A novel mode of DNA recognition by a B-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. Allen MD, Yamasaki K, Ohme-Takagi M, ... Significance: The solution structure determination of a novel.
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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 1998, 8:665-671 http:l/biomednet.com/elecref/O959440XO0800665 © Current BiologyLtd ISSN 0059-440X Contents (chosen by) 665 Protein-nucleic acid interactions (Converyet al.) 665 Folding and binding (Pielak) 666 Macromolecular assemblages (Engeland Heymann) 666 Theory and simulation (MichaelLevitt'slaboratory) 667 Nucleic acids (Neidleand Wood) 668 Engineering and design (Gilardi) 669 Lipids (Newman) 669 Carbohydrates and glycoconjugates (Flitschand Lowden) 670 Biophysical methods (Matthews) 670 Proteins (Kleywegt) 671 Catalysis and regulation (Stewart) • 0•
of special interest of outstanding interest
Protein-nucleic acid interactions Selected by M~.ireConvery*,CaitrionaDennis*and Si&n Rowsellt *Universityof Leeds, Leeds, UK tZeneca Pharmaceuticals,Macclesfield,UK A novel mode of DNA recognition by a B-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. Allen MD, Yamasaki K, Ohme-Takagi M, Tatento M, Suzuki M: EMBO J 1998, 17:5484-5496. • Significance: The solution structure determination of a novel DNA-binding motif, a three-stranded ~ sheet, in complex with DNA. Findings: The authors describe the structure of the DNAbinding domain of a ethylene responsive element-binding protein, which recognises the GCC-box DNA sequence and is related to path•genesis in plants. The domain contains a three-stranded ~ sheet, which has an interruption in the central strand that allows the sheet to follow the curve of the DNA groove and, thus, recognise the seven bases in the DNA sequence. Conventional ~-ribbon motifs cannot span this number of bases due to their intrinsic curvature. A novel DNA-binding motif in MarA: the first structure for an AreC family transcriptional activator. Rhee S, Martin RG, Rosner JL, Davies DR: Proc Nat/ Acad Sci USA 1998, 95:10413-10418. • Significance: The first DNA complex crystal structure of a prokaryotic transcriptional regulator bound to a bipartite helixturn-helix motif is described. Findings: The transcriptional activator MarA has a unique overall fold comprising two structurally homologous helixturn-helix DNA-binding motifs. Sequence specificity is provided by the two recognition helices that insert into adjacent major-groove segments on the same face of the DNA. The two DNA-binding motifs are linked by a helix that serves as a portion of the helix-turn-helix unit. In contrast, eukaryotic transcriptional regulatory factors containing two helix-turn-helix motifs per subunit have flexible loops as
linkers and the two motifs can therefore bind to DNA in various orientations relative to one another. Crystal structure of restriction endonuclease Bgll bound to its interrupted DNA recognition sequence. Newman M, Lunnun K, Wilson G, Greci J, Schildkraut I, Phillips SEV: EMBO J 1998 17:5466-5476. • • Significance: The first crystal structure determination of a class II restriction endonuclease. Bgll binds to an interrupted DNA sequence and cleaves it to yield 3' overhanging ends. Findings: Surprisingly, the core of Bgll, a central six-stranded sheet flanked by (~ helices, is similar to those of two other restriction endonucleases, EcoRV and Pvull; however, the dimerisation interface and subsequent arrangement of the protein subunits in the active dimer are significantly different. This difference in orientation and relative position results in the differing DNA specificities for both recognition and cleavage. Two Ca 2+ ions present in the structure invite speculation about the possible cleavage mechanism. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-~ signalling. Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Ikura Mh Ceil 1998, 94:585-594. • Significance: The recently determined structure of a transforming growth factor (TGF)-~ signalling protein reveals a novel DNA-binding motif. Findings: The 2.8 A structure of the N-terminal MH1 domain of Smad3, a protein involved in the mediation of TGF-~ signalling within the cell, in complex with the Smad-binding element (an eight base pair palindromic DNA sequence), reveals an exposed novel DNA-binding motif. A conserved 11 residue ~ hairpin is observed to make specific contacts to DNA within the major groove. Crystal structure of a human alkylase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Lau AY, Scharer OD, Samson L, Verdine GL, Ellenberger T: Ceil 1998, 95:249-258. 00 Significance: This DNA glycosylase-inhibitor complex structure, in which the glycosylase has a novel fold, provides mechanistic evidence for base excision-repair. Findings: The human alkylase repair enzyme comprises a unique, single domain of mixed o#~ structure and lacks the helix-hairpin-helixmotif common to other base excision-repair proteins. The pyrrolidine inhibitor has flipped out of the DNA duplex and sits in the active site, close to a bound water molecule that is poised for nucleophilic attack. The structure also shows a network of residues around the site that is necessary for catalysis of damaged base displacement from the DNA backbone. Folding and binding Selected by Gary J Pielak Universityof North Carolina,North Carolina,USA Osmolyte-driven contraction of a random coil protein. Qu Y, Bolen CL, Bolen DW: Proc Nat/ Acad Sci USA 1998, 95:9268-9273. • Significance: Osmolytes are important because their protein stabilising effects are completely general, but how do they
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work? The authors provide a clue by uncovering a correlation between the thermodynamics of osmolyte-induced stabilisation and their ability to induce the formation of compact states. Findings: Qu et aL use carboxymethylated RNase to show that several osmolytes induce compaction of the otherwise unfolded protein. Most importantly, the degree of compaction is proportional to the apparent free energy change for transferring the protein from water to an osmolyte solution. Thermal stability of proteins in aqueous polyol solutions: role of the surface tension of water in the stabilizing effect of polyols. Kaushik JK, Bhat R: J Phys Chem B 1998, 102:7058-7066. • Significance: The authors provide another clue to the mechanism of osmolyte-induced stabilization by uncovering a correlation between the surface tension of osmolyte solutions and their stabilizing effect. The transfer free energy effect observed by O.u et al. (see above) and this surface tension effect are related, but the relationship is unclear. Findings: Kaushik et aL quantify the effect of the presence of five sugar alcohols on the thermal stability of five proteins as a function of pH. Osmolyte solutions with the highest surface tensions have the largest stabilizing effect. The structural distribution of cooperative interactions in proteins: analysis of the native state ensemble. Hilser VJ, Dowdy D, Oas TG, Freire E: Proc Nat/Acad Sci USA 1998, 95:9903-9908. • Significance: Under conditions of increasing temperature or denaturant concentration, many proteins denature cooperatively. That is, each molecule is either folded or unfolded. Under native conditions, however, locally unfolded states are populated, so unfolding is no longer cooperative. The authors use a statisticalmechanical model to explain this apparent contradiction. Findings: The model used reproduces NMR-monitored amide proton exchange data for several proteins. Importantly, the model explains why some local stabilities are higher than the corresponding global stabilities. By introducing mutations into the model that affect the energy but not the structure, they identify the essence of the cores of the proteins and explain the experimentally observed onset of cooperativity with increasing temperature and denaturant concentration.
• Significance: The ability to predict the three-dimensional structure of a protein from its primary structure is essential for exploiting data from genome sequencing initiatives. Furthermore, to design useful proteins, we must understand nature's strategies, especially its ability to produce proteins that are tolerant to mutations. This paper sheds light on both issues. Findings: Using collections of low-energy, misfolded forms of 10 natural proteins, the authors show that misfolded conformations with the largest number of similar structures are most like the correctly folded structure.
Macromolecular assemblages Selected by Andreas Engel and J Bernard Heymann UniversitatBasel, Basel,Switzerland Group II chaperonin in an open conformation examined by electron tomography. Nitsch M, Walz J, Typke D, Klumpp M, Essen L-O, Baumeister W: Nat Struct Bio/1998, 5:855-857. • Significance: Chaperonins are key agents required for the correct folding of proteins in cell interiors. They are large complexes that exhibit large movements, allowing interaction with and the modification of substrates. In this paper, the chaperonin from Thermop/asma acidophilum, the thermosome, is shown in an open, nucleotide-deficient conformation. Findings: The three-dimensional structure of the open thermosome was reconstructed from a tilt series of single particles using cryoelectron tomography. The barrel-shaped molecule consists of two rings of eight subunits each, with a big central channel that is able to accommodate large substrates. Each subunit consists of three domains; the equatorial domain is located at the junction between the two rings, with the intermediate and apical domains extending towards the ends. The molecular structure was assembled from the intermediate and equatorial domains of the thermosome homologue, GroEL, together with the apical domain of the (x-thermosome, and fitted into the electron microscopic data. A comparison between the open and closed states of the thermosome indicates a 70° swing of each of the 16 apical domains, closing off the two ends of the central channel. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 .A resolution. Sutton RB, Fasshauer D, Jahn R, BrOnger AT: Nature 1998, 395:347-353. • Significance: The exocytosis of synaptic vesicles is a key event in signal propagation across synapses. The SNARE complex is the core synaptic fusion protein and consists of syntaxin-lA, synaptobrevin-II and SNAP-25B. The formation of this complex precedes the fusion of the synaptic vesicle with the presynaptic membrane and it is targeted by neurotoxins and drugs. Findings: The core complex was obtained by limited digestion of the whole complex, crystallised and solved to atomic resolution. The complex is a parallel four-helix bundle with a length of 120 ~. It forms a loose coiled-coil consisting of two SNAP-25B helices, one syntaxin-lA and one synaptobrevin-II. Syntaxin-lA is anchored in the presynaptic membrane and synaptobrevin is anchored in the vesicular membrane through their transmembrane regions (these were removed by the digestion). The formation of the intertwined bundle is therefore postulated to superimpose the lipid bilayers of the vesicle and the cell, preparing for fusion.
Protein denaturation: a small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c. Segel DJ, Fink AL, Hodgson KO, Doniach S: Biochemistry 1998, 23:12443-12451. • Significance: Chemical denaturation data are often consistent with a two-state model, but should we be concerned that most probes monitor only the native state? In other words, unless a probe is sensitive to non-native states, these states go undetected. Segel et aL show that concern is warranted by using a probe that is sensitive to both native and denatured states. Findings: The conservative analysis of scattering data acquired as a function of guanidinium chloride concentration shows that denaturation is best explained using a three-state model. On the other hand, circular dichroism and fluorescence-detected data are best fit using a conventional two-state model.
Theory and simulation
Clustering of low-energy conformations near the native structures of small proteins, Shortle D, Simons KT, Baker D: Proc Nat/Acad Sci USA 1998, 95:11158-11162.
Selected by membersof MichaelLevitt'slaboratory Stanford University,California,USA The early stage of folding of villin headpiece subdomain observed in a 200-nanosecond fully solvated molecular
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dynamics simulation. Duan Y, Wang L, Kollman PA: Proc Nat/ Acad Sci USA 1998, 95:9897-9902. • Significance: Understanding protein folding remains an experimental as well as a theoretical challenge. Molecular dynamics (MD) simulations possess a unique advantage in such investigations because of their atomic level resolution and accuracy. Due to restrictions of computing time, their scope, however, has so far been limited to short simulation (< 10 ns). This paper presents a new, highly parallel implementation of MD that enables simulations an order of magnitude longer than previous longest simulations. Applications to studying the folding of a short peptide are described. Findings: The folding of the villin headpiece subdomain, a 36 residue peptide, was studied in a single, fully solvated MD trajectory, starting from an unfolded state, over 200 ns. A "burst" phase was observed over the first 60 ns, in which both native contacts and the native helical content increase. A slow "adjustment" phase follows, during which large scale motions can occur. Burial of the hydrophobic surface appears to be the driving force of the collapse observed in the initial burst phase. Pathways to a protein folding intermediate observed in a 1-microsecond simulation in aqueous solution. Duan Y, Kollman PA: Science 1998, 282:740-744. • Significance: The authors extend their initial study (see above) and present a 1 I.l.s fully solvated molecular dynamics (MD) study of the folding of a small peptide, the villin headpiece. It is usually believed that small proteins can fold in the 1-10 tis range; this paper shows that such simulation times are now accessible to MD simulations. Findings: A single MD trajectory over 1 ~s of the folding of the villin headpiece is shown. After the initial "burst" over 60 ns, previously described by the same authors, the protein is shown to reach a marginally stable state, which has a lifetime of 150 ns and shows significant resemblance to the native structure. Over the course of the simulation, escapes from non-native conformations are observed, usually through unfolding of the corresponding structures. The only regret regarding this paper is that this considerable amount of supercomputer time led to just one trajectory. Tertiary structure prediction of mixed (x/I] proteins via energy minimisation. Standley D, Gunn J, Freisner R, McDermott A: Proteins 1998, 33:240-252. • Significance: The authors describe an improved algorithm for protein structure prediction that uses knowledge of exact secondary structure. Such methods are an important precursor of full ab initio protein structure prediction. Findings: The authors present an elaborate method for generating the ab initio fold of possible protein structures. The procedure involves two stages of minimisation. The first employs a simplified protein representation in which helices/strands and loops are represented, respectively, by rigid cylinders and spheres. The population is varied using, among other things, a genetic algorithm. The second minimisation uses a more detailed representation, selects Monte Carlo moves from a loop database and employs a full-atom, mean force energy function including hydrophobicity, a contact potential, steric constraints, a torsional term, a hydrogen bonding term and a disulphide pairing term. The resulting set of structures was clustered and compared with the native fold. The lowest energy conformation had a CA root mean square deviation (rsmd) of 9.05 ,~ for CTF and 9.31 A for BPTI. However, for CTF, the second lowest energy conformation had
a CA rsmd of 4.84 A and for BPTI, the third lowest conformation had a CA rsmd of 5.93 A. Transfer matrix method for enumeration and generation of compact self-avoiding walks. I1. Cubic lattice. Kloczkowski A, Jernigan RL: J Chem Phys 1998, 109:5147-5159. • Significance: Complete enumeration of self-avoiding compact walks on lattices is of great importance in theoretical protein studies. A solution to this problem will help in collecting proper statistics of protein conformations, studying thermodynamic and kinetic properties of protein folding, and predicting protein tertiary structures. Findings: Traditional methods grow lattice walks linearly and only a small fraction of all trials are self-avoiding. The new method constructs lattice walks plane by plane and is attrition free. At each build-up stage, the status of partially constructed walks is characterised by "connectivity", defined as the list of pairs of lattice sites within the current plane that are connected by a chain with a maximum of two ends. The current connectivity state and the bond distribution within the next plane uniquely determines the new connectivity state after the new plane is added. A lattice walk can be uniquely specified by a series of alternating connectivity states and bond distribution states. This method is very efficient for the enumeration of all possible Hamiltonian paths on cubic lattices and can be generalised to incorporate Monte Carlo sampling, irregular boundaries and different types of constraints. Clustering of low-energy conformations near the native structures of small proteins. Shortle D, Simons KT, Baker D: Proc Nat/Acad Sci USA 1998, 95:11158-11162. • Significance: The authors describe a method for selecting a native-like protein conformation from a set of conformations generated by simulation, independent of the energy function. The method uses the hypothesis that there are greater number of low-energy conformations surrounding the correct fold than there are surrounding low-energy incorrect folds. Findings: For 12 proteins, the authors used a set of 500-1000 conformations varying in root mean square deviation (rsmd) from the experimental structure and determined the conformation with the greatest number of neighbours (using a 4 ,a, rmsd cut-off) in the entire set. This conformation was found to be closer to the native structure than the majority of the conformations in the set and, in most cases, is in the top 5% of the conformations, as ranked by their rsmd to the experimental structure. The authors used this result to suggest that proteins in nature may have evolved a native structure surrounded by a large number of low-energy conformations Nucleic acids Selected by Stephen Neidle and Alex Wood The Institute of Cancer Research, Surrey, UK A structural basis for the recognition of A.T and T.A base pairs in the minor groove of DNA. Kielkopf CL, White S, Szewczyk JW, Turner JM, Baird EE, Dervan PB, Rees DC: Science 1998, 282:111-115. • Significance: Polyamide dimers containing pyrrole, imidazole and hydroxypyrrole aromatic rings afford a small molecule recognition code that will discriminate among all four Watson-Crick base pairs in the minor groove. The side-by-side pairing of the residues in an eight-ring polyamide dimer determines the DNA sequence recognised. The structural basis of how a hydroxypyrrole-pyrrole (HpPy) pair distinguishes T.A from A.T and both T.A and A.T from G.C and C.G was established by co-crystallisation
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of the ImHpPyPy-l~-Dp (Im=imidazole, ~ = ~ alanine and Dp = dimethylamino-propylamide) dimer with a self-complementary DNA duplex of sequence 5'-CCAGTACTGG-3'. The non-T.AJA.T selective polyamide ImPyPyPy-J3-Dp bound to the same DNA sequence was examined as a comparison. Findings: In both the ImHpPyPy and ImPyPyPy structures, the polyamides bind as antiparallel dimers centred over the target GTAC sequence in the minor groove of the B-form DNA duplex. Two factors were identified in the discrimination of T.A from A.T. Firstly, the hydroxyl group of Hp and the preceding amid• form hydrogen bonds to thymine 02. This cannot occur with the adenine N3 because it only possesses one free lone pair of electrons. Secondly, adenine is distinguished from thymine by a bulkier aromatic ring. The additional carbon results in an asymmetric cleft in the minor groove of the T.A base pair that is recognised by the Hp hydroxyl.
A preorganized active site in the crystal structure of the Tetrahymena ribozyme. Golden BL, Gooding AR, Podell ER, Cech TR: Science 1998, 282:259-264. =• Significance: This work describes the crystal structure determination at 5 h, resolution of the 247 nucleotide ribozyme of the Tetrahymena thermophila group I intron. Group I introns possess a single active site that catalyses the two sequential reactions of self-splicing. This large ribozyme is found to be largely preorganised for catalysis. This is the first RNA that has been shown to display catalytic activity. Findings: The two domains that form the ribozyme pack together closely to form a shallow cleft that is capable of binding the short helix that contains the 5' splice site. The helix that provides the binding site for the guanosine substrate deviates significantly from A-form geometry to provide a tight binding pocket. Both of these two binding pockets are formed and oriented in the absence of these substrates, leading to the conclusion that, in contrast to small ribozymes such as the hammerhead RNA, group I introns are preorganised to bind to their substrates.
=• Significance: This paper describes a novel development in engineering synthetic, functional molecular maquettesflavin and haem cofactors were both bound to the same synthetic protein scaffold. Key properties of their natural counterparts were observed. Findings: A synthetic tetrameric c~ helix protein of 62 amino acids was engineered to contain a bishistidine-coordinated haem cofactor and a cysteine-linked flavin (?-acetyl-10methylisoalloxazine) in each of the two protein subunits. Spectroscopic data indicate that both redox cofactors are buried within the hydrophobic core of the protein and that, due to the lower redox potential of the flavin, efficient photoinduced electron transfer can occur from the flavosemiquinone to the meso haem (100 ns timescale, -100 meV free energy). Two of the key features of flavoproteins, light activation of the flavin and intramolecular electron transfer from flavin to haem, were achieved in the flavocytochrome molecular maquette. New enzyme lineages by subdomain shuffling. Hopfner KP, Kopetzki E, KreBe GB, Bode W, Huber R, Engh RA: Proc Nat/ Acad Sci USA 1998, 95:9813-9818. == Significance: This is a novel and significant report in the field of generating new enzymes with novel substrate specificities. Findings: The $1 serine protease family has probably evolved by the recombination of domains yielding new functions associated with catalytic sites situated at their interfaces. The architecture of these proteins, comprising two homologous 13-barrel subdomains assembled to form a catalytic site between them, has been exploited for engineering new properties by swapping subdomains from different members of the family. A novel enzyme, fXYa, was generated by the recombination of the N-terminal subdomain from factor X with the C-terminal subdomain from trypsin. The hybrid protein fXYa was expressed in Escherichia coil as inclusion bodies, successfully folded in vitro and its structure was determined at 2.15 h, resolution by X-ray crystallography. Functional characterisation showed high catalytic activity towards a variety of synthetic substrates.
Crystal structure of a hepatitis delta virus ribozyme. FerreD'Amare AR, Zhou K, Doudna JA: Nature 1998, 395:567-574. • • Significance: This self-cleaving ribozyme is the only known catalytic RNA found to date to be involved in the function of a human virus. The structure shows this RNA to have a unique double pseudoknot fold, representing a new category of RNA fold. Findings: The hepatitis delta virus ribozyme was co-crystallised as a complex with the RNA-binding domain of the U1A splice•some, which does not affect its catalytic function, but acts to improve the ordering of the crystals. The crystal structure, determined to 2.3 ,~, resolution, shows five folded helical segments, held together by a double pseudoknot. No metal ions were found in the structure and are not implicated in the selfcleavage reaction. The catalytic site is highly buried and is inaccessible to solvent, in striking contrast to the catalytic site of the hammerhead ribozyme. Positioning of the substrate helix is facilitated by a G.U wobble base pair, which, in turn, is supported by two strand cross-overs from a pseudoknot.
Switching the amino acid specificity of an aminoacyl-tRNA synthetase. Agou F, Quevillon S, Kerjan P, Mirande M: Biochemistry 1998, 37:11309-11314. • Significance: A novel and interesting example of an engineered molecular switch in the amino acid specificity of an aminoacyl-tRNA synthetase. Findings: Aminoacyl-tRNA synthetases recognise their specific substrate through a proofreading reaction that ensures accuracy in protein biosynthesis. Glutamyl and glutaminyl tRNA synthetases (GluRS and GInRS, respectively) are derived from genes that evolved through the duplication of an ancestral gene and further specialised into the two related enzyme activities. In this work, a mutant GInRS was engineered to preferentially aminoacylate tRNA with glutamate instead of glutamine; not only the transition state but also the proofreading was switched by the mutation.
Engineering and design Selected by Gianfranco Gilardi ImperialCollegeof Science, Technologyand Medicine,London, UK Design, synthesis and characterisation of a photoactivatable flavocytochrome molecular maquette. Sharp RE, Moser CC, Rabanal F, Dutton PL: Proc Nat/Acad Sci USA 1998, 95:10465-10470.
Identification of a meander region proline residue critical for heme binding to cytochrome P4so: implications for the catalytic function of human CYP4BI. 7heng YM, Fisher MB, Yokotani N, Fujii-Kuriyama Y, Rettie AE: Biochemistry 1998, 37:12847-12851. -= Significance: This work is an excellent example of the potential of "analytical" protein engineering in studying the
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structure-function relationship of extremely relevant enzymes, such as cytochrome P45o. Findings: A conserved proline residue was identified by sequence alignment of P45o genes. This is situated two residues prior to the Glu-Arg-Arg triad of the meander region, which is thought to be important for haem binding. Reversion of a serine present in this position in the human P45o gene from lung and placenta (CYP4B1) into the conserved proline resulted in enzymes that are able to incorporate haem. Similarly, conversion of this proline into serine in rabbit CYP4B1 produced an enzyme that is unable to bind haem. The haem competent CYP4B1 was found to catalyse the hydroxylation of fatty acids preferentially at the (o position, with rates of hydroxylation in the order C1 2>C10>C9>C8; 1 2-hydroxylauric acid is formed at rates of 17-21 min-1. This work proves that the consensus sequence Pro-X-Arg is critical for haem incorporation.
Engineering an intertwined form of CD2 for stability and assembly. Murray AJ, Head JG, Barker JJ, Brady RL: Nat Struct Biol 1998, 5:778-782. == Significance: A novel and significant work whereby a natural protein domain, the N terminus of a cell adhesion molecule, can be engineered through a delicate balance of noncovalent interactions and the design of hinges to give different foldings through monomeric, dimeric and tetrameric forms. Findings: The N-terminal domain of the cell adhesion molecule from the surface of T-lymphocytes, CD2, can fold into a monomeric or intertwined head-to-tail dimeric form. Only 150/o of the recombinant protein is found to be the latter form, but drastic changes in the relative proportions of the two forms occurred by mutating residues within the hydrophilic core at the interface between the two monomers. In addition, X-ray crystallography revealed that the hinge deletion mutant A46A47, which is stable as an intertwined dimer, assembles into a tetramer. This study shows how a single polypeptide chain can be differently stabilised towards different folding pathways through simple mutations. This may reproduce evolutionary strategies, through delicate balances that could bear important biological relevance, offering new opportunities for supramolecular engineering.
Lipids Selected by Richard Newman ImperialCancer Research Fund, London, UK Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late step of the conformational maturation of the polytopic membrane protein lactose permease. Bogdanov M, Dowham W: EMBO J 1998, 17:5255-5264. • Significance: This paper gives evidence that phosphatidylethanolamine (PE) acts as a molecular chaperone for lactose permease (LacY), leading to speculation that specific phospholipids can effect membrane protein folding. Findings: The insertion of LacY into the membrane prevents its irreversible aggregation. PE participates in a late step of its conformational aggregation. The temporal requirement for PE was shown using in vitro coupled translation-membrane insertion assays, which distinguish between membrane insertion and phospholipid-assisted folding. LacY folded properly when synthesised in the presence of PE-containing inside out membrane vesicles, but not when synthesised in vesicles lacking PE. Critical folding steps occurring after membrane insertion
are dependent on the interaction of LacY with PE to prevent illicit interactions that lead to the misfolding of LacY.
The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties, Zawadki J, Scholz C, Currie G, Coombs GH, McConville MJ: J Mol Bio/ 1998, 282:287-299. • Significance: Sandfly-transmitted protozoa of the genus Leishmania cause a wide spectrum of human diseases. Their survival in mammalian hosts is linked to coat glycoinositolphospholipids (GIPLs) and glycosylophosphatidylinositol (GPI)-anchored proteins. The L. panamensis GIPLs are useful reagents for understanding the role of parasite GPIs in mammalian cells. Findings: The cell surface of Leishmania parasites is coated by G PI-anchored macromolecules and a polymorphic family of free GPI glycolipids or GIPLs. The structures of these glycolipids were determined using gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences with galactose extensions, and the phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features of the GIPLs of Leishmania spp.
Carbohydrates and glycoconjugates Selected by Sabine L Flitsch and PhilipAS Lowden Edinburgh University,Edinburgh, UK Insights into transition state stabilisation of the 13-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants. Notenboom V, Birsan C, Nitz M, Rose DR, Warren A J, Withers SG: Nat Struct Biol 1998, 5:812-818. • Significance: This paper provides the first structure determination of a retaining glycosidase in which a covalent glycosyl-enzyme intermediate is observed with a natural substrate. This reveals a potentially important role for the 2-hydroxyl in transition-state stabilisation. Findings: Previous structural studies of a fluorinated glycosyl-enzyme intermediate suggested a crucial role for His205 in the active site of Cex. Kinetic analysis of His205 mutants confirmed that this residue plays a key role in the hydrolysis of the glycosyl-enzyme intermediate. Further stabilisation of the intermediate in the H205N/E127A double mutant allowed the determination of its crystal structure, in which a short hydrogen bond is observed between the 2-hydrexyl and the glutamic acid nucleophile.
The polysialic acid units of the neural cell adhesion molecule N-CAM form filament bundle networks, Toikka J, Aalto J, H~yrinen, Pelliniemi LJ, Finne J: J Bio/Chem 1998, 273:2855728559. • Significance: The first and unexpected observation of the assembly of cell-surface polysialic acid into filamentous bundles. This suggests an associative role for polysialic acid in cell-cell interactions, in addition to their previously suggested repulsive roles. Findings: Oligomers of 6, 9, 12, 15 and 18 residues of (x-2,8 linked N-acetylneuraminic acid (dried on mica surfaces) were imaged by atomic force microscopy. Filamentous structures were observed for oligomers of 12 or more sugar units, with extensive branching occurring as the chain length increased.
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Biophysical methods Selected by Steve Matthews Imperial College of Science, Technologyand Medicine, London, UK Identification of protein folding patterns using site-directed spin labelling. Structural characterisation of a B-sheet and putative substrate binding regions in the conserved domain of (xA-crystallin, Koteiche HA, Berengian AR, Mchaourab HS: Biochemistry 1998, 37:12681-12688. • Significance: A major obstacle to understanding the mechanisms by which ~-crystallins and heat-shock proteins mediate their chaperone-like function is the lack of structural information on the oligomeric protein scaffold. A strategy involving sitedirected spin labelling (SDSL) and electron spin resonance (ESR) is described that enables recognition of the topology. Findings: A nitroxide scan was performed in order to search for putative binding sites within (xA-crystallin. Nitroxide mobility and solvent accessibility were measured and analysed. The data reveal a triple-stranded, antiparallel ~ sheet. One of the substrate-binding sites overlaps a buried loop, suggesting that the transient exposure of this loop may be responsible for the thermal initiation of binding. These results provide, for the first time, structural insight into the molecular recognition processes that are responsible for o~-crystallin function. Sedimentation analysis of noninteracting and self-associating solutes using numerical solutions to the Lamm equation. Schuck P: Biophys J 1998, 75:1503-1512. • Significance: Analytical ultracentrifugation has re-emerged as powerful technique for the characterisation of protein-protein and protein-nucleic acid interactions in solution; however, the dynamic information it provides has not been exploited to its full potential. The authors describe a method for obtaining this information. Findings: This paper describes the numerical solution of the Lamm equation using a moving grid approach. Only this method has the potential to obtain hydrodynamic shape information from analytical ultracentrifugation experiments on complexes. NOE pumping: a novel NMR technique for identification of compounds with binding activity to macromolecules. Chen A, Shapiro MJ: J Am Chem Soc 1998, 120:10258-10259. • Significance: The drug discovery process involves the screening of compound libraries in order to identify potent binding to macromolecules. NMR spectroscopy has recently emerged as an invaluable tool for the rapid screening for biological activity. A novel extension to the affinity NMR technique is proposed. Findings: Traditional affinity NMR requires that there be a large difference between the translational diffusion of the ligand and target macromolecule. This criterion is not always satisfied because the diffusion coefficients represent a weighted average of the bound and free states. This new experiment uses the nuclear Overhauser effect (NOE) to transfer signal from the receptor to the bound ligands. The method was used to identify a bound ligand, salicylic acid, from unbound ligands on human serum albumin.
Proteins Selected by Gerard J Kleywegt Uppsala University, Uppsala, Sweden The atomic structure of the bluetongue virus core, Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, Zi6ntara S, Mertens PPC, Stuart Dh Nature 1998, 395:470-478. • o Significance: The structure of the bluetongue virus (B'I'V) core extends the classical conceptual framework of virus assembly.
Findings: The BTV core, 700 ,~ in diameter, represents the largest structure determined by crystallographic methods to date. The 3.5 ,~, structure reveals how roughly 1000 protein components self-assemble. The fact that 120 copies of one protein, VP3(T2), can assemble into an icosahedral shell is incongruous with the classical framework of Caspar and Klug. The structure reveals that this assembly, in addition to the classical mechanism of quasi-equivalent contacts, is achieved through a new mechanism called geometrical quasi-equivalence. (Protein Data Bank code 2BTV.) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Sutton RB, Fasshauer D, Jahn R, Br~inger AT: Nature 1998, 395:347-353. • Significance: This paper sheds light on the structural role of SNARE complexes in the fusion of vesicles with their target membranes. Findings: The term SNARE refers to a set of conserved proteins that mediates the fusion of a vesicle with its target membrane. The structure of this complex is a heterotetramer of four SNARE protein fragments. This synaptic fusion complex folds into a highly twisted, parallel four-helix bundle with a length of 120 A. The surface of the complex is grooved and reveals charged, hydrophilic and hydrophobic areas that may be important for membrane fusion. A hypothetical model is proposed for the synaptic fusion complex as it joins two membranes. (Protein Data Bank code 1SFC.) Structure of a glutamate-receptor ligand-binding core in complex with kainate. Armstrong N, Sun Y, Chen GQ, Gouaux E: Nature 1998, 395:913-917. • Significance: The structure determination of the glutamate receptor ligand-binding core provides a framework for the design of new drugs. Findings: Ionotropic glutamate receptors play an important role in the development of the nervous system and in memory and learning. They have been implicated in a variety of disorders, including schizophrenia, epilepsy, Alzheimer's, Huntington's and Parkinson's disease. The ligand-binding core consists of two ~/~ domains, with the ligand-binding site situated between them, an overall structure similar to that of glutamine-binding protein. Surprisingly, the ligand-binding site has a negative electrostatic potential. The structure reveals the major determinants of receptor-ligand interactions, as well as the role of remote residues in determining specificity and affinity. (Protein Data Bank code 1GR2.) Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Curry S, Mandelkow H, Brick P, Franks N: Nat Struct Biol 1998, 5:827-835. • Significance: This structure determination answers the longstanding question of where fatty acids bind on serum albumin. Findings: Serum albumin is the most abundant protein in the circulatory system. It is the major transport protein for unesterifled fatty acids, but it also binds a wide range of drugs, metabolites and organic compounds. Although the structure of serum albumin has been known since 1992, the number and location of its many fatty-acid-binding sites remained uncertain. This current structure reveals the location of five myristate-binding sites. Although serum albumin consists of three similar and homologous domains, the distribution of the binding sites turns out to be surprisingly asymmetric. The carboxylate moieties.of
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the fatty acids all form salt links to arginine or lysine residues, and hydrogen bonds with serine or tyrosine residues. (Protein Data Bank codes 1BJ5 and 1BKE.)
Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction. Banfield MJ, Barker JJ, Perry ACF, Brady RL: Structure 1998, 6:1245-1254. • Significance: An attempt is made to obtain clues regarding protein function from the determination and analysis of the crystal structure. Findings: In the future, sequence and structure information may well become more abundant than information regarding the functional role of gene products. This paper is an attempt to shed light on the function of phosphatidylethanolamine-binding protein (PEBP) through the analysis of its structure. The PEBP family consists of highly conserved proteins that do not resemble any other proteins of known structure or function and whose function is unclear. PEBP turns out to have a novel fold. It occurs as a dimer that has an appreciable dipole moment, suggesting how the dimer might interact with the membrane. A ligand-binding site is identified (occupied by cacodylate), which could accommodate the phosphate head groups of membrane lipids. Hence, the protein may play a role in relaying signals from the membrane to the cytoplasm. (Protein Data Bank codes 1BD9 and 1BEll.) Catalysis and regulation Selected by Jon D Stewart Universityof Florida,Gainesville,Florida,USA Snapshots along an enzymatic reaction coordinate: analysis of a retaining (x-glycoside hydrolase. Davies G J, Mackenzie L, Varrot A, Dauter M, Brzozowski AM, Sch(Jlein M, Withers SG: Biochemistry 1998, 37:11707-11713. *- Significance: This study brings together very high resolution X-ray crystal structure data and kinetic studies in order to provide a highly detailed description of the catalytic mechanism employed by a prototypical glycosidase. This mechanism is likely to be very similar to those employed by other enzymes in this family. Findings: In common with many retaining (x-glycosidases, the Cel5A enzyme from Bacillus agarahaerens forms a covalent intermediate involving an o~linkage with an active site carboxylate (Glu228), following a transition state that involves a high degree of charge build up at the anomeric carbon. The crystal structure of the Michaelis complex showed that the substrate was distorted into a skew boat conformation that allows for better stabilisation of the positively charged transition state by neighbouring groups. In contrast, the crystal structure of the covalent intermediate revealed a typical chair conformation for the pyran ring, as well as a water molecule that is positioned properly for nucleophilic attack above the anomeric carbon to liberate the final product. Structures of the free enzyme and enzyme-product complexes were also determined. This collection of structures thereby surveys the entire reaction coordinate for glycosyl hydrolysis and includes data for all isolateable species. Chemical modifications at a single site can induce significant shifts in the pH profiles of a serine protease. DeSantis G, Jones JB: J Am Chern Soc 1998, 120:8582-8586.. • Significance: This paper describes one of the simplest general approaches for tailoring the pKa values of specific enzyme functional groups by altering their local environment.
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Findings: Bacillus lentus subtilisin features a typical catalytic triad, in which Ser221 (the key nucleophite) is hydrogen bonded to His64, which also forms a hydrogen bond with Asp32. The pH-rate profile for the wildtype enzyme (as measured by kcat/KM) features a pKa of 701 that was assigned to the ionisation of His64. By replacing Asn62 with cysteine and then derivatising it with long chain alkylthiol reagents that formed mixed disulphides with the cysteine sidechain, the local environment of His64 was altered and the macroscopic pKa values of the derivatised enzymes were lowered relative to the wildtype (maximum difference of 0.72 pH units). Moreover, there was a linear correlation between the macroscopic pKa values and the calculated log P values (partition coefficient between water and n-octanol) for the derivatising reagents, consistent with the notion that hydrophobicity largely accounted for the variations in pKa values. A method for directed evolution and functional cloning of enzymes. Pedersen H, H~lder S, Sutherlin DP, Schwitter U, King DS, Schultz PG: Proc Natl Acad Sci USA 1998, 95:10523-10528. • Significance: This paper describes methodology that allows bacteriophage display libraries to be screened directly for catalytic efficiency. The approach is generally applicable provided that the catalyst can be expressed in its active form as a fusion protein with the gene III minor coat protein (gplll) of M13 phage. Findings: In order to show that the widely used phage display approach could be extended to allow direct selection for catalysis, Staphylococcal nuclease was fused to the gene III minor coat protein of M13 phage. The helper phage used to produce the nuclease-containing phage particles had been modified by fusing an acidic peptide to the N terminus of the gene III protein. The resulting phage particles each contained one or no copies of the acidic peptide-gene III fusion, as well as four to five copies of the nuclease-gene III fusion. These phage particles were mixed with a basic peptide known to form a stable heterodimeric coiled-coil with the acidic peptide and a biotinylated DNA substrate was covalently linked to the basic peptide. Immobilised streptavidin captured only those phage particles that had not cleaved the DNA substrate attached to their surface. Model studies showed that this strategy allowed a 100-fold enrichment of catalytic versus noncatalytic phage particles. Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria. Koizumi S, Endo T, Tabata K, Ozaki A: Nat Biotech 1998, 16:847-850. • Significance: Many glycosyltransferases suitable for largescale oligosaccharide synthesis have been cloned and overexpressed; however, these syntheses are not yet practical because of the high cost of most sugar nucleotides. This paper describes a simple method that solves this problem and makes UDP-galactose available in large quantities. Findings: Two recombinant bacteria were prepared - an Escherichia coli strain overexpressing the U DP-galactose biosynthetic enzymes (galactose-l-phosphate uridyltransferase, galactokinase, glucose-l-phosphate uridyltransferase, and pyrophosphatase) and a Cornyebacterium ammoniagnes strain that overproduced UTP from orotic acid. The membranes of both cell types were permeabilised and then they were mixed together, along with orotic acid, galactose and glucose (to serve as an energy source for UTP production). On a 2.51 scale, this system accumulated 4? g/I of UDP-galactose in 26 hrs. The methodology was extended to the production of globotriose, which accumulated to levels of 188 g/I after 36 hrs.
