of maintaining a three-dimensional structure conducive to catalysis.
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Findings: The hypothesis that a pre-existing RNA domain derived from the Tetrahymena ribozyme could serve as a ‘scaffold’ for evolving new ribozyme catalysts was tested by appending three loops whose sequences were completely randomised (total of 85 positions). A library of 10 16 different sequences was screened for the ability to undergo self-ligation and 10 rounds of selection yielded catalysts with rate constants of 0.26 min–1 (>106-fold greater than the background rate). A parallel experiment using a simple 85-nucleotide RNA in which all positions were varied failed to yield catalysts for the self-ligation reaction, perhaps because the total size of the individual ribozymes was insufficient for both substrate binding and catalysis for this sophisticated reaction. The major advantage of the strategy outlined above is that it allows one to access high sequence diversity and yet retain a common core that provides a useful overall structure, similar to the situation in antibodies. One polypeptide with two aminoacyl-tRNA synthetase activities. Stathopoulos C, Li T, Longman R, Vothknecht UC, Becker HD, Ibba M, Söll D: Science 2000, 287:479-482. •• Significance: The first demonstration that a single tRNA synthetase can accept two different amino acids and two different tRNAs while forming only the cognate aminoacylated tRNAs in both cases. Findings: The genomes of the thermophilic methanogens Methanococcus jannaschii and Methanobacterium thermoautotrophicum both appear to lack homologues of cysteinyl-tRNA synthetase. A single protein was purified from M. jannaschii that catalysed the aminoacylation of tRNACys; unexpectedly, the sequence of this protein suggested that it was a homologue of prolyl-tRNA synthetase. A variety of kinetic experiments established that this single protein catalysed the aminoacylation of tRNACys and both tRNAPro molecules, but only with the correct amino acid (cysteine and proline, respectively). Apart from accepting two very different amino acid substrates and tRNAs, the other properties of the M. jannaschii enzyme were very similar to those of other prolyl-tRNA synthetases. Phylogenetic analyses were not useful in detecting the expanded substrate specificity of the M. jannaschii synthetase and it is not clear how widespread this phenomenon may be. Assessment of protein-tyrosine phosphatase 1B substrate specificity using “inverse alanine scanning”. Vetter SW,
Keng Y-F, Lawrence DS, Zhang Z-Y: J Biol Chem 2000, 275:2265-2268. • Significance: A simple and generally applicable approach to determining the specificity of enzymes that accept peptide substrates is described. The method provides information on individual sequences and requires a relatively small number of library members. Findings: From the crystal structure of murine protein-tyrosine phosphatase 1B, it was predicted that Ac–Ala4–Tyr(OPO3)– Ala4–CONH2 would be an acceptable substrate and this was subsequently confirmed experimentally. A series of 152 additional peptides was synthesised in which each alanine residue was replaced separately and sequentially with each of the 19 nonalanine residues. The kinetic parameters for each peptide were determined individually, which allowed the substrate preferences to be investigated at each position in a physiologically relevant reaction. The results from the library screening reiterated known substrate preferences for protein-tyrosine phosphatase 1B, but also revealed several other previously unsuspected peptide sequences that were also excellent substrates. Conformational effects in biological catalysis: an antibodycatalyzed oxy-Cope rearrangement. Mundorff EC, Hanson MA, Varvak A, Ulrich H, Schultz PG, Stevens RC: Biochemistry 2000, 39:627-632. • Significance: These crystal structures clearly illustrate how selection for tighter binding to a transition-state analogue by the immune system can actually compromise catalytic activity for a reaction involving a flexible substrate. Findings: A catalytic antibody was raised against a flexible antigen that mimicked the transition-state structure for an oxy-Cope rearrangement, a sigmatropic reaction related to that catalysed by chorismate mutase. Surprisingly, although the germ-line precursor antibody that gave rise to the mature catalytic antibody possessed 40-fold lower affinity for the transition-state analogue, it catalysed the reaction with 35-fold higher efficiency. X-ray crystal structures of the germ-line precursor and the mature antibody, in the presence and absence of transition-state analogue, were therefore obtained to probe the origin of this phenomenon. These data revealed that the mature antibody bound the flexible transition-state analogue in a conformation that disfavoured the sigmatropic rearrangement; by contrast, the binding site of the germ-line antibody was more flexible, which may allow the substrate to more easily reach the reactive conformation.
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Structural biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology. Current Opinion in Structural Biology 2000, 10:129–134 Contents (chosen by) 129 Protein–nucleic acid interactions (Convery et al.) 129 Folding and binding (Kuhlman et al.) 130 Macromolecular assemblages (Engel and Heymann) 131 Theory and simulation (Melo) 131 Nucleic acids (Parkinson) 132 Sequences and topology (Russell) 132 Lipids (Newman) 132 Carbohydrates and glycoconjugates (Flitsch and Lowden) 133 Biophysical methods (Matthews) 133 Proteins (Kleywegt) 133 Catalysis and regulation (Stewart)
• ••
of special interest of outstanding interest
Protein–nucleic acid interactions Selected by Máire Convery, Caitríona Dennis and Siân Rowsell* University of Leeds, Leeds, UK *AstraZeneca, Macclesfield, UK
Structural basis of preinitiation complex assembly on human Pol II promoters. Tsai FTF, Sigler PB: EMBO J 2000, 19:25-36. •• Significance: The first structural evidence that the Pol II preinitiation complex has directionality conferred by the binding of transcription initiation factor TFIIB to a sequence flanking the TATA-box. Findings: The structure described consists of a ternary complex between the cores of the human TATA-box-binding protein and TFIIB, and a DNA sequence. The structure is unique because of the length of the DNA promoter sequence, which allows the TFIIB core to make more extensive interactions with the DNA. TFIIB differentiates between the major groove upstream and the minor grove immediately downstream of the bound TATA-box sequence, thus conferring polarity on the complex. DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination. Mol CD, Izumi T, Mitra S, Tainer JA: Nature 2000, 403:451-456. • Significance: Three structures that reveal a structure-based catalytic mechanism for phosphate bond cleavage. Findings: Upon binding, the protein engulfs the DNA with a preformed, positively rigid surface. The DNA is strongly kinked and has the abasic site flipped out. There is an unusual interaction between an arginine and the phosphate 3′ to the abasic site. When this arginine is mutated to alanine, the catalytic rate of the enzyme is increased, suggesting that the protein is optimised to retain the cleaved product, possibly in order to coordinate DNA repair.
Understanding the immutability of restriction enzymes: crystal structure of BglII and its DNA substrate at 1.5 Å resolution. Lukacs CM, Kucera R, Schildkraut I, Aggarwal AK: Nat Struct Biol 2000, 7:134-140. • Significance: The highest resolution protein–DNA complex solved to date yields insight into the specificity of restriction endonuclease target recognition. Findings: The crystal structure of the restriction endonuclease BglII in complex with its target DNA has been determined to 1.5 Å resolution. A comparison of the structure with that of endonuclease BamHI, which recognises a closely related DNA sequence, reveals the different strategies for site-specific recognition employed by the restriction endonuclease family. Base pairs in common in the two structures are recognised in rather different ways as a result of different distortions of the DNA. The new structure suggests a novel mechanism for DNA hydrolysis. Structure of a transcribing T7 RNA polymerase initiation complex. Cheetham GMT, Steitz TA: Science 1999, 286:2305-2309. •• Significance: The crystal structure of a T7 RNA polymerase initiation complex transcribing a trinucleotide of RNA from a DNA promoter has been determined for the first time. Findings: The structure of a transcribing DNA-dependent RNA polymerase initiation complex reveals that, during synthesis, the template strand scrunches into the active site pocket of the enzyme in order to place the +4 base at the catalytic site. Only the observed three base pairs of DNA–RNA heteroduplex are likely to form before the RNA peels off the template. Numerous hydrogen bonds between the 2′-OH of the incoming nucleotide and the transcript account for the specificity of the polymerase for RNA, rather than DNA, synthesis.
Folding and binding Selected by Brian Kuhlman, Tanja Kortemme, Viara Grantcharova and Alex Watters University of Washington, Seattle, Washington, USA
Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Merkel R, Nassoy P, Leung A, Ritchie K, Evans E: Nature 2000, 397:50-53. •• Significance: Atomic force microscopy can be used to map the energy landscape of a receptor–ligand dissociation pathway. Findings: By varying the rate at which biotin is pulled from streptavidin (or avidin), it is possible to probe activation barriers at different distances along the reaction coordinate. Two prominent barriers were detected for streptavidin–biotin dissociation, whereas three barriers were detected for avidin–biotin dissociation. The location of these barriers agrees approximately with the location of transition states identified in molecular dynamics simulations. Redistribution and loss of side chain entropy upon formation of a calmodulin-peptide complex. Lee AL, Kinnear SA, Wand AJ: Nat Struct Biol 2000, 7:72-77. AND
Increased protein backbone conformational entropy upon hydrophobic ligand binding. Zídek L, Novotny MV, Stone MJ: Nat Struct Biol 1999, 6:1118-1121.
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• Significance: NMR relaxation methods can yield information on changes in protein sidechain and backbone dynamics upon ligand binding and provide a high-resolution picture of entropic effects occurring during molecular recognition. Findings: Zídek et al. observed an increase in backbone flexibility when a small hydrophobic ligand binds mouse major urinary protein and propose that the resulting increase in conformational entropy could be a general mechanism favouring complex formation. Lee at al. found that peptide binding to calmodulin is accompanied by both increases and decreases in sidechain methyl group dynamics, with largely unchanged backbone mobility. This suggests site-specific enthalpy/entropy compensation at the interface. Evidence concerning rate-limiting steps in protein folding from the effects of trifluoroethanol. Hamada D, Chiti F, Guijarro JI, Kataoka M, Taddei N, Dobson CM: Nat Struct Biol 2000, 7:58-61. • Significance: The effect of the co-solvent trifluoroethanol (TFE) on protein folding was previously found to be very proteindependent. In this paper, by examining the folding kinetics of 13 proteins (both two-state and multistate folding proteins), the authors have been able to correlate the effect of different concentrations of TFE to structural properties of the transition state. Findings: The ability of low concentrations of TFE to increase the rate of folding has been quantitatively correlated to the number of local hydrogen bonds in the native protein, whereas the decrease in folding rate with high concentrations of TFE was found to be proportional to the change in the solvent-accessible surface area of polar residues. The effect of TFE on folding kinetics can, therefore, serve as a probe of the rate-limiting step in folding. The introduction of strain and its effects on the structure and stability of T4 lysozyme. Lio R, Walter AB, Matthews BW: J Mol Biol 2000, 295:127-145. • Significance: The mutation of core residues to have larger sidechains has been observed to cause varying amounts of strain on the structure and stability of a protein. Understanding how a protein accommodates such strain may prove to be important in understanding structural differences between related proteins. Findings: Lio et al. show that structural strain due to mutation is usually accommodated by many small changes in the torsion angles of neighbouring sidechains or backbone atoms and, very rarely, by relaxation as a result of a small number of large changes in torsion angles. In addition, the results suggest that the elasticity of the protein to such substitutions, although correlated to increases in sidechain size at an individual site, can differ significantly throughout the core.
Macromolecular assemblages Selected by Andreas Engel and J Bernard Heymann Universitat Basel, Basel, Switzerland
Maturation dynamics of a viral capsid: visualization of transitional intermediate states. Lata R, Conway JF, Cheng N, Duda RL, Hendrix RW, Wikoff WR, Johnson JE, Tsuruta H, Steven AC: Cell 2000, 100:253-263. •• Significance: The maturation of eicosahedral viruses involves large conformational changes in their capsid proteins. Bacteriophage HK97, studied here, is a useful model system to understand the expansion from the relatively unstable prohead formed from soluble capsid proteins to the much more stable head. This expansion occurs concurrent with or just before
packaging of the DNA into the head. This has implications for protein folding and pathways of viral proliferation, allowing the development of agents against viral infection. Findings: The expansion of the prohead II stage is induced at a pH of around 4. Low-angle X-ray scattering revealed an endstate at low pH with a size that is intermediate between the prohead II and head particles. Cryo-electron microscopy further showed three distinct expansion intermediates (E-I, E-II and EIII), where E-III corresponds to the end-state at low pH. Prohead II has a very short lifetime at pH 4.18 (> 30 s), whereas E-I and E-II have lifetimes of about 100 min. E-III is stable at low pH, but converts rapidly to head particles at neutral pH. During the E-II to E-III to head transition, the particle expands by about 25%, increasing the available space for DNA packaging. The result is that the capsid wall becomes much thinner and surface features become less pronounced. 15 Å resolution model of the monomeric kinesin motor, KIF1A. Kikkawa M, Okada Y, Hirokawa N: Cell 2000, 100:241-252. • Significance: Kinesins are (mostly) dimeric motor proteins that move that along microtubules. It is commonly thought that both heads of a kinesin dimer are required for processive movement. However, the authors describe a monomeric kinesin with a higher affinity for microtubules and much better processive movement than normal dimeric kinesins. This intriguing situation is elegantly explained by the presence of an additional loop region in monomeric kinesin that has a high affinity for a region of the microtubule. Findings: The increased affinity and processivity of monomeric kinesin KIF1A compared with the dimeric kinesins is due to the presence of a 12 amino acid insert in the L12 loop region (the ‘K loop’). Deletion of the K loop in C351, the catalytic core of KIF1A, abolishes processivity and decreases its affinity for microtubules. The K loop cross-links with both α- and β-tubulin in the microtubule. Cryo-electron microscopy of the C351–microtubule complex clearly shows an additional protrusion on the kinesin compared with normal human dimeric kinesin. Docking the atomic structures of human kinesin and tubulin into the cryo-electron microscopy 3D reconstructions placed the K loop in this protrusion. Gold cluster labelling further established the correct placement of the K loop. The K loop appears to interact with the very flexible C terminus of tubulin, which would allow it to move a significant distance along the microtubule surface. Therefore, the K loop is proposed to play the role of the dimeric second head in the monomeric kinesins. The crystal structure of a GroEL/peptide complex: plasticity as a basis for substrate diversity. Chen L, Sigler PB: Cell 1999, 99:757-768. • Significance: GroEL binds a large variety of unfolded cytosolic proteins in E. coli to accelerate their folding. This X-ray analysis suggests that various modes of molecular plasticity warrant promiscuous binding of non-native substrates. Findings: GroEL is a cylindrical complex comprising two heptameric rings, each one forming a cavity where nascent or non-native proteins fold. These are trapped by interactions with the apical domains (AD) at the cavity entrance and are pushed into the cavity by the co-chaperonin GroES, which binds even more strongly to the AD. To assess the nature of this process, the binding of a phage peptide display library to the AD was tested and a strongly binding peptide (SBP) was selected.
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SBP–AD complexes were crystallised and their structure solved to 2 Å. The AD consists of three helices (H, I and J) and seven β strands. The binding site for SBP consists of parallel helices H and I, which form a hydrophobic cleft accommodating bulky hydrophobic sidechains of SBP. GroES also binds to this site, but more strongly as result of the perfect steric match of heptameric GroES to the heptameric GroEL half-cylinder. A comparison of unliganded AD, the SBP–AD complex and GroES–AD reveals structural plasticity mainly in helices H and I, but also in two other regions. These four regions have also been found to differ in different X-ray structures of AD.
Theory and simulation Selected by Francisco Melo Rockefeller University, New York, New York, USA
Increased protein backbone conformational entropy upon hydrophobic ligand binding. Zídek L, Novotny MV, Stone MJ: Nat Struct Biol 1999, 6:1118-1121. •• Significance: This paper provides evidence that the motion of a protein can increase upon ligand binding. Moreover, these results may provide a general mechanism for the binding of small hydrophobic ligands. Findings: The results presented in this paper indicate that the conventional assumption, that ligand binding reduces motion, may be incorrect. The authors show that the backbone flexibility of mouse major urinary protein increases upon binding a small hydrophobic ligand. The associated entropy appears to make a substantial contribution towards the stabilisation of the protein–ligand complex. kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction. Pilpel Y, Ben-Tal N, Lancet D: J Mol Biol 1999, 294:921-935. • Significance: The authors have generated a new and more accurate method for modelling and predicting the orientation of transmembrane segments in integral membrane proteins. Findings: The authors have developed a new scale for the propensity of residue orientation in transmembrane segments. They used this scale to predict angular helical orientation in a benchmark test of several multispan α-helical transmembrane proteins, showing that their method yielded an average angular error that is significantly lower than the error observed with alternative propensity scales. Filtered neighbors threading. Bienkowska JR, Rogers RG Jr, Smith TF: Proteins 1999, 37:346-359. • Significance: The authors developed a function that gives a significant improvement in the accuracy of sequence-to-structure alignment in fold recognition prediction. Findings: The authors developed a new statistical scoring function and have used it to filter the sequence-to-structure alignment scores by eliminating stereochemically improbable physical contacts for each possible alignment. They show that the accuracy of the sequence-to-structure alignments generated by this method is approximately twofold better than those obtained with unfiltered threading alignments. Analysis and prediction of inter-strand packing distances between β-sheets of globular proteins. Nagarajaram HA, Reddy BVB, Blundell TL: Protein Eng 1999, 12:1055-1062. •• Significance: The authors show how to improve the prediction of rigid-body shifts in the modelling of homologous proteins.
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Findings: The authors found a correlation between the packing of β strands and the weighted sum of the volumes of interacting residues at the packing interface. Using this correlation, the authors show that they can often improve predictions of the distances between β strands, thus making their protein models closer to the target structure. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. Jones DT: J Mol Biol 1999, 287:797-815. • Significance: The author has developed a fast and reliable method for fold recognition that can be applied to complete genomes. Findings: This method has been applied to the whole genome of Mycoplasma genitalium. This algorithm correlated 46% of the genes with a known protein fold. In several instances, however, only one domain of a protein can be assigned, thus giving a total fold assignment coverage of about 30% of the number of residues in the whole proteome.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Sutton, UK
Structure of a triple helical DNA with a triplex-duplex junction. Rhee S, Han Z, Liu K, Miles TH, Davies DR: Biochemistry 1999, 38:16810-16815. • Significance: Using the design of novel DNA sequences, the authors have determined the first single crystal structure of a DNA triplex and its junction with a duplex. Triplex DNA structures have been proposed to play a role in gene expression and recombination. Recent interest has focused on antisense inhibitors that block transcription and, thus, control gene expression. This 1.7 Å resolution crystal structure complements several recent NMR solution structures of intermolecular DNA triplexes. Findings: The structure was determined using multiple-wavelength anomalous diffraction with bromine–uracil bases. The asymmetric unit consists of two 1:1 complexes of a 12-mer and a 9-mer related by a noncrystallographic twofold axis. This arrangement generates a palindromic sequence with two independent triple and double helical regions in the asymmetric unit. The two triple helix segments consist of three base triplets that have canonical dimensions between A- and B-form DNA. The triplex to duplex step shows only modest changes in helical conformation. The Hoogsteen strand is positioned within the major groove of the B-DNA structure, with the Hoogsteen pairs between cytosine and guanine protonated on the N3 atom of cytosine. A typical spine of hydration is seen in the minor groove of the Watson–Crick duplex. This spine of hydration is not observed in the Crick–Hoogsteen region because of a reduced groove width that results from electrostatic interactions between cytosine+ and the phosphate group of the Crick strand. The hydration structure and groove width may have implications for the recognition of triplex DNA by protein molecules. Structure and recognition of sheared tandem G·A base pairs associated with human centromere DNA sequence atomic resolution. Gao Y-G, Robinson H, Sanishvili R, Joachimiak A, Wang AH-J: Biochemistry 1999, 38:16452-16460. • Significance: The structure of a DNA sequence [d(CCGAATGAGG)] in which the centromere core sequence motif GAATG is embedded has been determined. Near atomic resolution reveals the detailed conformation of tandem sheared G·A mismatched base pairs. These mismatches, frequently found in
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nucleic acids, help the purine-rich strand of the centromere to form duplex and hairpin structures with unusual stability. These results are important for further understanding centromere structure and the role played by mismatches in ribozymes and other nucleic acids. Findings: The structures of three crystal forms were determined using the multiple-wavelength anomalous diffraction method and refined to near atomic resolution. The incorporation of two tandem GpA steps causes the decamer duplex to bend towards the major groove and produces unusual interstrand base stacking, which contributes to the overall stability. Examination of the six independent CGAA/TGAG tetranucleotide motifs obtained reveals consistent conformations, with large propeller twist and buckle for the sheared G·A base pairs. This produces uneven helical twists, resulting in an irregular double helix. Water, spermine, magnesium ion and cobalt (III) hexaammine ion interactions with the tandem G·A motif are presented. The observation of tertiary base pair interactions is suggested to have implications for the biological activity of ribozymes.
Sequences and topology Selected by Rob Russell SmithKline Beecham Pharmaceuticals, Harlow, UK
A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Uetz P, Giot L, Cagne G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P et al.: Nature 2000, 403:623-627. •• Significance: The first large-scale experimental analysis of protein–protein interactions within yeast. Findings: Despite the current availability of complete genomic sequence data for several organisms, many genes are still of unknown function. Other experimental and/or theoretical techniques must thus be employed to elucidate gene function. These authors have performed yeast two-hybrid experiments on a large scale to determine the interactions between the 6000 proteins in Saccharomyces cerevisiae. The idea behind the two-hybrid experiments is simple: proteins are fused to either the activation or the DNA-binding domain of Gal4, which is essential for galactose metabolism. The yeast are denied galactose and only those cells containing interacting proteins (fused to either of the two domains of Gal4) survive, thus providing experimental evidence that the two proteins interact. Using two screens, the authors identified a total of 957 putative protein–protein interactions in yeast. Moreover, they have developed a new bioinformatics platform for interrogating the data (http://portal.curagen.com). Several interesting results are discussed, ranging from interactions between proteins of unknown function and those of known function, to insights into the complex interactions between proteins known to be involved in common pathways (e.g. autophagy, cyclin-mediated signalling, translation, cross-overs during meiosis etc). The data provide an incredibly useful resource; the results described in this paper are likely to be just the tip of the iceberg.
Lipids Selected by Richard Newman Imperial Cancer Research Fund, London, UK
Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-bisphosphate-specific PH domain. Hodgkin MN, Masson MR, Powner D, Saqib KM, Ponting CR, Wakelam JO: Curr Biol 2000, 10:43-46. • Significance: The signalling pathway leading to actin cytoskeletal reorganisation, secretion or superoxide generation
involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. The localisation and regulation of PLD is shown to be dependent upon its phosphatidylinositol 4,5-bisphosphate-specific pleckstrin homology (PH) domain. Findings: Sequence analysis indicating the presence of a PH domain was used to instigate studies, using surface plasmon resonance on supported lipid monolayers, of the binding of PLD1b to phosphatidylethanolamine, phosphatidylcholine and an activating lipid, phosphatidylinositol 4,5-bisphosphate. Results suggesting a key role for the PH domain in PLD function were obtained. Confocal microscopy results confirmed that human PLD1b containing a functional PH domain is critical in regulating its subcellular localisation. Its interaction with polyphosphoinositide-containing membranes is mediated by inducing a conformational change in the enzyme, thereby regulating catalytic activity. Trienoic fatty acids and plant tolerance of high temperatures. Murakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K: Science 2000, 287:476-479. •• Significance: Photosynthesis is inhibited by moderate to high temperatures, but the causes of this inhibition are not clear. This study shows that the number of unsaturated lipids in the thylakoid membrane of chloroplasts, which contain the lightabsorbing system, electron import chain and ATP synthase, is important in determining a plant’s ability for growth and photosynthesis at temperatures of 35°C or more. This report may provide valuable information about the best approach to engineering plants that can carry out photosynthesis in the face of heat stress. Findings: Transgenic tobacco plants in which the gene encoding chloroplast omega-3 fatty acid desaturase, which synthesises trienoic fatty acids, was silenced contained a lower level of trienoic fatty acids than wild-type plants and grew much better than controls at higher temperatures.
Carbohydrates and glycoconjugates Selected by Sabine L Flitsch and Philip AS Lowden* Edinburgh University, Edinburgh, UK *University of Exeter, Exeter, UK
Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes. Shpak E, Leykam JF, Kiesliszewski MJ: Proc Natl Acad Sci USA 1999, 96:14736-14741. • Significance: Hydroxyproline-O-glycosylation is an important feature of plant cell wall structural glycoproteins. The authors provide the first documentation that the extent and type of this glycosylation is determined by codes of specific polypeptide sequences. Findings: Analysis of hydroxyproline-O-glycosylated glycoproteins in the cell walls of plants had previously suggested that glycosylation is dependent on the polypeptide sequence. To test this hypothesis, synthetic genes that encode proposed glycosylation sites were constructed fused to green fluorescent protein. As predicted, a repetitive (serine–hydroxyproline) polypeptide sequence was highly decorated with arabinogalactan polysaccharides, whereas a polypeptide sequence containing contiguous hydroxyproline blocks contained arabinoside sidechains. These experiments not only confirm the existence of a glycosylation polypeptide code, but also open the way to the design and synthesis of unnatural glycoproteins with defined hydroxyproline glycosylation patterns.
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On the meaning of affinity: cluster glycoside effects and concanavalin A. Dimmick SM, Powell SC, McMahon SA, Moothoo DN, Naismith JH, Toone EJ: J Am Chem Soc 1999, 121:10286-10296. •• Significance: One of the first thorough structural investigations of multivalency effects in protein–carbohydrate interactions, this paper suggests that the behaviour of polyvalent ligands is more complicated than previously assumed. This should have important implications for the design of therapeutically useful polyvalent ligands. Findings: The interaction of the plant lectin concanavalin A with synthetic multivalent dendritic saccharides was investigated by agglutination assays, calorimetric titration and low-angle dynamic light scattering, and by the determination of crystal structures of multivalent ligands cross-linking concanavalin A dimers. Although a 30-fold enhancement in the affinity of polyvalent ligands was observed from the agglutination assays, the results obtained from the other measurements suggest that, in this case, this is not due to increased protein–ligand binding (which is unchanged), but is a result of the ability of the ligand to drive aggregation processes. Statements concerning increased binding that are based on agglutination assays must therefore be treated with caution.
Biophysical methods Selected by Steve Matthews Imperial College of Science, Technology and Medicine, London, UK
Characterisation of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid. Chalk SG, MacMillan AM: Biochemistry 2000, 39:2-8. • Significance: A description of the folding pathways by which three-dimensional RNA structures are formed is critical for an indepth understanding of their biological activities. This study demonstrates the use of a readily available reagent, peroxynitrous acid, to kinetically footprint the Tetrahymena ribozyme during folding. Findings: Freshly prepared potassium peroxynitrite was added at various time points to a solution of folding 32P-labelled RNA. In agreement with results from other studies, this work suggested an ordered hierarchical folding pathway for the ribozyme. A ratedetermining step for folding was not observed, which suggests that the formation of the global, protected structure is followed by slow local arrangements to produce the final active species.
Proteins Selected by Gerard J Kleywegt Uppsala University, Uppsala, Sweden
βDirected evolution of new catalytic activity using the α/β barrel scaffold. Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Nature 2000, 403:617-622. •• Significance: A new catalytic function was successfully evolved on the scaffold of an α/β-barrel protein using a combination of rational design, in vitro mutation, recombination and in vivo selection. Findings: The α/β barrel has its substrate-binding residues predominantly within the barrel itself and its catalytic residues mainly in the loop regions. This structural separation suggests that evolution might be mimicked in vitro by grafting a new catalytic function onto an existing binding site (or the other way around). This principle is demonstrated using indole-3-glycerolphosphate synthase (IGPS) as a scaffold and switching its activity to that of phosphoribosyl-anthranilate isomerase (PRAI).
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In E. coli, IGPS and PRAI form two covalently linked domains of a bifunctional enzyme and they share a ligand (the product of PRAI is the substrate of IGPS). The engineered enzyme shares only 28% sequence identity with PRAI (and 90% with IGPS), but has catalytic properties similar to those of PRAI, with an even higher specificity constant. Specific chemical and structural damage to proteins produced by synchrotron radiation. Weik M, Ravelli RBG, Kryger G, McSweeney S, Raves ML, Harel M, Gros P, Silman I, Kroon J, Sussman J: Proc Natl Acad Sci USA 2000, 97:623-628. • Significance: The experiments described in this paper show how intense synchrotron radiation can cause specific structural damage to proteins in frozen crystals. Findings: Radiation damage, a problem inherent to X-ray crystallography, is usually presumed to be a nonspecific phenomenon, manifested as a gradual decay of the quality of the diffraction data. In this work, rapid collection of a time series of datasets at cryogenic temperatures at a third-generation synchrotron facility was applied to two different enzymes (acetylcholinesterase and lysozyme). Analysis of the experimental data reveals that synchrotron radiation can cause highly specific damage, in addition to the loss of diffractive power of the crystal and an overall increase in the atomic B factors. In particular, breakage of certain disulfide bonds is observed, as well as the loss of definition (in the electron density) of the carboxyl groups of acidic residues.
Catalysis and regulation Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA
The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA. Gu X, Liu Y, Santi DV: Proc Natl Acad Sci USA 1999, 96:14270-14275. • Significance: The mechanistic course of an important tRNA modification enzyme has been established, along with its relationship to other enzyme-catalysed reactions involving the pyrimidine ring of uracil. Findings: Incubation of pseudouridine synthase I with yeast tRNAPhe containing 5-fluorouracil at the site modified by this enzyme (position 39) led to the formation of a covalent intermediate that was stable to detergent and urea, but heat labile. By incorporating radioactive isotopes at various positions in the 5-fluorouracil-modified tRNA, it was established that the covalent linkage involved the pyrimidine of 5-fluorouracil. All of the data were consistent with an intermediate formed by conjugate addition of the sidechain carboxylate of Asp60 to the 6-position of the fluorouracil ring, with protonation occurring on the opposite face to yield the cis-5S,6R stereochemistry. This intermediate is believed to be analogous to that formed during the normal catalytic cycle, in which the pyrimidine of U39 is isomerised to form ψ39, and it provides a logical chemical mechanism for this conversion. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Jaeger L, Wright MC, Joyce GF: Proc Natl Acad Sci USA 1999, 96:14712-14717. •• Significance: This paper represents a strategy for more completely exploring the sequence space associated with the randomised portion of a ribozyme, while still affording a high probability of maintaining a three-dimensional structure conducive to catalysis.
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Findings: The hypothesis that a pre-existing RNA domain derived from the Tetrahymena ribozyme could serve as a ‘scaffold’ for evolving new ribozyme catalysts was tested by appending three loops whose sequences were completely randomised (total of 85 positions). A library of 10 16 different sequences was screened for the ability to undergo self-ligation and 10 rounds of selection yielded catalysts with rate constants of 0.26 min–1 (>106-fold greater than the background rate). A parallel experiment using a simple 85-nucleotide RNA in which all positions were varied failed to yield catalysts for the self-ligation reaction, perhaps because the total size of the individual ribozymes was insufficient for both substrate binding and catalysis for this sophisticated reaction. The major advantage of the strategy outlined above is that it allows one to access high sequence diversity and yet retain a common core that provides a useful overall structure, similar to the situation in antibodies. One polypeptide with two aminoacyl-tRNA synthetase activities. Stathopoulos C, Li T, Longman R, Vothknecht UC, Becker HD, Ibba M, Söll D: Science 2000, 287:479-482. •• Significance: The first demonstration that a single tRNA synthetase can accept two different amino acids and two different tRNAs while forming only the cognate aminoacylated tRNAs in both cases. Findings: The genomes of the thermophilic methanogens Methanococcus jannaschii and Methanobacterium thermoautotrophicum both appear to lack homologues of cysteinyl-tRNA synthetase. A single protein was purified from M. jannaschii that catalysed the aminoacylation of tRNACys; unexpectedly, the sequence of this protein suggested that it was a homologue of prolyl-tRNA synthetase. A variety of kinetic experiments established that this single protein catalysed the aminoacylation of tRNACys and both tRNAPro molecules, but only with the correct amino acid (cysteine and proline, respectively). Apart from accepting two very different amino acid substrates and tRNAs, the other properties of the M. jannaschii enzyme were very similar to those of other prolyl-tRNA synthetases. Phylogenetic analyses were not useful in detecting the expanded substrate specificity of the M. jannaschii synthetase and it is not clear how widespread this phenomenon may be. Assessment of protein-tyrosine phosphatase 1B substrate specificity using “inverse alanine scanning”. Vetter SW,
Keng Y-F, Lawrence DS, Zhang Z-Y: J Biol Chem 2000, 275:2265-2268. • Significance: A simple and generally applicable approach to determining the specificity of enzymes that accept peptide substrates is described. The method provides information on individual sequences and requires a relatively small number of library members. Findings: From the crystal structure of murine protein-tyrosine phosphatase 1B, it was predicted that Ac–Ala4–Tyr(OPO3)– Ala4–CONH2 would be an acceptable substrate and this was subsequently confirmed experimentally. A series of 152 additional peptides was synthesised in which each alanine residue was replaced separately and sequentially with each of the 19 nonalanine residues. The kinetic parameters for each peptide were determined individually, which allowed the substrate preferences to be investigated at each position in a physiologically relevant reaction. The results from the library screening reiterated known substrate preferences for protein-tyrosine phosphatase 1B, but also revealed several other previously unsuspected peptide sequences that were also excellent substrates. Conformational effects in biological catalysis: an antibodycatalyzed oxy-Cope rearrangement. Mundorff EC, Hanson MA, Varvak A, Ulrich H, Schultz PG, Stevens RC: Biochemistry 2000, 39:627-632. • Significance: These crystal structures clearly illustrate how selection for tighter binding to a transition-state analogue by the immune system can actually compromise catalytic activity for a reaction involving a flexible substrate. Findings: A catalytic antibody was raised against a flexible antigen that mimicked the transition-state structure for an oxy-Cope rearrangement, a sigmatropic reaction related to that catalysed by chorismate mutase. Surprisingly, although the germ-line precursor antibody that gave rise to the mature catalytic antibody possessed 40-fold lower affinity for the transition-state analogue, it catalysed the reaction with 35-fold higher efficiency. X-ray crystal structures of the germ-line precursor and the mature antibody, in the presence and absence of transition-state analogue, were therefore obtained to probe the origin of this phenomenon. These data revealed that the mature antibody bound the flexible transition-state analogue in a conformation that disfavoured the sigmatropic rearrangement; by contrast, the binding site of the germ-line antibody was more flexible, which may allow the substrate to more easily reach the reactive conformation.