- 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. • ••
of special interest of outstanding interest
Current Opinion in Structural Biology 2002, 12:685–691 Contents (chosen by) 685 Protein–nucleic acid interactions (Convery et al.) 685 Folding and binding (Sood and Kortemme) 685 Theory and simulation (Fiser) 686 Nucleic acids (Parkinson) 686 Sequences and topology (Copley) 687 Engineering and design (Gilardi) 688 Membranes (Newman and Sinning) 689 Biophysical methods (McCoy and Dafforn) 690 Proteins (Miele) 690 Catalysis and regulation (Stewart)
Protein–nucleic acid interactions Selected by Stephen Carr*, Máire Convery† and Caitríona Dennis* *University of Leeds, Leeds, UK †GlaxoSmithKline, Stevenage, UK e-mail: [email protected]
• The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. Vannini A, Volpari C, Garfioli C, Muraglia E, Cortese R, De Francesco R, Neddermann P, Di Marco S: EMBO J 2002, 17:4393-4401. Significance: The first ternary complex structure comprising a transcriptional regulator, TraR, its autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone, and its target DNA. Findings: TraR forms a two-domain asymmetric homodimer, with both domains showing structural similarity to previously determined structures. The autoinducer is completely buried within an enclosed cavity of the ligand-binding domain, suggesting that binding of inducer is required for correct folding of the regulator. The DNA-binding domain interacts with DNA through a HTH motif from each protein of the dimer. •• BRCA2 function in DNA binding and recombination from BCRA2-DSS1-ssDNA structure. Yang H, Jeffrey PD, Miller J, Kinnucan E, Song Y, Thomä NH, Zheng N, Chen P-L, Lee W-H, Pavletich NP: Science 2002, 297:1837-1848. Significance: The structure of BRCA2 (breast cancer susceptibility gene 2) bound to DNA provides a basis for understanding the loss of recombination-mediated repair of DNA in BRCA-associated cancers. Findings: BRCA2 tumour suppressor mutants are defective in the repair of dsDNA breaks (DSBs); the crystal structure reveals that BRCA2 plays a direct role in this process. The structure reveals three oligonucleotide-binding (OB) folds and a HTH motif. The OB folds are shown to bind ssDNA in an extended conformation, with the bases located in a channel at the surface of the protein.
Biochemical analysis also reveals that the HTH motif interacts with dsDNA and that BRCA2 stimulates RAD-51-mediated recombination in vitro. Most mutations leading to BRCA-linked cancers map to the DNA-binding interfaces within the protein, suggesting a direct role for BRCA in the detection of DSBs in the chromosome.
Folding and binding Selected by Vanita D Sood* and Tanja Kortemme† University of Washington, Seattle, Washington, USA *e-mail: [email protected] †e-mail: [email protected]
•• Modular recognition of RNA by a human pumilio-homology domain. Wang X, McLachlan J, Zamore PD, Hall TM: Cell 2002, 110:501-512. Significance: Sequence-specific interactions with the untranslated regions of mRNAs are critical for many developmental and regulatory processes in the cell. This structure demonstrates how sequence specificity is achieved for an important group of RNA-binding proteins, the Puf family of proteins. Findings: The crystal structure of human Pumilio in complex with a Nanos response element reveals that each base in the RNA is recognised by three amino acids via a sequence-specific combination of stacking, hydrogen bonding and van der Waals interactions. These amino acid ‘triplets’ are each situated on a separate repeat of Pumilio, which consists of eight structural repeats. Substituting one repeat for another changes the sequence specificity in a predictable manner, opening the possibility of ‘designer’ proteins that recognise specific messages. • The rate-limiting step in the folding of a large ribozyme without kinetic traps. Fang X-W, Thiyagarajan P, Sosnick TR, Pan T: Proc Natl Acad Sci USA 2002, 99:8518-8523. Significance: The study of RNA folding has been hampered by the fact that most large RNAs fold very slowly because of the formation of kinetic traps. The authors use the catalytic domain of RNase P, which folds without kinetic traps, to perform a detailed study of RNA folding kinetics. Findings: Adopting the traditional tools used to study protein folding kinetics, Fang et al. determine that the rate-limiting step in C-domain folding is a small conformational change involving, most probably, a small-scale rearrangement or dehydration of prebound metal ions. This is in contrast to other RNAs, whose folding rate is limited by kinetic traps or by the formation of long-range tertiary contacts.
Theory and simulation Selected by Andras Fiser Rockefeller University, New York, New York, USA e-mail: [email protected]
•• Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. Day R, Bennion BJ, Ham S, Daggett V: J Mol Biol 2002, 322:189-203.
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Significance: An extensive molecular dynamics (MD) simulation study established that high-temperature simulations are relevant in describing the energy landscape of protein unfolding processes. Findings: Traditionally, MD studies of unfolding events use a drastically high temperature to speed up simulations. This approach is necessary to compensate for the difference between the experimental timescale and the much shorter computer simulation timescale. However, it is often questioned whether unrealistically high temperature simulations are at all relevant to the study of the energy landscape. The authors investigated this question in an extensive MD simulation study of chymotrypsin inhibitor using seven different unfolding temperatures between 298 and 495K, and up to 94 ns simulation time. All the simulations followed a similar order of unfolding events, regardless of temperature, which affected only the timescale of transitions. Simulations below the experimentally established melting temperature retained the native conformation of the protein. The authors concluded that using elevated temperatures in unfolding simulations only activates the process, but does not affect its pathway. • MAMOTH (Matching molecular models obtained from theory): an automated method for model comparison. Ortiz AR, Strauss CEM, Olmea O: Protein Sci 2002, 11:2606-2621 Significance: A sequence-independent, fast and accurate method is presented that compares low-resolution protein structures. Findings: In the post-genomics era, when protein structures and models are produced by high-throughput experimental and theoretical methods, it is necessary to have fast, flexible and accurate methods to compare vast numbers of three-dimensional molecular models. The authors introduce a new such method, MAMOTH. The underlying idea of the approach is that a prediction is successful if the modelled structure is significantly more similar to the target fold than any other known fold. Consequently, the evaluation of a superposition needs to be consistent with the classification of experimental protein structures to folds. To assign a probability score to the quality of the superposition, it is compared with scores of randomly superposed alignments. The superposition method is robust, independent of sequence, secondary structure and contact map, and also insensitive to sequence length differences. Although it is much faster, its performance is competitive with other known approaches, such as DALI, VAST and PRISM. • A model binding site for testing scoring functions in molecular docking. Wei BQ, Baase WA, Weaver LH, Matthews BW, Schoichet BK: J Mol Biol 2002, 322:339-355. Significance: Although theoretical docking studies are becoming increasingly important, the development and evaluation of their scoring function are confronted with the problem of accurately testing their effect. This study uses a prototype model binding system to test modifications to a docking scoring function. Findings: The docking of small molecules on protein-binding sites remains a major challenge. Large-scale studies are often facilitated by computational screening of potential ligand candidates. The current study focused on the well-characterised experimental system of T4 lysozyme. Mutation-created binding sites were introduced to test how atomic charges and solvation energies affect the molecular docking of 172 118 small molecules. Various currently available scoring functions were employed. The best seven docking predictions were tested experimentally on artificially created apolar and polar binding
sites. All seven ligands bound to the polar cavity, but did not detectably bind to the apolar one. Five ligand-bound structures were determined by X-ray crystallography and the models were predicted within 0.4 Å rmsd of the experimental solutions.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Fulham, UK e-mail: [email protected]
• Specificity of RNA-RNA helix recognition. Battle DJ, Doudna JA: Proc Natl Acad Sci USA 2002, 99:11676-11681. Significance: A thermodynamic and crystallographic investigation, within the Tetrahymena group I self-splicing intron, into A-minor motif specificity. This motif involves interactions between unpaired adenosine residues and the minor groove of receptor helices, and may play a role in RNA architecture and recognition. Findings: Thermodynamic data showed that the A-minor interaction is not greatly disrupted by the substitution of various canonical base pairs in the receptor helix, thus showing little selectivity. Crystallographic analysis of a P4–P6 domain variant, C•G to G•C, also reveals no significant structural rearrangement upon transversion. The use of native gel electrophoresis on all 12 possible base mismatches revealed pyrimidine•pyrimidine mismatches as the least preferred. Canonical base pairs are strongly preferred over base mismatches in the A-minor interaction through recognition of Watson–Crick minor groove geometry.
Sequences and topology Selected by Richard Copley Wellcome Trust Centre for Human Genetics, Oxford, UK e-mail: [email protected]
• De novo prediction of three-dimensional structures for major protein families. Bonneau R, Strauss CEM, Rohl CA, Chivian D, Bradley P, Malmström L, Robertson T, Baker D: J Mol Biol 2002, 322:65-78. Significance: The application of a good fold prediction method that uses no structural information to a large number of protein families. Findings: The Baker group’s ‘Rosetta’ method for de novo protein fold prediction has performed well in Critical Assessment of Structure Prediction (CASP) trials. In this paper, the authors first assess the probable reliability of the method using known protein structures and then go on to perform a large-scale structure prediction exercise on all protein families shorter than 150 amino acids in the PFAM-A database. In the absence of experimental confirmation of structure, it is difficult to assess how accurate the results are, but several examples are presented that appear plausible and the authors envisage that they may help frame functional hypotheses. The value of the method is that it can be applied in cases in which all other techniques have failed. • Loopy proteins appear conserved in evolution. Liu J, Tan H, Rost B: J Mol Biol 2002, 322:53-64. Significance: An unusual analysis of regions of proteins containing no regular secondary structure. Findings: ‘Loopy’ regions were defined as those that contain 70 or more consecutive residues with no regular secondary
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structure (NORS). Such regions were identified in proteins of known 3D structure and a method developed to predict them from sequences. This method was then applied to the predicted protein sets of complete genomes. Five times as many NORS were found in eukaryotes than in prokaryotes. These regions were found to be just as conserved as flanking protein sequences, typically had more interaction partners than other proteins (based on two-hybrid data) and often had functions related to regulation and transcription. The authors speculate that their conformational flexibility makes them amenable to new kinds of regulation.
Engineering and design Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK e-mail: [email protected]
• Structure-based Combinatorial Protein Engineering (SCOPE). O’Maille PE, Bakhtina M, Tsai MD: J Mol Biol 2002, 321:677-691. Significance: This paper proposes a new protein engineering approach based on structural information combined with existing directed evolution methods. Findings: In this work, the authors take two distantly related DNA polymerase enzymes, Pol X and Pol β, which share low sequence homology but have some similar secondary structure, and create a cross-over library from the two genes, based on equivalent secondary structure elements. The resulting library was tested using complementation in E. coli and several novel polymerases were found. It is seen that both the secondary structure elements and the linkages between them are important in determining activity. This approach could be useful for completing the range of application of directed evolution techniques, as it is homology independent but does not rely simply on enzymatic digestion of the gene. • Stable self-assembly of a protein engineering scaffold on gold surfaces. Terrettaz S, Ulrich WP, Vogel H, Hong Q, Dover LG, Lakey JH: Protein Sci 2002, 11:1917-1925. Significance: A mutant of the E. coli protein OmpF is created that binds directly to gold surfaces and provides an interesting anchor to link other proteins by further engineering. Findings: The pore-forming outer membrane protein OmpF is engineered by the introduction of a single cysteine residue on the periplasmic surface. The resulting mutant is deposited onto a gold surface that had been previously treated with β-mercaptoethanol. Once the protein is immobilised, thiolipids are added, forming a proteolipid layer at the gold surface. The conformation of OmpF was tested using binding of the R-domain of the toxin colicin N, which is monitored by surface plasmon resonance and ion channel blockage. The gold surface denatures some of the otherwise highly robust OmpF, but functional protein is still present at high density. Although this technique could be applied to other outer membrane proteins, the interest here from a protein engineering standpoint lies in the possibility of creating OmpF fusions that can be easily purified and assembled onto gold surface. This could have applications in the field of biosensor construction. • A positive charge preservation at position 116 of αA-crystallin is critical for its structural and functional
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integrity. Bera S, Thampi P, Cho WJ, Abraham E: Biochemistry 2002, 41:12421-12426. Significance: Reports of autosomal dominant congenital cataract associated with a missense mutation (R116C) in the sequence of human αA-crystallin are further examined in this study. The study demonstrated that preservation of a positive charge at position 116 is critical in preserving the structure and function of the protein. Findings: R116 in human αA-crystallin was mutated to lysine (R116K), cysteine (R116C), glycine (R116G) and aspartic acid (R116D). Wild-type and mutant proteins were expressed, purified and characterised by measurements of circular dichroism, tryptophan fluorescence, TNS fluorescence and chaperone function. The mutant R116K had similar structure and function to the wild type. However, mutation of the residue to an acidic residue (R116D) led to drastic changes in protein structure. Mutation of the arginine to cysteine or glycine showed similar changes in structure, oligomerisation and chaperone function, thereby suggesting that the presence of the cysteine does not cause the changes. Therefore, a positive charge at residue 116 is essential for preserving the structure and function of αA-crystallin. • Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding. Xu Y, Dewanti AR, Mitra B: Biochemistry 2002, 41:12313-12319. Significance: Using site-directed mutagenesis, the role of the conserved residue Arg165 in (S)-mandelate dehydrogenase and the overall importance of the Arg165/Arg277 pair were examined. It was found that, although Arg165 plays a role in substrate binding and catalysis, its role is not as important as that of Arg277. Findings: Single mutants at Arg165, as well as double mutants at Arg165 and Arg277, were characterised kinetically and functionally. The results showed that Arg165 stabilises the transition state through its positive charge and the ground state through a charge-independent interaction. The kcat for the R165K/R277K mutant was found to be ~350-fold lower than that of the wild type. This demonstrated that at least one arginine residue is required for optimal substrate orientation and catalysis. pH studies showed that a pKa of 9.3 in the free wild-type enzyme did not belong to Arg165 and suggested that it belongs to Arg277. Therefore, Arg277 is the more critical residue in terms of catalysis and substrate binding in (S)-mandelate dehydrogenase. • Rational cytokine design for increased lifetime and enhanced potency using pH-activated ‘histidine switching’. Sarkar CA, Lowenhaupt K, Horan T, Boone TC, Tidor B, Lauffenburger D: Nat Biotechnol 2002, 20:908-913. Significance: The authors provide a rational strategy through which more potent analogues of specific protein therapeutics might be designed. Findings: Drugs typically rely on the ability of ligands to bind tightly to their target. However, the sorting of complexes involving growth factors or cytokines is related to the endosomal ligand–receptor binding affinity, with complexes that remain bound to the receptor generally becoming degraded and those that dissociate being recycled. Redesigning a cytokine to decrease endosomal affinity after internalisation could reduce receptor down-regulation and ligand depletion, enhancing drug effectiveness. The rationale takes advantage of the pH decrease from 7 to 5–6 in the endosomes, and is based on the
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principle that neutral histidine could maintain relatively tight binding on the cell surface, but protonated histidine could lead to weaker binding in the endosomal compartment. Candidate mutation sites were identified in areas of excessive negative charge density and electrostatic complementarity of the granulocyte colony-stimulating factor (GCSF) and its receptor. Calculation of the electrostatic contribution for the wild type and each of the histidine mutants shows that, for three of them (E20H, D110H and D113H), the charged form has about 5 kcal/mol higher binding free energy than the neutral form. The Kd determined for the selected candidates (D110H and D113H) shows that, although binding is unaffected at pH 7.4, it weakens at pH 5.5 (Kd up to sevenfold higher at pH 5.5 for D113H). Tests on ligand depletion demonstrate that each of the mutants shows a much longer half-life (>500 hr) than the wild type (50 hr). Finally, an enhancement of ligand recycling in the case of the two mutants was found, validating the goal set by the authors. • Conversion of a transmembrane to a water-soluble protein complex by a single point mutation. Tsitrin Y, Morton CJ, El Bez C, Paumard P, Velluz MC, Adrian M, Dubochet J, Parker MW, Lanzavecchia S, Van Der Goot FG: Nat Struct Biol 2002, 9:729-733. Significance: This paper presents the conversion of the pore-forming toxin aerolysin into a soluble complex by simple point mutation. Findings: Pore-forming aerolysin is a four-domain protein that undergoes C-terminal proteolytic cleavage and oligomerisation into a ring-like heptameric structure with amphipathic properties that allow it to insert into the lipid membrane. The upper boundary of domain 4 is delineated by a row of three aromatic residues (Y298, F410 and Y221), which are reminiscent of the ‘aromatic belt’ found in other membrane proteins that is thought to anchor and stabilise the proteins in the bilayer. The authors analyse the importance of these aromatic residues by mutating each to a glycine and show that the Y221G mutation completely blocked haemolysis. CD spectra of Y221G show no difference to those of the wild type. On the contrary, the near-UV CD spectrum of Y221G heptamers differed from that of the wild type, indicating that their tertiary structures must differ, despite the similarity of their secondary structures. Cryonegative staining EM shows that the wild-type heptamers formed irregular, mostly aggregated structures and micelle-like structures. In contrast, regular structures were observed for the Y221G mutant, suggesting that these heptamers do not expose hydrophobic surface, as further confirmed by ANS binding experiments. The 13.5 Å model generated from the EM data provides some clues on the conversion of the toxin to a membrane-bound state. • Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin. Goh YH, Frecer V, Ho B, Ding JL: Protein Eng 2002, 15:493-502. Significance: Virtual mutagenesis was used to design mutants of enhanced green fluorescent protein (EGFP) with high affinities for bacterial endotoxin, lipopolysaccharide (LPS) and its bioactive component, lipid A (LA). The most successful rationally designed EGFP mutant, G10, captured LA with a dissociation constant of 8.5 µm and exhibited the highest attenuation of fluorescence intensity in the presence of LPS/LA. Findings: Previous work by this group has shown that LPS or LA can interact with and bind to short cationic sequences
containing symmetrical amphipathic β-sheet motifs composed of alternating basic, hydrophobic and polar residues with dissociation constants in the micromolar range. Virtual mutagenesis of an EGFP model suggested the most likely position of this motif for successful LPS/LA detection. Several mutants of EGFP (G10–G12) were then made and mutant G10, which has amino acids 200–204 (YLSTQ) mutated to KLKTK, was found to have the highest level of fluorescence quenching in the presence of LPS/LA. Further development of the G10 mutant may provide the basis for a fluorescent biosensor of bacterial endotoxin. • Excretion of human β-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli. Jeong KJ, Lee SY: Appl Environ Microbiol 2002, 68:4979-4985. Significance: A novel OmpF fusion system was developed for the production of recombinant proteins in E. coli BL21 to be excreted into the culture medium in large amounts. To show the validity of the system, human β-endorphin was used as a model protein. Using the OmpF fusion system, 545 mg of β-endorphin protein were obtained from 2.7 litres of culture supernatant. Findings: A variety of methods have been developed for the excretion of recombinant proteins from E. coli; unfortunately, most give poor yields of the target protein. This group have developed a system using a combination of an OmpF fusion protein and an OmpF knock-out strain of E. coli BL21(DE3). Human β-endorphin was used as a test case for the system, with good results. The protein was found to be excreted into the culture medium, from which it was successfully purified. OmpF was then cleaved from β-endorphin using an engineered factor Xa cleavage site. This novel system provides high yields of a biologically important protein and should be applicable to the production of other recombinant proteins.
Membranes Selected by Richard Newman EMBL-EBI, Cambridge, UK e-mail: [email protected]
•• Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Gordeliy VI, Labahn J, Moukhametzianov R, Efremov R, Granzin J, Schlesinger R, Bueldt G, Savopol T, Scheidig AJ, Klare JP, Engelhard M: Nature 2002, 419:484-487. Significance: The authors present structural evidence of a common mechanism for light-driven ion transport and phototaxis. The microbial rhodopsins constitute a family of seven-helix membrane proteins with retinal as a prosthetic group and are distributed throughout bacteria, archaea and eukaryota. The high-resolution structure of the sensory rhodopsin II (SRII)–transducer complex provides the foundation for understanding transmembrane signalling at a molecular level. It is possible to solve the structures of intermediates, as has been done for bacteriorhodopsin. This new information will be significant not only for bacterial phototaxis and chemotaxis but also for other dimeric receptors, and might lead finally to a general model for transmembrane signal transduction. Findings: Sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the resulting cytoplasmic signal cascade is formed by a transducer molecule, which
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binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). Crystallisation of the SRII–transducer complex, a member of the two-component signalling cascade, has been achieved successfully using a shortened transducer (residues 1–114 of N. pharaonis HtrII) comprising TM1, TM2 and an additional small cytoplasmic fragment. The thin orange crystals of SRII in complex with HtrII114 grown in the lipidic cubic phase displayed an orthorhombic shape of about 140 µm and diffracted to 1.8 Å. The asymmetric unit contains one complex. It is thought that light excitation of SRII from N. pharaonis in complex with its transducer (HtrII) induces an outward movement of its helix F, which in turn triggers a rotation of TM2. It is unclear how this TM2 transition is converted into a cellular signal. This structure and the observation that the flap-like movement of helix F induces a rotation of the cytoplasmic end of TM2 give rise to a probable mechanism of transmembrane signalling. Selected by Irmgard Sinning BZH, Heidelberg, Germany e-mail: [email protected]
•• Crystal structure of bacterial multidrug efflux transporter AcrB. Murakami S, Nakashima R, Yamashita E, Yamaguchi A: Nature 2002, 419:587-593. Significance: Bacterial multidrug resistance is an increasing problem in treating infectious diseases. This paper describes the first three-dimensional structure of a multidrug transporter, which provides the basis for understanding the transport mechanism. Findings: AcrB is a multidrug exporter in E. coli that works in conjunction with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. The structure of AcrB was determined at 3.5 Å resolution. AcrB is a homotrimer of monomers consisting of more than 1000 amino acids. Each protomer contains a transmembrane region with 12 α helices and a head domain, which protrudes about 70 Å from the membrane. The top of this headpiece opens like a funnel and is proposed to interact with TolC. A central cavity at the bottom of the headpiece is connected to the funnel by a pore formed by three α helices. In the structure, the pore seems to be closed. The structure implies that substrates pass into the central cavity and are then actively transported through the pore into the TolC tunnel.
Biophysical methods Selected by Airlie McCoy* and Tim Dafforn† University of Cambridge, Cambridge, UK *e-mail: [email protected] †e-mail: [email protected]
•• An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Ando R,
Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A: Proc Natl Acad Sci USA 2002, 99:12651-12656. Significance: A new fluorescent protein (named Kaede) from a stony coral has been found that converts from green to red in response to UV irradiation. Findings: The serendipitous observation that a sample of Kaede protein left on the bench overnight turned from green to red led to the finding that Kaede irreversibly photoconverts — after exposure to UV radiation, the fluorescence peak moves from 518 nm to 582 nm. The red fluorescence is comparable in intensity to that of the green and is stable under aerobic conditions. Kaede
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was used as an optical marker for a single neuron in a complex culture. Marking was achieved by exposing a small region of the cytosol to a focused UV pulse (power density 1.3 W/cm2) for 10 s to create a source of red Kaede, which then diffused throughout the cytosol within a few minutes and allowed visualisation of contact sites between the marked (red) neuron and neighbouring green neurons. Differential marking of cells could be achieved by differences in irradiation times. • Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces. Thess A, Hutschenreiter S, Hofmann M, Tampe R, Baumeister W, Guckenberger R: J Biol Chem 2002, 277:36321-36328. Significance: Recombinant T. acidophilum 20S proteosomes were engineered to be immobilised in a predictable orientation at metal-chelating lipid interfaces by inserting a His-tag at various positions. Findings: Proteosomes were engineered to include His-tags at the C termini of the β subunits, at the N termini of the α subunits, and at a solvent-accessible turn of the α subunits connecting helix H and strand S1. Proteosomes were then absorbed for either 1 hr or overnight to lipid films containing 10% Ni-nitrilotriacetic acid-Nα-Nα-bis(carboxymethyl)-Nα[(di-octadecuylamino)-succinyl]-L-lysine. The lipid films were then transferred onto carbon grids and examined by transmission EM. Proteosomes His-tagged at the C termini of the β subunits displayed exclusively ‘side-on’ views and those His-tagged at the N termini of the α subunits displayed exclusively ‘end-on’ views. However, those His-tagged at the solvent-accessible turn of α subunits connecting helix H and strand S1 were randomly oriented, which correlated with the extremely low binding of this protein to nickel nitrilotriacetic resin. The uniform orientation on the lipid films promoted two-dimensional crystallisation. • Reaction path of protein farnesyltransferase at atomic resolution. Long SB, Casey PJ, Beese LS: Nature 2002, 419:645-650. Significance: The reaction mechanism of farnesyltransferase (FTase) is elucidated by the solution of a number of X-ray crystal structures. Findings: FTase participates in the transfer of farnesyl lipid groups to cysteine residues in a number of proteins. This process is important in directing proteins to the cell membrane. However, the process can also result in the modification of members of the Ras oncoprotein family, leading to transformation and hence cancer. Long et al. present the crystal structures of FTase with product and FTase with product and bound substrate (farnesyl diphosphate). Using these structures alongside those already solved for apo FTase, FTase with bound farnesyl diphosphate, and FTase with an inactive farnesyl diphosphate analogue and bound protein substrate, the authors unravel the molecular events that occur during the reaction mechanism. The structures suggest that, upon formation of a ternary complex containing enzyme, substrate protein and farnesyl diphosphate, the first two isoprenoid units of the farnesyl moiety rotate to bring the substrates into close proximity. This structure is proposed to be the transition state for the reaction. Interestingly, in this case, this movement is likely to represent the only major conformational change during the reaction mechanism, with only small motions of the enzyme sidechains being seen. The structures also provide information on the product-release mechanism of the enzyme. Upon formation of the farnesyl–protein product, a second farnesyl diphosphate binds to the enzyme,
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forming contacts with the product. It is only now that the product can be released. This gives the enzyme a processivity like that observed in the ribosome. The authors propose that this may be more important in the analogous enzyme geranylgeranyl transferase, which catalyses the sequential addition of geranyl moieties. • Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Schuler B, Lipman EA, Eaton WA: Nature 2002, 419:743-747. Significance: Changes in the distance between the N and C termini that occur during the folding of single protein molecules are measured using Förster resonance energy transfer (FRET). Findings: A bold combination of FRET and single-molecule methods is used to measure protein folding. Fluorescent dyes are attached to the N and C termini of the cold shock protein CspTm. These dyes are used to make FRET measurements during the folding of CspTm. In the folded protein, the termini are separated by only 1 Å. In the folded state, the FRET transfer efficiency is high due to the closeness of the termini. However, upon unfolding, the efficiency drops. Using single-molecule fluorescence techniques, the folding of individual protein molecules is examined. Polyproline is used as a control throughout the experiments. The results provide information on the free energy surface of folding that is unobtainable using traditional ensemble techniques. In particular, the experiments provide information on the polypeptide reconfiguration time. This information allows direct comparison with theoretical models of protein folding, yielding the conclusion that these results are closer to those obtained by statistical mechanical models than they are to the more complex molecular dynamics models of folding. • Molecular topographical imaging by intermembrane fluorescence resonance energy transfer. Wong AP, Groves JT: Proc Natl Acad Sci USA 2002, 99:4147-14152. Significance: An elegant system involving fluorescence resonance energy transfer (FRET) is used to examine membrane topography. Findings: A system is developed that uses two overlaid membranes doped with fluorescent probes to examine their topography. The technique relies on overlaying a membrane from a ruptured giant vesicle on a second membrane supported on a glass substrate. The top and bottom membranes contain different populations of fluorescent probes that together can undergo interlayer FRET. This allows the distances between the membranes to be measured from the FRET efficiency. In a simple example, membranes containing lipid-linked PEG show a punctate FRET pattern that may correspond to complex packing within the membrane. Measurements of the FRET efficiency and hence intermembrane spacing are made for three systems, including simple phosphotidylinositol, ganglioside GM1 and cholera-toxin-doped membranes. The intermembrane distances obtained for these systems closely match figures obtained from other techniques, proving the system may have important applications in the study of membrane topography
Miles CS, Leys D, Walkinshaw MD, Reid GA, Chapman SK: Biochemistry 2002, 41:11990-11996. Significance: The authors show how it is possible to engineer the active site of an enzyme and substitute a catalytic amino acid with a water molecule, provided that there is extensive knowledge of the enzyme’s function. Findings: Bacterial fumarate reductases belong to a well-known family of enzymes that use an arginine both as a Lewis acid to stabilise the transition state and as a Brønsted acid in proton delivery. This enables the microorganisms to use fumarate as a terminal electron acceptor in anaerobic respiration. Mowat et al. were able, with just two substitutions, to trap a water molecule in the active site and to make it work as the wild-type arginine. The resulting enzyme is fully functional, but has a less high kcat due to the inability of the water to perform the dual role. • Structure of domain III of the blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1). Nair M, Hinds MG, Coley AM, Hodder A, Foley M, Anders RF, Norton RS: J Mol Biol 2002, 322:741-753. Significance: The spread of drug-resistant forms of malaria accelerates the search for a safe and effective vaccine. One very promising candidate is the protein studied by Nair and co-workers (AMA1). The NMR structure of the protein reveals the region that is potentially interesting as antigenic. Findings: The authors correlate very cleverly the structural data on the only structured domain of AMA1 with data from epidemologists and from immunisation experiments on mice and monkeys. The recombinant domain produced from both baculovirus and E. coli is able to raise protective antibodies in the animal trials and to efficiently block the invasion of the plasmodium. • Structure and catalytic mechanism of a SET domain protein methyltransferase. Trievel RC, Beach BM, Dirk LMA, Houtz RL, Hurley JH: Cell 2002, 111:91-103. • Crystal structure and functional analysis of the histone methyltransferase SET7/9. Wilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackbourn GM, Gamblin SJ, Xiao B: Cell 2002, 111:103-115. • Structure of the Neurospora SET domain protein DIM-5, a histone methyltransferase. Zhang X, Tamaru H, Khan SI, Horton JR, Keefe LJ, Selker EU, Cheng X: Cell 2002, 111:117-127. Significance: Histone methylation is yet another method of regulation of gene expression and has become very important lately. Three groups present crystal structures of three different proteins that carry the domain with the catalytic activity. Findings: All three groups have solved the structure of a methyltransferase, revealing the active site. They also give a very convincing and independently consistent explanation of the mechanism of substrate and cofactor recognition, and prove the mechanism of action by site-directed mutagenesis.
Catalysis and regulation Proteins Selected by Adriana E Miele MRC Laboratory of Molecular Biology, Cambridge, UK e-mail: [email protected]
• Engineering water to act as an active site acid catalyst in a soluble fumarate reductase. Mowat CG, Pankhurst KL,
Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA e-mail: [email protected]
• A clogged gutter mechanism for protease inhibitors. Radisky ES, Koshland DE: Proc Natl Acad Sci USA 2002, 99:10316-10321.
Paper alert
Significance: This work demonstrates that naturally occurring serine protease inhibitors are in fact good substrates, forming the acyl-enzyme intermediate rapidly. However, their highly stable tertiary structures maintain the amine leaving group in close proximity to the ester linkage, which leads to facile reformation of the starting peptide bond. Findings: Evidence for rapid formation of a covalent acyl-enzyme intermediate was obtained by SDS-PAGE analysis of reactions containing subtilisin and chymotrypsin inhibitor 2. A new band with a molecular weight consistent with the expected adduct was observed by gel electrophoresis and this intermediate reached its steady-state level within ~1 min. The exact mass of the new species was determined by electrospray MS and the data were consistent with those expected for the acyl-enzyme intermediate. On the other hand, the high molecular weight adduct was not observed if the reactions contained 0.1% SDS, which disrupts the structure of chymotrypsin inhibitor 2 (but not subtilisin), or if the protease was inactivated before incubation with the inhibitor. A high-resolution X-ray crystal structure (1.5 Å) of the covalent intermediate was obtained. Although there were few interactions between the protease and the leaving-group portion of the inhibitor protein, there were extensive contacts between the two halves of the inhibitor protein, even after cleavage of the central bond. These interactions are the probable reason that, despite the rapid formation of a cleaved acyl-enzyme intermediate, the subsequent hydrolysis of the ester linkage is disfavoured relative to religation by the liberated amine, which leads back to the noncovalent Michaelis complex. This is said to be “…analogous to the clogging of a gutter drain by a combination of twigs and leaves”. • Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases. Fu H, Park J, Pei D: Biochemistry 2002, 41:10700-10709. Significance: A novel strategy for inhibiting a key class of cellular control enzymes is reported. The approach should be broadly applicable and the structures can be easily tailored to suit specific phosphatases. Findings: A series of aromatic aldehydes was assayed for the ability to inhibit some representative protein tyrosine phosphatases; 4-carboxycinnamaldehyde was the most effective of those examined. To add affinity and specificity, the inhibitor was linked via its carboxyl group to the tripeptide Gly-Glu-Glu-NH2. The improved molecule was a reversible, slow-binding inhibitor
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of all three phosphatases examined, with KI values in the micromolar range. All three enzymes employ an active site cysteine residue that forms a covalent phospho-enzyme intermediate and it was reasonable to suppose that a stable hemithioacetal was formed with the inhibitor, analogous to those of cysteine proteases inhibited by peptidyl aldehydes. This was not what was observed. Instead, NMR studies utilising 13C-labeled inhibitors indicated that these compounds formed adducts with the active site arginine residue whose normal function is to assist phosphate binding and stabilise the high-energy pentacoordinate species that form during the cleavage reaction. •• Glutamate racemase is an endogenous DNA gyrase inhibitor. Ashiuchi M, Kuwana E, Yamamoto T, Komatsu K, Soda K, Misono H: J Biol Chem 2002, 277:39070-39073. Significance: That an enzyme whose sole role was believed to be providing a building block for cell wall biosynthesis is also a specific inhibitor of DNA gyrase was completely unexpected and suggests that this connection should be explored in other cell types in which D-glutamate racemase is expressed, including human carcinomas. Findings: Escherichia coli D-glutamate racemase is representative of a ubiquitous bacterial enzyme and strains lacking this enzyme are D-glutamate auxotrophs, indicating that this protein does provide an essential metabolite for the cell. However, D-glutamate racemases are unusual enzymes in that they do not utilise pyridoxal phosphate as a cofactor, they do not share sequence similarity with other amino acid racemases and they catalyse glutamate racemisation only in the presence of the peptidoglycan precursor UDP-MurNAc-L-Ala. When E. coli D-glutamate racemase was overexpressed in E. coli, the copy number of intracellular plasmids was reduced significantly (from ~20 to 1 copy per cell). Several lines of evidence pointed to inhibition of DNA gyrase as the cause of this copy number decrease and this was confirmed by in vitro assays in which supercoiling by GyrAB was measured in the presence and absence of D-glutamate racemase. Although the racemase alone had no effect on supercoiling activity, addition of UDP-MurNAc-L-Ala along with D-glutamate racemase completely abolished GyrAB activity, with a KI value of 6.8 nM. Attempts to inhibit E. coli topoisomerase IV under similar conditions were unsuccessful, implying that GyrAB is the key target for inhibition.
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. • ••
of special interest of outstanding interest
Current Opinion in Structural Biology 2002, 12:685–691 Contents (chosen by) 685 Protein–nucleic acid interactions (Convery et al.) 685 Folding and binding (Sood and Kortemme) 685 Theory and simulation (Fiser) 686 Nucleic acids (Parkinson) 686 Sequences and topology (Copley) 687 Engineering and design (Gilardi) 688 Membranes (Newman and Sinning) 689 Biophysical methods (McCoy and Dafforn) 690 Proteins (Miele) 690 Catalysis and regulation (Stewart)
Protein–nucleic acid interactions Selected by Stephen Carr*, Máire Convery† and Caitríona Dennis* *University of Leeds, Leeds, UK †GlaxoSmithKline, Stevenage, UK e-mail: [email protected]
• The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. Vannini A, Volpari C, Garfioli C, Muraglia E, Cortese R, De Francesco R, Neddermann P, Di Marco S: EMBO J 2002, 17:4393-4401. Significance: The first ternary complex structure comprising a transcriptional regulator, TraR, its autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone, and its target DNA. Findings: TraR forms a two-domain asymmetric homodimer, with both domains showing structural similarity to previously determined structures. The autoinducer is completely buried within an enclosed cavity of the ligand-binding domain, suggesting that binding of inducer is required for correct folding of the regulator. The DNA-binding domain interacts with DNA through a HTH motif from each protein of the dimer. •• BRCA2 function in DNA binding and recombination from BCRA2-DSS1-ssDNA structure. Yang H, Jeffrey PD, Miller J, Kinnucan E, Song Y, Thomä NH, Zheng N, Chen P-L, Lee W-H, Pavletich NP: Science 2002, 297:1837-1848. Significance: The structure of BRCA2 (breast cancer susceptibility gene 2) bound to DNA provides a basis for understanding the loss of recombination-mediated repair of DNA in BRCA-associated cancers. Findings: BRCA2 tumour suppressor mutants are defective in the repair of dsDNA breaks (DSBs); the crystal structure reveals that BRCA2 plays a direct role in this process. The structure reveals three oligonucleotide-binding (OB) folds and a HTH motif. The OB folds are shown to bind ssDNA in an extended conformation, with the bases located in a channel at the surface of the protein.
Biochemical analysis also reveals that the HTH motif interacts with dsDNA and that BRCA2 stimulates RAD-51-mediated recombination in vitro. Most mutations leading to BRCA-linked cancers map to the DNA-binding interfaces within the protein, suggesting a direct role for BRCA in the detection of DSBs in the chromosome.
Folding and binding Selected by Vanita D Sood* and Tanja Kortemme† University of Washington, Seattle, Washington, USA *e-mail: [email protected] †e-mail: [email protected]
•• Modular recognition of RNA by a human pumilio-homology domain. Wang X, McLachlan J, Zamore PD, Hall TM: Cell 2002, 110:501-512. Significance: Sequence-specific interactions with the untranslated regions of mRNAs are critical for many developmental and regulatory processes in the cell. This structure demonstrates how sequence specificity is achieved for an important group of RNA-binding proteins, the Puf family of proteins. Findings: The crystal structure of human Pumilio in complex with a Nanos response element reveals that each base in the RNA is recognised by three amino acids via a sequence-specific combination of stacking, hydrogen bonding and van der Waals interactions. These amino acid ‘triplets’ are each situated on a separate repeat of Pumilio, which consists of eight structural repeats. Substituting one repeat for another changes the sequence specificity in a predictable manner, opening the possibility of ‘designer’ proteins that recognise specific messages. • The rate-limiting step in the folding of a large ribozyme without kinetic traps. Fang X-W, Thiyagarajan P, Sosnick TR, Pan T: Proc Natl Acad Sci USA 2002, 99:8518-8523. Significance: The study of RNA folding has been hampered by the fact that most large RNAs fold very slowly because of the formation of kinetic traps. The authors use the catalytic domain of RNase P, which folds without kinetic traps, to perform a detailed study of RNA folding kinetics. Findings: Adopting the traditional tools used to study protein folding kinetics, Fang et al. determine that the rate-limiting step in C-domain folding is a small conformational change involving, most probably, a small-scale rearrangement or dehydration of prebound metal ions. This is in contrast to other RNAs, whose folding rate is limited by kinetic traps or by the formation of long-range tertiary contacts.
Theory and simulation Selected by Andras Fiser Rockefeller University, New York, New York, USA e-mail: [email protected]
•• Increasing temperature accelerates protein unfolding without changing the pathway of unfolding. Day R, Bennion BJ, Ham S, Daggett V: J Mol Biol 2002, 322:189-203.
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Significance: An extensive molecular dynamics (MD) simulation study established that high-temperature simulations are relevant in describing the energy landscape of protein unfolding processes. Findings: Traditionally, MD studies of unfolding events use a drastically high temperature to speed up simulations. This approach is necessary to compensate for the difference between the experimental timescale and the much shorter computer simulation timescale. However, it is often questioned whether unrealistically high temperature simulations are at all relevant to the study of the energy landscape. The authors investigated this question in an extensive MD simulation study of chymotrypsin inhibitor using seven different unfolding temperatures between 298 and 495K, and up to 94 ns simulation time. All the simulations followed a similar order of unfolding events, regardless of temperature, which affected only the timescale of transitions. Simulations below the experimentally established melting temperature retained the native conformation of the protein. The authors concluded that using elevated temperatures in unfolding simulations only activates the process, but does not affect its pathway. • MAMOTH (Matching molecular models obtained from theory): an automated method for model comparison. Ortiz AR, Strauss CEM, Olmea O: Protein Sci 2002, 11:2606-2621 Significance: A sequence-independent, fast and accurate method is presented that compares low-resolution protein structures. Findings: In the post-genomics era, when protein structures and models are produced by high-throughput experimental and theoretical methods, it is necessary to have fast, flexible and accurate methods to compare vast numbers of three-dimensional molecular models. The authors introduce a new such method, MAMOTH. The underlying idea of the approach is that a prediction is successful if the modelled structure is significantly more similar to the target fold than any other known fold. Consequently, the evaluation of a superposition needs to be consistent with the classification of experimental protein structures to folds. To assign a probability score to the quality of the superposition, it is compared with scores of randomly superposed alignments. The superposition method is robust, independent of sequence, secondary structure and contact map, and also insensitive to sequence length differences. Although it is much faster, its performance is competitive with other known approaches, such as DALI, VAST and PRISM. • A model binding site for testing scoring functions in molecular docking. Wei BQ, Baase WA, Weaver LH, Matthews BW, Schoichet BK: J Mol Biol 2002, 322:339-355. Significance: Although theoretical docking studies are becoming increasingly important, the development and evaluation of their scoring function are confronted with the problem of accurately testing their effect. This study uses a prototype model binding system to test modifications to a docking scoring function. Findings: The docking of small molecules on protein-binding sites remains a major challenge. Large-scale studies are often facilitated by computational screening of potential ligand candidates. The current study focused on the well-characterised experimental system of T4 lysozyme. Mutation-created binding sites were introduced to test how atomic charges and solvation energies affect the molecular docking of 172 118 small molecules. Various currently available scoring functions were employed. The best seven docking predictions were tested experimentally on artificially created apolar and polar binding
sites. All seven ligands bound to the polar cavity, but did not detectably bind to the apolar one. Five ligand-bound structures were determined by X-ray crystallography and the models were predicted within 0.4 Å rmsd of the experimental solutions.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Fulham, UK e-mail: [email protected]
• Specificity of RNA-RNA helix recognition. Battle DJ, Doudna JA: Proc Natl Acad Sci USA 2002, 99:11676-11681. Significance: A thermodynamic and crystallographic investigation, within the Tetrahymena group I self-splicing intron, into A-minor motif specificity. This motif involves interactions between unpaired adenosine residues and the minor groove of receptor helices, and may play a role in RNA architecture and recognition. Findings: Thermodynamic data showed that the A-minor interaction is not greatly disrupted by the substitution of various canonical base pairs in the receptor helix, thus showing little selectivity. Crystallographic analysis of a P4–P6 domain variant, C•G to G•C, also reveals no significant structural rearrangement upon transversion. The use of native gel electrophoresis on all 12 possible base mismatches revealed pyrimidine•pyrimidine mismatches as the least preferred. Canonical base pairs are strongly preferred over base mismatches in the A-minor interaction through recognition of Watson–Crick minor groove geometry.
Sequences and topology Selected by Richard Copley Wellcome Trust Centre for Human Genetics, Oxford, UK e-mail: [email protected]
• De novo prediction of three-dimensional structures for major protein families. Bonneau R, Strauss CEM, Rohl CA, Chivian D, Bradley P, Malmström L, Robertson T, Baker D: J Mol Biol 2002, 322:65-78. Significance: The application of a good fold prediction method that uses no structural information to a large number of protein families. Findings: The Baker group’s ‘Rosetta’ method for de novo protein fold prediction has performed well in Critical Assessment of Structure Prediction (CASP) trials. In this paper, the authors first assess the probable reliability of the method using known protein structures and then go on to perform a large-scale structure prediction exercise on all protein families shorter than 150 amino acids in the PFAM-A database. In the absence of experimental confirmation of structure, it is difficult to assess how accurate the results are, but several examples are presented that appear plausible and the authors envisage that they may help frame functional hypotheses. The value of the method is that it can be applied in cases in which all other techniques have failed. • Loopy proteins appear conserved in evolution. Liu J, Tan H, Rost B: J Mol Biol 2002, 322:53-64. Significance: An unusual analysis of regions of proteins containing no regular secondary structure. Findings: ‘Loopy’ regions were defined as those that contain 70 or more consecutive residues with no regular secondary
Paper alert
structure (NORS). Such regions were identified in proteins of known 3D structure and a method developed to predict them from sequences. This method was then applied to the predicted protein sets of complete genomes. Five times as many NORS were found in eukaryotes than in prokaryotes. These regions were found to be just as conserved as flanking protein sequences, typically had more interaction partners than other proteins (based on two-hybrid data) and often had functions related to regulation and transcription. The authors speculate that their conformational flexibility makes them amenable to new kinds of regulation.
Engineering and design Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK e-mail: [email protected]
• Structure-based Combinatorial Protein Engineering (SCOPE). O’Maille PE, Bakhtina M, Tsai MD: J Mol Biol 2002, 321:677-691. Significance: This paper proposes a new protein engineering approach based on structural information combined with existing directed evolution methods. Findings: In this work, the authors take two distantly related DNA polymerase enzymes, Pol X and Pol β, which share low sequence homology but have some similar secondary structure, and create a cross-over library from the two genes, based on equivalent secondary structure elements. The resulting library was tested using complementation in E. coli and several novel polymerases were found. It is seen that both the secondary structure elements and the linkages between them are important in determining activity. This approach could be useful for completing the range of application of directed evolution techniques, as it is homology independent but does not rely simply on enzymatic digestion of the gene. • Stable self-assembly of a protein engineering scaffold on gold surfaces. Terrettaz S, Ulrich WP, Vogel H, Hong Q, Dover LG, Lakey JH: Protein Sci 2002, 11:1917-1925. Significance: A mutant of the E. coli protein OmpF is created that binds directly to gold surfaces and provides an interesting anchor to link other proteins by further engineering. Findings: The pore-forming outer membrane protein OmpF is engineered by the introduction of a single cysteine residue on the periplasmic surface. The resulting mutant is deposited onto a gold surface that had been previously treated with β-mercaptoethanol. Once the protein is immobilised, thiolipids are added, forming a proteolipid layer at the gold surface. The conformation of OmpF was tested using binding of the R-domain of the toxin colicin N, which is monitored by surface plasmon resonance and ion channel blockage. The gold surface denatures some of the otherwise highly robust OmpF, but functional protein is still present at high density. Although this technique could be applied to other outer membrane proteins, the interest here from a protein engineering standpoint lies in the possibility of creating OmpF fusions that can be easily purified and assembled onto gold surface. This could have applications in the field of biosensor construction. • A positive charge preservation at position 116 of αA-crystallin is critical for its structural and functional
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integrity. Bera S, Thampi P, Cho WJ, Abraham E: Biochemistry 2002, 41:12421-12426. Significance: Reports of autosomal dominant congenital cataract associated with a missense mutation (R116C) in the sequence of human αA-crystallin are further examined in this study. The study demonstrated that preservation of a positive charge at position 116 is critical in preserving the structure and function of the protein. Findings: R116 in human αA-crystallin was mutated to lysine (R116K), cysteine (R116C), glycine (R116G) and aspartic acid (R116D). Wild-type and mutant proteins were expressed, purified and characterised by measurements of circular dichroism, tryptophan fluorescence, TNS fluorescence and chaperone function. The mutant R116K had similar structure and function to the wild type. However, mutation of the residue to an acidic residue (R116D) led to drastic changes in protein structure. Mutation of the arginine to cysteine or glycine showed similar changes in structure, oligomerisation and chaperone function, thereby suggesting that the presence of the cysteine does not cause the changes. Therefore, a positive charge at residue 116 is essential for preserving the structure and function of αA-crystallin. • Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding. Xu Y, Dewanti AR, Mitra B: Biochemistry 2002, 41:12313-12319. Significance: Using site-directed mutagenesis, the role of the conserved residue Arg165 in (S)-mandelate dehydrogenase and the overall importance of the Arg165/Arg277 pair were examined. It was found that, although Arg165 plays a role in substrate binding and catalysis, its role is not as important as that of Arg277. Findings: Single mutants at Arg165, as well as double mutants at Arg165 and Arg277, were characterised kinetically and functionally. The results showed that Arg165 stabilises the transition state through its positive charge and the ground state through a charge-independent interaction. The kcat for the R165K/R277K mutant was found to be ~350-fold lower than that of the wild type. This demonstrated that at least one arginine residue is required for optimal substrate orientation and catalysis. pH studies showed that a pKa of 9.3 in the free wild-type enzyme did not belong to Arg165 and suggested that it belongs to Arg277. Therefore, Arg277 is the more critical residue in terms of catalysis and substrate binding in (S)-mandelate dehydrogenase. • Rational cytokine design for increased lifetime and enhanced potency using pH-activated ‘histidine switching’. Sarkar CA, Lowenhaupt K, Horan T, Boone TC, Tidor B, Lauffenburger D: Nat Biotechnol 2002, 20:908-913. Significance: The authors provide a rational strategy through which more potent analogues of specific protein therapeutics might be designed. Findings: Drugs typically rely on the ability of ligands to bind tightly to their target. However, the sorting of complexes involving growth factors or cytokines is related to the endosomal ligand–receptor binding affinity, with complexes that remain bound to the receptor generally becoming degraded and those that dissociate being recycled. Redesigning a cytokine to decrease endosomal affinity after internalisation could reduce receptor down-regulation and ligand depletion, enhancing drug effectiveness. The rationale takes advantage of the pH decrease from 7 to 5–6 in the endosomes, and is based on the
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Paper alert
principle that neutral histidine could maintain relatively tight binding on the cell surface, but protonated histidine could lead to weaker binding in the endosomal compartment. Candidate mutation sites were identified in areas of excessive negative charge density and electrostatic complementarity of the granulocyte colony-stimulating factor (GCSF) and its receptor. Calculation of the electrostatic contribution for the wild type and each of the histidine mutants shows that, for three of them (E20H, D110H and D113H), the charged form has about 5 kcal/mol higher binding free energy than the neutral form. The Kd determined for the selected candidates (D110H and D113H) shows that, although binding is unaffected at pH 7.4, it weakens at pH 5.5 (Kd up to sevenfold higher at pH 5.5 for D113H). Tests on ligand depletion demonstrate that each of the mutants shows a much longer half-life (>500 hr) than the wild type (50 hr). Finally, an enhancement of ligand recycling in the case of the two mutants was found, validating the goal set by the authors. • Conversion of a transmembrane to a water-soluble protein complex by a single point mutation. Tsitrin Y, Morton CJ, El Bez C, Paumard P, Velluz MC, Adrian M, Dubochet J, Parker MW, Lanzavecchia S, Van Der Goot FG: Nat Struct Biol 2002, 9:729-733. Significance: This paper presents the conversion of the pore-forming toxin aerolysin into a soluble complex by simple point mutation. Findings: Pore-forming aerolysin is a four-domain protein that undergoes C-terminal proteolytic cleavage and oligomerisation into a ring-like heptameric structure with amphipathic properties that allow it to insert into the lipid membrane. The upper boundary of domain 4 is delineated by a row of three aromatic residues (Y298, F410 and Y221), which are reminiscent of the ‘aromatic belt’ found in other membrane proteins that is thought to anchor and stabilise the proteins in the bilayer. The authors analyse the importance of these aromatic residues by mutating each to a glycine and show that the Y221G mutation completely blocked haemolysis. CD spectra of Y221G show no difference to those of the wild type. On the contrary, the near-UV CD spectrum of Y221G heptamers differed from that of the wild type, indicating that their tertiary structures must differ, despite the similarity of their secondary structures. Cryonegative staining EM shows that the wild-type heptamers formed irregular, mostly aggregated structures and micelle-like structures. In contrast, regular structures were observed for the Y221G mutant, suggesting that these heptamers do not expose hydrophobic surface, as further confirmed by ANS binding experiments. The 13.5 Å model generated from the EM data provides some clues on the conversion of the toxin to a membrane-bound state. • Rational design of green fluorescent protein mutants as biosensor for bacterial endotoxin. Goh YH, Frecer V, Ho B, Ding JL: Protein Eng 2002, 15:493-502. Significance: Virtual mutagenesis was used to design mutants of enhanced green fluorescent protein (EGFP) with high affinities for bacterial endotoxin, lipopolysaccharide (LPS) and its bioactive component, lipid A (LA). The most successful rationally designed EGFP mutant, G10, captured LA with a dissociation constant of 8.5 µm and exhibited the highest attenuation of fluorescence intensity in the presence of LPS/LA. Findings: Previous work by this group has shown that LPS or LA can interact with and bind to short cationic sequences
containing symmetrical amphipathic β-sheet motifs composed of alternating basic, hydrophobic and polar residues with dissociation constants in the micromolar range. Virtual mutagenesis of an EGFP model suggested the most likely position of this motif for successful LPS/LA detection. Several mutants of EGFP (G10–G12) were then made and mutant G10, which has amino acids 200–204 (YLSTQ) mutated to KLKTK, was found to have the highest level of fluorescence quenching in the presence of LPS/LA. Further development of the G10 mutant may provide the basis for a fluorescent biosensor of bacterial endotoxin. • Excretion of human β-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli. Jeong KJ, Lee SY: Appl Environ Microbiol 2002, 68:4979-4985. Significance: A novel OmpF fusion system was developed for the production of recombinant proteins in E. coli BL21 to be excreted into the culture medium in large amounts. To show the validity of the system, human β-endorphin was used as a model protein. Using the OmpF fusion system, 545 mg of β-endorphin protein were obtained from 2.7 litres of culture supernatant. Findings: A variety of methods have been developed for the excretion of recombinant proteins from E. coli; unfortunately, most give poor yields of the target protein. This group have developed a system using a combination of an OmpF fusion protein and an OmpF knock-out strain of E. coli BL21(DE3). Human β-endorphin was used as a test case for the system, with good results. The protein was found to be excreted into the culture medium, from which it was successfully purified. OmpF was then cleaved from β-endorphin using an engineered factor Xa cleavage site. This novel system provides high yields of a biologically important protein and should be applicable to the production of other recombinant proteins.
Membranes Selected by Richard Newman EMBL-EBI, Cambridge, UK e-mail: [email protected]
•• Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Gordeliy VI, Labahn J, Moukhametzianov R, Efremov R, Granzin J, Schlesinger R, Bueldt G, Savopol T, Scheidig AJ, Klare JP, Engelhard M: Nature 2002, 419:484-487. Significance: The authors present structural evidence of a common mechanism for light-driven ion transport and phototaxis. The microbial rhodopsins constitute a family of seven-helix membrane proteins with retinal as a prosthetic group and are distributed throughout bacteria, archaea and eukaryota. The high-resolution structure of the sensory rhodopsin II (SRII)–transducer complex provides the foundation for understanding transmembrane signalling at a molecular level. It is possible to solve the structures of intermediates, as has been done for bacteriorhodopsin. This new information will be significant not only for bacterial phototaxis and chemotaxis but also for other dimeric receptors, and might lead finally to a general model for transmembrane signal transduction. Findings: Sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the resulting cytoplasmic signal cascade is formed by a transducer molecule, which
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binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). Crystallisation of the SRII–transducer complex, a member of the two-component signalling cascade, has been achieved successfully using a shortened transducer (residues 1–114 of N. pharaonis HtrII) comprising TM1, TM2 and an additional small cytoplasmic fragment. The thin orange crystals of SRII in complex with HtrII114 grown in the lipidic cubic phase displayed an orthorhombic shape of about 140 µm and diffracted to 1.8 Å. The asymmetric unit contains one complex. It is thought that light excitation of SRII from N. pharaonis in complex with its transducer (HtrII) induces an outward movement of its helix F, which in turn triggers a rotation of TM2. It is unclear how this TM2 transition is converted into a cellular signal. This structure and the observation that the flap-like movement of helix F induces a rotation of the cytoplasmic end of TM2 give rise to a probable mechanism of transmembrane signalling. Selected by Irmgard Sinning BZH, Heidelberg, Germany e-mail: [email protected]
•• Crystal structure of bacterial multidrug efflux transporter AcrB. Murakami S, Nakashima R, Yamashita E, Yamaguchi A: Nature 2002, 419:587-593. Significance: Bacterial multidrug resistance is an increasing problem in treating infectious diseases. This paper describes the first three-dimensional structure of a multidrug transporter, which provides the basis for understanding the transport mechanism. Findings: AcrB is a multidrug exporter in E. coli that works in conjunction with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. The structure of AcrB was determined at 3.5 Å resolution. AcrB is a homotrimer of monomers consisting of more than 1000 amino acids. Each protomer contains a transmembrane region with 12 α helices and a head domain, which protrudes about 70 Å from the membrane. The top of this headpiece opens like a funnel and is proposed to interact with TolC. A central cavity at the bottom of the headpiece is connected to the funnel by a pore formed by three α helices. In the structure, the pore seems to be closed. The structure implies that substrates pass into the central cavity and are then actively transported through the pore into the TolC tunnel.
Biophysical methods Selected by Airlie McCoy* and Tim Dafforn† University of Cambridge, Cambridge, UK *e-mail: [email protected] †e-mail: [email protected]
•• An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Ando R,
Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A: Proc Natl Acad Sci USA 2002, 99:12651-12656. Significance: A new fluorescent protein (named Kaede) from a stony coral has been found that converts from green to red in response to UV irradiation. Findings: The serendipitous observation that a sample of Kaede protein left on the bench overnight turned from green to red led to the finding that Kaede irreversibly photoconverts — after exposure to UV radiation, the fluorescence peak moves from 518 nm to 582 nm. The red fluorescence is comparable in intensity to that of the green and is stable under aerobic conditions. Kaede
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was used as an optical marker for a single neuron in a complex culture. Marking was achieved by exposing a small region of the cytosol to a focused UV pulse (power density 1.3 W/cm2) for 10 s to create a source of red Kaede, which then diffused throughout the cytosol within a few minutes and allowed visualisation of contact sites between the marked (red) neuron and neighbouring green neurons. Differential marking of cells could be achieved by differences in irradiation times. • Specific orientation and two-dimensional crystallization of the proteasome at metal-chelating lipid interfaces. Thess A, Hutschenreiter S, Hofmann M, Tampe R, Baumeister W, Guckenberger R: J Biol Chem 2002, 277:36321-36328. Significance: Recombinant T. acidophilum 20S proteosomes were engineered to be immobilised in a predictable orientation at metal-chelating lipid interfaces by inserting a His-tag at various positions. Findings: Proteosomes were engineered to include His-tags at the C termini of the β subunits, at the N termini of the α subunits, and at a solvent-accessible turn of the α subunits connecting helix H and strand S1. Proteosomes were then absorbed for either 1 hr or overnight to lipid films containing 10% Ni-nitrilotriacetic acid-Nα-Nα-bis(carboxymethyl)-Nα[(di-octadecuylamino)-succinyl]-L-lysine. The lipid films were then transferred onto carbon grids and examined by transmission EM. Proteosomes His-tagged at the C termini of the β subunits displayed exclusively ‘side-on’ views and those His-tagged at the N termini of the α subunits displayed exclusively ‘end-on’ views. However, those His-tagged at the solvent-accessible turn of α subunits connecting helix H and strand S1 were randomly oriented, which correlated with the extremely low binding of this protein to nickel nitrilotriacetic resin. The uniform orientation on the lipid films promoted two-dimensional crystallisation. • Reaction path of protein farnesyltransferase at atomic resolution. Long SB, Casey PJ, Beese LS: Nature 2002, 419:645-650. Significance: The reaction mechanism of farnesyltransferase (FTase) is elucidated by the solution of a number of X-ray crystal structures. Findings: FTase participates in the transfer of farnesyl lipid groups to cysteine residues in a number of proteins. This process is important in directing proteins to the cell membrane. However, the process can also result in the modification of members of the Ras oncoprotein family, leading to transformation and hence cancer. Long et al. present the crystal structures of FTase with product and FTase with product and bound substrate (farnesyl diphosphate). Using these structures alongside those already solved for apo FTase, FTase with bound farnesyl diphosphate, and FTase with an inactive farnesyl diphosphate analogue and bound protein substrate, the authors unravel the molecular events that occur during the reaction mechanism. The structures suggest that, upon formation of a ternary complex containing enzyme, substrate protein and farnesyl diphosphate, the first two isoprenoid units of the farnesyl moiety rotate to bring the substrates into close proximity. This structure is proposed to be the transition state for the reaction. Interestingly, in this case, this movement is likely to represent the only major conformational change during the reaction mechanism, with only small motions of the enzyme sidechains being seen. The structures also provide information on the product-release mechanism of the enzyme. Upon formation of the farnesyl–protein product, a second farnesyl diphosphate binds to the enzyme,
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forming contacts with the product. It is only now that the product can be released. This gives the enzyme a processivity like that observed in the ribosome. The authors propose that this may be more important in the analogous enzyme geranylgeranyl transferase, which catalyses the sequential addition of geranyl moieties. • Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Schuler B, Lipman EA, Eaton WA: Nature 2002, 419:743-747. Significance: Changes in the distance between the N and C termini that occur during the folding of single protein molecules are measured using Förster resonance energy transfer (FRET). Findings: A bold combination of FRET and single-molecule methods is used to measure protein folding. Fluorescent dyes are attached to the N and C termini of the cold shock protein CspTm. These dyes are used to make FRET measurements during the folding of CspTm. In the folded protein, the termini are separated by only 1 Å. In the folded state, the FRET transfer efficiency is high due to the closeness of the termini. However, upon unfolding, the efficiency drops. Using single-molecule fluorescence techniques, the folding of individual protein molecules is examined. Polyproline is used as a control throughout the experiments. The results provide information on the free energy surface of folding that is unobtainable using traditional ensemble techniques. In particular, the experiments provide information on the polypeptide reconfiguration time. This information allows direct comparison with theoretical models of protein folding, yielding the conclusion that these results are closer to those obtained by statistical mechanical models than they are to the more complex molecular dynamics models of folding. • Molecular topographical imaging by intermembrane fluorescence resonance energy transfer. Wong AP, Groves JT: Proc Natl Acad Sci USA 2002, 99:4147-14152. Significance: An elegant system involving fluorescence resonance energy transfer (FRET) is used to examine membrane topography. Findings: A system is developed that uses two overlaid membranes doped with fluorescent probes to examine their topography. The technique relies on overlaying a membrane from a ruptured giant vesicle on a second membrane supported on a glass substrate. The top and bottom membranes contain different populations of fluorescent probes that together can undergo interlayer FRET. This allows the distances between the membranes to be measured from the FRET efficiency. In a simple example, membranes containing lipid-linked PEG show a punctate FRET pattern that may correspond to complex packing within the membrane. Measurements of the FRET efficiency and hence intermembrane spacing are made for three systems, including simple phosphotidylinositol, ganglioside GM1 and cholera-toxin-doped membranes. The intermembrane distances obtained for these systems closely match figures obtained from other techniques, proving the system may have important applications in the study of membrane topography
Miles CS, Leys D, Walkinshaw MD, Reid GA, Chapman SK: Biochemistry 2002, 41:11990-11996. Significance: The authors show how it is possible to engineer the active site of an enzyme and substitute a catalytic amino acid with a water molecule, provided that there is extensive knowledge of the enzyme’s function. Findings: Bacterial fumarate reductases belong to a well-known family of enzymes that use an arginine both as a Lewis acid to stabilise the transition state and as a Brønsted acid in proton delivery. This enables the microorganisms to use fumarate as a terminal electron acceptor in anaerobic respiration. Mowat et al. were able, with just two substitutions, to trap a water molecule in the active site and to make it work as the wild-type arginine. The resulting enzyme is fully functional, but has a less high kcat due to the inability of the water to perform the dual role. • Structure of domain III of the blood-stage malaria vaccine candidate, Plasmodium falciparum apical membrane antigen 1 (AMA1). Nair M, Hinds MG, Coley AM, Hodder A, Foley M, Anders RF, Norton RS: J Mol Biol 2002, 322:741-753. Significance: The spread of drug-resistant forms of malaria accelerates the search for a safe and effective vaccine. One very promising candidate is the protein studied by Nair and co-workers (AMA1). The NMR structure of the protein reveals the region that is potentially interesting as antigenic. Findings: The authors correlate very cleverly the structural data on the only structured domain of AMA1 with data from epidemologists and from immunisation experiments on mice and monkeys. The recombinant domain produced from both baculovirus and E. coli is able to raise protective antibodies in the animal trials and to efficiently block the invasion of the plasmodium. • Structure and catalytic mechanism of a SET domain protein methyltransferase. Trievel RC, Beach BM, Dirk LMA, Houtz RL, Hurley JH: Cell 2002, 111:91-103. • Crystal structure and functional analysis of the histone methyltransferase SET7/9. Wilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackbourn GM, Gamblin SJ, Xiao B: Cell 2002, 111:103-115. • Structure of the Neurospora SET domain protein DIM-5, a histone methyltransferase. Zhang X, Tamaru H, Khan SI, Horton JR, Keefe LJ, Selker EU, Cheng X: Cell 2002, 111:117-127. Significance: Histone methylation is yet another method of regulation of gene expression and has become very important lately. Three groups present crystal structures of three different proteins that carry the domain with the catalytic activity. Findings: All three groups have solved the structure of a methyltransferase, revealing the active site. They also give a very convincing and independently consistent explanation of the mechanism of substrate and cofactor recognition, and prove the mechanism of action by site-directed mutagenesis.
Catalysis and regulation Proteins Selected by Adriana E Miele MRC Laboratory of Molecular Biology, Cambridge, UK e-mail: [email protected]
• Engineering water to act as an active site acid catalyst in a soluble fumarate reductase. Mowat CG, Pankhurst KL,
Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA e-mail: [email protected]
• A clogged gutter mechanism for protease inhibitors. Radisky ES, Koshland DE: Proc Natl Acad Sci USA 2002, 99:10316-10321.
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Significance: This work demonstrates that naturally occurring serine protease inhibitors are in fact good substrates, forming the acyl-enzyme intermediate rapidly. However, their highly stable tertiary structures maintain the amine leaving group in close proximity to the ester linkage, which leads to facile reformation of the starting peptide bond. Findings: Evidence for rapid formation of a covalent acyl-enzyme intermediate was obtained by SDS-PAGE analysis of reactions containing subtilisin and chymotrypsin inhibitor 2. A new band with a molecular weight consistent with the expected adduct was observed by gel electrophoresis and this intermediate reached its steady-state level within ~1 min. The exact mass of the new species was determined by electrospray MS and the data were consistent with those expected for the acyl-enzyme intermediate. On the other hand, the high molecular weight adduct was not observed if the reactions contained 0.1% SDS, which disrupts the structure of chymotrypsin inhibitor 2 (but not subtilisin), or if the protease was inactivated before incubation with the inhibitor. A high-resolution X-ray crystal structure (1.5 Å) of the covalent intermediate was obtained. Although there were few interactions between the protease and the leaving-group portion of the inhibitor protein, there were extensive contacts between the two halves of the inhibitor protein, even after cleavage of the central bond. These interactions are the probable reason that, despite the rapid formation of a cleaved acyl-enzyme intermediate, the subsequent hydrolysis of the ester linkage is disfavoured relative to religation by the liberated amine, which leads back to the noncovalent Michaelis complex. This is said to be “…analogous to the clogging of a gutter drain by a combination of twigs and leaves”. • Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases. Fu H, Park J, Pei D: Biochemistry 2002, 41:10700-10709. Significance: A novel strategy for inhibiting a key class of cellular control enzymes is reported. The approach should be broadly applicable and the structures can be easily tailored to suit specific phosphatases. Findings: A series of aromatic aldehydes was assayed for the ability to inhibit some representative protein tyrosine phosphatases; 4-carboxycinnamaldehyde was the most effective of those examined. To add affinity and specificity, the inhibitor was linked via its carboxyl group to the tripeptide Gly-Glu-Glu-NH2. The improved molecule was a reversible, slow-binding inhibitor
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of all three phosphatases examined, with KI values in the micromolar range. All three enzymes employ an active site cysteine residue that forms a covalent phospho-enzyme intermediate and it was reasonable to suppose that a stable hemithioacetal was formed with the inhibitor, analogous to those of cysteine proteases inhibited by peptidyl aldehydes. This was not what was observed. Instead, NMR studies utilising 13C-labeled inhibitors indicated that these compounds formed adducts with the active site arginine residue whose normal function is to assist phosphate binding and stabilise the high-energy pentacoordinate species that form during the cleavage reaction. •• Glutamate racemase is an endogenous DNA gyrase inhibitor. Ashiuchi M, Kuwana E, Yamamoto T, Komatsu K, Soda K, Misono H: J Biol Chem 2002, 277:39070-39073. Significance: That an enzyme whose sole role was believed to be providing a building block for cell wall biosynthesis is also a specific inhibitor of DNA gyrase was completely unexpected and suggests that this connection should be explored in other cell types in which D-glutamate racemase is expressed, including human carcinomas. Findings: Escherichia coli D-glutamate racemase is representative of a ubiquitous bacterial enzyme and strains lacking this enzyme are D-glutamate auxotrophs, indicating that this protein does provide an essential metabolite for the cell. However, D-glutamate racemases are unusual enzymes in that they do not utilise pyridoxal phosphate as a cofactor, they do not share sequence similarity with other amino acid racemases and they catalyse glutamate racemisation only in the presence of the peptidoglycan precursor UDP-MurNAc-L-Ala. When E. coli D-glutamate racemase was overexpressed in E. coli, the copy number of intracellular plasmids was reduced significantly (from ~20 to 1 copy per cell). Several lines of evidence pointed to inhibition of DNA gyrase as the cause of this copy number decrease and this was confirmed by in vitro assays in which supercoiling by GyrAB was measured in the presence and absence of D-glutamate racemase. Although the racemase alone had no effect on supercoiling activity, addition of UDP-MurNAc-L-Ala along with D-glutamate racemase completely abolished GyrAB activity, with a KI value of 6.8 nM. Attempts to inhibit E. coli topoisomerase IV under similar conditions were unsuccessful, implying that GyrAB is the key target for inhibition.