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Structural biology
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Structural biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology. Current Opinion in Structural Biology 2001, 11:131–138 Contents (chosen by) 131 Protein–nucleic acid interactions (Convery et al.) 132 Folding and binding (Kortemme et al.) 132 Theory and simulation (Fiser) 132 Nucleic acids (Parkinson) 133 Sequences and topology (Copley) 133 Engineering and design (Gilardi) 135 Lipids (Newman) 135 Membrane proteins (Sinning) 136 136 136 137 • ••
Carbohydrates and glycoconjugates (Flitsch and Lowden) Biophysical methods (Matthews) Proteins (Noble) Catalysis and regulation (Stewart) of special interest of outstanding interest
analogue reveals stabilisation of the extrahelical target base and provides a basis for the general mechanism of AdoMetdependent DNA N-methylation. To catalyse the transfer of the methyl group from the cofactor to the DNA, the target nucleoside is rotated out of the DNA helix. This extrahelical conformation is stabilised by compression of the DNA helix at the target base pair position and relocation of the partner thymine base. Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Carter AP, Clemons WM Jr, Brodersen DE, Morgan-Warren RJ, Hartsch T, Wimberly BT, Ramakrishnan V: Science 2001, 291:498-501. • Significance: The 3.2 Å crystal structure of initiation factor IF1 bound to the small ribosomal subunit reveals initiation-factor-induced conformational changes in the ribosomal subunit. Findings: The structure of the complex reveals how localised changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit. Binding of IF1 occludes the ribosomal A site (blocking tRNA binding to this site) and ‘flips out’ functionally important 16S rRNA bases, burying them in pockets in the initiation factor. Binding of IF1 also causes longrange changes in the conformation of helix 44 of 16S rRNA and leads to movement of the domains of the 30S subunit with respect to each other.
Protein–nucleic acid interactions Selected by Máire Convery*, Caitríona Dennis† and Siân Rowsell‡ *GlaxoSmithKline, Stevenage, UK †University of Leeds, Leeds, UK ‡AstraZeneca, Macclesfield, UK
The structure of Pariacoto virus reveals a dodecahedral cage of duplex RNA. Tang L, Johnson KN, Ball LA, Lin T, Yeager M, Johnson JE: Nat Struct Biol 2001, 8:77-83. • Significance: The structure of Pariacoto virus reveals extensive interactions between the viral RNA genome and the protein capsid. Findings: The crystal structure of Pariacoto virus has been determined at 3.0 Å resolution and a three-dimensional cryo-EM reconstruction has been determined to better than 23 Å resolution; both were obtained from the same sample. The structures reveal a dodecahedral cage of 30 RNA duplexes inside the icosahedral virus capsid. The ordered portion of the viral RNA associates closely with the capsid and, through its interactions with the protein shell, acquires icosahedral symmetry. The protein capsid shows remarkable complementarity to the RNA, both in shape and in electrostatic environment. Structure of the N6-adenine DNA methyltransferase M••TaqI in complex with DNA and a cofactor analog. Goedecke K, Pignot M, Goody RS, Scheidig AJ, Weinhold E: Nat Struct Biol 2001, 8:121-125. • Significance: The co-crystal structure of M•TaqI in complex with its DNA substrate reveals a novel method of stabilisation of the ‘flipped-out’ base. Findings: The 2.0 Å crystal structure of the N6-adenine DNA methyltransferase M•TaqI in complex with DNA and a cofactor
Arrangement of RNA and protein in the spliceosomal U1 small nuclear ribonucleoprotein particle. Stark H, Dube P, Luhrmann R, Kastner B: Nature 2001, 409:539-542. •• Significance: The smallest structure determined to date by single-particle electron cryomicroscopy. Findings: The U1 small nuclear ribonucleoprotein (snRNP) is the first particle of the spliceosome to assemble on the mRNA. It contains elements common to all the snRNPs, along with protein and RNA unique to the U1 particle. This reconstruction at 10 Å shows that the seven Sm proteins, common to all the snRNPs, form a central doughnut-shaped core with an outer diameter of 80 Å. The other three proteins and the RNA make extensive contacts with the core and with each other. Architecture of nucleotide excision repair complexes: DNA is wrapped by UrvB before and after damage recognition. Verhoeven EE, Wyman C, Moolenaar GF, Hoeijmakers JHJ, Goosen N: EMBO J 2001, 20:601-611. • Significance: An insight into the role UvrA, UvrB and UvrA2B complexes with DNA play in damage recognition and repair in Escherichia coli. Findings: This study was carried out using atomic force microscopy (AFM) methods. The lengths of DNA arms on either side of the protein bound to the DNA at a defined damage site were measured. The amount of DNA involved in the protein–DNA complex could therefore be ascertained. It was shown that, in both the pre-incision UvrB–DNA complex and UvrA2B–DNA complexes on undamaged DNA, the DNA is wrapped around UvrB. The initial presence of UvrA imposes asymmetry on the complex and is postulated as being important for damage recognition.
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Folding and binding
Theory and simulation
Selected by Tanja Kortemme, Sehat Nauli and Alex Watters University of Washington, Seattle, Washington, USA
Selected by Andras Fiser Rockefeller University, New York, New York, USA
Design of three-dimensional domain-swapped dimers and fibrous oligomers. Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D: Proc Natl Acad Sci USA 2001, 98:1404-1409. • Significance: The authors rationally designed proteins that form domain-swapped dimers and fibrillar aggregates. Findings: The domain-swapped dimers and aggregates were designed using an α-helical bundle structure as the basic motif. Carefully placed charged residues determine the topology of the monomers by their electrostatic interactions. X-ray diffraction shows that the domain-swapped dimer is formed and a combination of electron microscopy with other biophysical studies shows that the fibrillar aggregates are formed as designed. Biomaterial design using synthetic fibrous proteins is one exciting possibility suggested by the authors.
Automated discovery of structural signatures of protein fold and function. Turcotte M, Muggleton SH, Sternberg JE: J Mol Biol 2001, 306:591-605. • Significance: An automatic approach is presented to search systematically for protein fold signatures. Findings: The authors applied Inductive Logic Programming, a machine learning technique that derives rules from observations and encoded principles. The paper shows that, applying this approach for protein fold recognition, fold signatures do exist and superfamilies can be automatically and systematically discovered. The derived new rules were partly tested by presenting them in a form of a questionnaire to the world expert Murzin, who identified virtually all of them. The approach aims to replace the need for human expertise, used so far in constructing protein family databases such as SCOP.
Barstar is electrostatically optimized for tight binding to barnase. Lee L-P, Tidor B: Nat Struct Biol 2001, 8:73-76. • Significance: The importance of electrostatic interactions for protein stability and binding is unclear, as the replacement of polar with hydrophobic sidechains can be energetically favourable. Here, the authors present evidence that the optimisation of electrostatic interactions may have been used in naturally occurring proteins to achieve tight binding. Findings: Lee and Tidor developed an algorithm to design a charge distribution on a protein ligand that optimises the electrostatic contribution to binding to its receptor. For a complex between a protein inhibitor, barstar, and its receptor, barnase, the computed charge distribution is very similar to the actual one. Furthermore, mutation of interface sidechains on barstar with each of the naturally occurring amino acids is not predicted to increase binding affinity, suggesting that this interface is electrostatically optimized.
Protein structural alignments and functional genomics. Irving JA, Whisstock JC, Lesk AM: Proteins 2001, 42:378-382. • Significance: A new approach is introduced to locate functional sites in proteins using pairwise structural alignments alone. Findings: The strong conservation of active site conformations suggests a method for identifying active sites. This depends on the relationship between size and goodness-of-fit of aligned substructures in homologous proteins. This relationship was well established before, but not analysed in quantitative detail. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins.
Tailoring in vitro evolution for protein affinity or stability. Jermutus L, Honegger A, Schwesinger F, Hanes J, Pluckthun A: Proc Natl Acad Sci USA 2001, 98:75-80. • Significance: Due to limited knowledge of structure/function relationships, rational design of proteins with specific physical properties has not been consistently achieved. Directed evolution using display techniques that specifically optimise certain properties, such as protein stabilities and affinities, may provide a convenient method to overcome current limitations in rational design. Findings: Using single-chain Fv antibody fragments (scFvs), the authors were able to use a series of PCR mutagenesis and directional selection rounds to generate variants with either higher overall stability or greater affinity for a specific antigen, fluorescein. scFv phage libraries bound to biotin-tagged fluorescein were incubated with saturating levels of untagged fluorescein for increasing lengths of time, such that only individual scFvs with low dissociation rates would remain bound to the biotin-labelled fluorescein. These phage were then recovered by binding to streptavidin-coated magnetic beads. This gave a variant with a 30-fold decrease in the dissociation rate. Stability selections were selected in increased reducing environments to produce stable scFvs without disulfide bonds. When disulfide bonds were reintroduced by returning the selected proteins to an oxidising environment, the selected proteins had a stability twice that of the starting protein.
A Gaussian statistical mechanics model for the equilibrium thermodynamics of barnase folding. Crippen MG: J Mol Biol 2001, 306:565-573. • Significance: A new statistical mechanical model is presented that is able to reproduce the experimental free energy of a real protein during unfolding. Findings: It was possible to adjust a potential function to give the correct free energies for the isothermal denaturation of the protein barnase. By extending a statistical mechanical model with the empirical estimate of the partial molar heat capacity of denatured proteins as a function of amino acid composition, it could quantitatively account for experimental observations during barnase denaturation, such as melting temperature, width of thermal transition and cooperativity. The model described in this paper makes an attempt to bridge experimental observations of a real protein and theory, at least in the case of one protein and at least for the equilibrium thermodynamics.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Sutton, UK
Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection. van Buuren BN, Overmars FJ, Ippel JH, Altona C, Wijmenga SS: J Mol Biol 2000, 304:371-383. • Significance: The authors report the first high-resolution NMR solution structure of a DNA three-way junction (3H). Three-way junctions are a recurring motif in nucleic acid structures; such DNA motifs are associated with recombination involving phage, with replication and with site-specific integration of viral DNA
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into the host genome. Using this structure and previous data, the authors developed rules for the predictive stacking preferences of a three-way junction, of any sequence, with two unpaired bases (3HS2). Findings: Arms A and B are co-axially stacked. Arm C is in an antiparallel orientation with respect to the A–B-stacked helix. The interhelix angle of 53(±11)° is in agreement with previous FRET studies on three-way junctions. Both the A–B-stacked helices and arm C show an overall B-DNA geometry. Only small deviations from this standard helical geometry are observed, such as a reduced twist in the A–B-stacked helix at the junction with arm C. The two unpaired bases are stacked onto arm C reminiscent of a type I hairpin fold. The authors propose two rules, the ‘pyrimidine rule’ and the ‘loop rule’, that, when combined, can reproduce experimentally known conformational preferences for 3H2S structures.
• Significance: An interesting approach to the evolution of gene context, using a local alignment algorithm to detect conserved gene order in different organisms. Findings: The authors’ claims of the value of genome context for function prediction are relatively modest. Predictions for proteins of unknown function or major clarifications of function were found for 4% of the orthologous groups analysed. As is pointed out, however, the true value of such approaches is that the information provided is completely complementary to homology-based methods, helping place proteins within systems and complexes. Some of the examples found include new components of the translation machinery and proteins having replication- and repair-related functions.
Sequences and topology
Protein design of an HIV-1 entry inhibitor. Root MJ, Kay MS, Kim PS: Science 2001, 291:884-888. •• Significance: The design of a small protein may serve as the basis for a new class of antiviral agents displaying potent inhibitory activity against diverse HIV-1 variants. Findings: Infection by HIV-1 requires fusion of the viral and cellular membranes via the formation of a trimer of hairpin structures in gp41. The core of gp41 is a bundle of six α helices. The authors designed a protein, denoted 5-Helix, in which five of the six helices of the core of gp41 are connected with short peptide linkers. The resulting protein is shown not only to be well folded, soluble and stable under physiological conditions, but also to potently inhibit HIV-1 membrane fusion (nanomolar IC50), as measured by viral infectivity and cell–cell fusion assays. It also inhibits infection by viruses pseudo-typed with a variety of HIV-1 envelope proteins with similar potency. Finally, the authors suggest that, as a potent broad-spectrum inhibitor of viral entry, 5-Helix may serve as the basis for development of a new class of therapeutic agents against HIV-1.
Selected by Richard Copley EMBL, Heidelberg, Germany
KH domain: one motif, two folds. Grishin NV: Nucleic Acids Res 2001, 29:638-643. •• Significance: A rare example of homologous sequences adopting folds with different topologies. Findings: The KH domain was originally detected in various proteins using sequence-profile-based methods. With the availability of 3D structures, Grishin has noticed that these original sequence definitions fall into two distinct fold topologies. The differences between the two types of fold are not minor — the central β sheet undergoes major rearrangement, in a manner reminiscent of the two forms of serpin. On the other hand, the sequence evidence that the two types of folds are indeed homologous is persuasive. Grishin goes on to discuss evolutionary scenarios that could have led to this situation. It will be interesting to see how many such results are uncovered in the coming years and whether they can be drawn into a consistent theoretical framework. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: J Mol Biol 2001, 305:567-580. • Significance: A method to predict 97–98% of transmembrane helices from single sequences. Findings: Transmembrane helix prediction is important not only for understanding the function of proteins, but also from a practical point of view in determining domain boundaries and guiding procedures that aim to express the soluble portions of proteins. The method presented here is particularly accurate, although (in common with other approaches) it can occasionally confuse signal peptides with transmembrane helices (particularly for some bacterial proteins). Depending on the organism, between 20 and 30% of genes are predicted to encode transmembrane-domain-containing proteins and proteins with both the N and C termini on the cytoplasmic side of the membrane are preferred. Organisms with large genomes do not seem to be relatively enriched in transmembrane-domain-containing proteins compared with organisms encoding fewer genes. Genome alignment, evolution of prokaryotic genome organization and prediction of gene function using genomic context. Wolf YI, Rogozin IB, Kondrashov AS, Koonin EV: Genome Res 2001, 11:356-372.
Engineering and design Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK
Thermostabilization of a chimeric enzyme by residue substitution: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase. Numatu K, Hayashi-Iwasaki Y, Kawaguchi J, Sakurai M, Moriyama H, Tanaka N, Oshima T: Biochem Biophys Acta 2001, 1545:174-183. • Significance: Thermostability of proteins is a key factor in the development of enzymes for industry. The authors investigated the effect on thermostability of single amino acid changes within a thermophilic–mesophilic chimeric enzyme that restore the thermophilic sequence. By following the temperature at which 50% irreversible thermal denaturation (Th) occurs, they were able to quantify the effect of different changes. Findings: The chimeric enzyme had 22 amino acid changes relative to the thermophilic enzyme in a domain comprising 20% of the entire protein and a Th of 63ºC compared with 83ºC. The authors found four amino acid reversions that led to a significant increase in Th. The changes resulted in tightening hydrophobic packing by removing cavities (e.g. A115L: 6ºC increase in Th), neutralising a surface charge (e.g. S62R: 6ºC increase in Th and kcat increases from 121 to 149 s—1) and placing glycine in a position requiring strained torsion angles (N125G: 3ºC increase in Th and kcat increases from 121 to 133 s–1). All effective changes were found to be in loops. Investigations on double mutations showed that the effects are additive. Changes that were predicted to result in similar
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effects failed to increase thermostability, probably as a result of steric effects. Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent proteins through zinc-site engineering. Jensen KK, Martini L, Schwartz TW: Biochemistry 2001, 40:938-945. • Significance: The authors exploited metal-binding site design principles to stabilise the interaction of two protein fluorescence partners. Close proximity of the fluorophores buried within the partners allowed enhanced fluorescence resonance energy transfer (FRET) signal. The authors suggest that such a system could be adapted to the study of protein–protein interactions in vivo. Findings: Partial zinc-binding sites were incorporated into cyan fluorescent protein (CFP) with complementary sites in yellow fluorescent protein (YFP), such that a stable heterodimer will form by binding two zinc ions in symmetrical sites. A zinc-dependent FRET signal was observed for heterodimers of CFP and YFP (excitation of CFP at 433 nm leads to emission at 475 nm, which, in turn, excites YFP, leading to emission at 533 nm). For a bidendate zinc-binding site design, a complete YFP signal was observed in 10–2.5 M ZnCl2. The authors investigate both bidendate and tridendate zinc-binding site designs; the apparent enhancement in the tridendate constructs was attributed to an increased availability of potential metal ligands, rather than tridendate metal binding. An approximately 100-fold increase in dimer formation was observed for zinc-binding mutants compared with autodimerisation in the wildtype. By designing proteins with only a single potential zinc-binding site, an enhancement in the FRET signal was observed (signal ratio I530:I475 increased from 4.8 to 8.1). This was attributed to a reduction in the propensity of nonproductive homodimer formation. A limitation of the constructs was a low zinc affinity (10–3 M), which would prevent the use of the current system in vivo. Structure of a genetically engineered molecular motor. Kliche W, Fujita-Becker S, Kollmar M, Manstein DJ, Kull FJ: EMBO J 2001, 20:40-46. •• Significance: Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. This work shows the possibility of engineering single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Findings: An active molecular motor was created by fusing a domain of myosin with an artificial lever arm composed of α-actinin. The artificial lever arm showed a structure similar to that of human α-actinin, with both repeats folded into a triple helix and connected by a continuous helical linker. Interestingly, the engineered linker connecting the myosin converter domain to α-actinin was also found to form an α helix. As the bending of helix 1 in the first α-actinin repeat dictates the position of the end of the lever arm, changing the helical register in the motor–lever linker region allowed accurate repositioning of the C-terminal end of the lever arm. Previous studies had shown a linear relationship between lever arm length and in vitro sliding velocity in artificial motors with lever arms composed of one or two α-actinin repeats. As two α-actinin repeats are about 13 nm long, it is argued that removing a single α-actinin repeat would shorten the lever arm by 6.5 nm. If the lever were coupled to an identical rotational displacement, the predicted step size would be about 55% of the dual repeat construct. This is
in agreement with a previously measured step size ratio, showing the possibility of having molecular motors with different properties. The authors also investigated the disruption of a salt bridge that appears to interfere with the formation of myosin’s catalytically active ‘closed’ conformation. Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation. Isalan M, Patel SD, Balasubramanian S, Choo Y: Biochemistry 2001, 40:830-836. • Significance: Telomeres are highly conserved DNA repeat sequences associated with proteins that are found at the end of chromosomes in nearly all eukaryotes. This paper shows the successful design of a Zn-finger protein with high specificity for binding to the G-quadruplex structure formed by the human telomere sequence (5′-GTTAGG-3′)n for potential therapeutic applications. Findings: A phage display library containing three Zn-finger modules was designed to maximise the chance of finding roteins that bind the single-stranded human telomeric G-quadruplex. This led to the isolation of four clones with a significant degree of sequence similarity. The library was screened with 5′biotin—(5′-GTTAGG-3′)5 (Biotin-Htelo) and the structure of the single-stranded G-quadruplex was tested using a DMS protection assay in order to confirm the specificity for the three-dimensional conformation adopted by the human G-quadruplex. The analysis of the binding properties of the selected Zn-finger, using analogues of a Biotin-Htelo oligonucleotide containing adenine or inosine in place of the critical guanine bases and of a double-stranded Htelo oligonucleotide, showed that the selected clone (Gq1) had high sequence and/or structure specificity for the human G-quadruplex, with an apparent Kd of approximately 26 nM. Gq1, cloned and overexpressed as a glutathione-S-transferase fusion protein, was tested for electromobility shift and DMS protection of the DNA–protein complex. The Kd of 34 nM for the Gq1–Htelo complex, obtained by analysis of the mobility shift in nondenaturing gel, was in good agreement with the one measured by the biotin assay. Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase. Gutierrez A, Yoshimura T, Fuchikami Y, Esaki N: Eur J Biochem 2000, 267:7218-7223. • Significance: The activity of an important family of enzymes could be modified towards different substrates. The method of inserting a short glycine loop may have wider applications to other enzymes. Findings: A loop of one, three or five glycines was inserted between the two domains of the D-amino acid aminotransferase homodimer. The activity of the mutant enzymes towards amino acid substrates of different sizes was found to have a different pattern compared with the wildtype. In particular, the five-glycine insertion allowed the protein to turn over small-chain amino acids more efficiently. Also, the one glycine mutant could use larger bulkier amino acids as substrates. The specific activity of the protein could be tuned by the insertion of a small glycine loop of different lengths. Critical role of the residue six at position 87 in H2O2-dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3. Li Q-S, Ogawa J, Shimizu S: Biochem Biophys Res Commun 2000, 280:1258-1261.
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•• Significance: The driving of the cytochrome P450 catalytic cycle using hydrogen peroxide presents a feasible and inexpensive alternative to the usual cofactor, NADPH. Findings: The group replaced a single phenylalanine in the active site of cytochrome P450BM-3 with either glycine or alanine. These single point mutations allowed the enzyme to hydroxylate substrates using hydrogen peroxide at around 40% of the wildtype enzyme’s rate when utilising NADPH. The mutations also improved the enzyme’s stability in hydrogen peroxide. These studies provide another important step forward for the commercial use of P450s. Engineering of a functional human NADH-dependent cytochrome P450 system. Dohr O, Paine MJI, Friedberg T, Roberts GCK, Wolf CR: Proc Natl Acad Sci USA 2001, 98:81-86. •• Significance: The residue responsible for distinguishing an enzyme’s normal cofactor, NADPH, has been identified and replaced so that NADH can be utilised instead. Findings: By replacing Trp676 of human cytochrome P450 NADPH-dependent oxido-reductase (CPR) with an alanine, the enzyme was engineered to use NADH instead of NADPH as the source for its reducing equivalents. The wildtype CPR has essentially no affinity for NADH and exhibits very little reducing activity in its presence. The W676A mutant was able to utilise NADH and exhibited around 90% of the reducing (NADPHdependent) activity of the wildtype CPR. These findings open up the possibility for the application of this approach to other oxido-reductases, aiding our understanding of how they distinguish between different cofactors.
Lipids Selected by Richard Newman EMBL-European Bioinformatics Institute, Cambridge, UK
Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Peters C, Bayer MJ, Buehler S, Andersen JS, Mann M, Mayer A: Nature 2001, 409:581-588. •• Significance: The mechanism of membrane fusion, without which all membrane traffic within a cell would grind to a halt, was thought to be mediated solely by SNAREs (proteins that inhabit all fusion-competent membranes of a cell). The authors describe a situation in which SNAREs do not cause fusion directly, but instead enable an ion-permeable membrane protein, the vacuolar H+ATPase, to form a bridge between membranes, allowing fusion to proceed. The important implication of these observations is that the mechanism of membrane fusion is explained in terms of the radial expansion of a proteolipid-lined protein channel. Findings: The authors show that two yeast vacuoles fuse by forming a head-to-head dimer of the V0 part of the vacuolar proton pump, which is found in most intracellular membranes and vesicles. This pump contains two multisubunit sectors — V1, which hydrolyses ATP, and V0, a complex of six so-called proteolipids — together with associated subunits, through which proteins are transported. The dimerisation of V0 is proposed to allow the two proteolipid hexamers to form a closed-off channel from one vacuole into the other, suggesting a mechanism for fusion. Calcium-bound calmodulin is believed to cause the proteolipids to separate within the plane of the membrane, so that lipids invade the space between them and the aqueous channel in the centre opens. The separating proteolipids expose surfaces between them that have a hydrophobic portion sandwiched between two hydrophilic
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strips. Such surfaces mimic lipid bilayers and allow lipids to invade the proteolipid ring without leaving their bilayer configuration. This model is attractive because it explains fusion without invoking temporary lipid intermediates.
Membrane proteins Selected by Irmgard Sinning EMBL, Heidelberg, Germany
Projection structure of a ClC-type chloride channel at 6.5 Å resolution. Mindell JA, Maduke M, Miller C, Grigorieff N: Nature 2001, 409:219-223. •• Significance: Chloride channels are present in virtually all eukaryotic cells. They serve a wide range of functions, such as controlling electrical excitability in skeletal muscle and maintaining systemic blood pressure. Two fundamentally different models for the channel architecture were proposed previously: a double-barrelled homodimer with two pores per dimer and a conventional barrel-stave architecture with a single pore on the homodimer twofold axis. The projection structure from twodimensional crystals of a prokaryotic ClC-channel homologue (EriC) allows a first glimpse at this dimeric channel complex with water-filled pores and can serve as a prototype, at least for the muscle subfamily. Findings: EriC was crystallised by reconstituting the purified channel into phospholipid membranes. The crystals have P22121 symmetry with a unit cell of 140 × 80 Å. Two EriC channels per unit cell are related by crystallographic twofold axes, confirming the homodimeric nature of the complex. EriC shows two pairs of two low-density troughs, each pair separated by a density peak of about 7 Å across. The authors interpret their data such that each pair of ‘density wells’ represents a single pore that is kinked roughly halfway through the membrane. The intracellular and extracellular openings are offset when viewed normal to the membrane plane. The model with two pores in the dimeric channel explains the double-barrelled gating behaviour of eukaryotic ClC channels. The distance of about 25–40 Å between the two pores would be far enough for them to be electrostatically independent. Although the authors count 12 peaks in all, the projection structure does not allow the assignment of transmembrane helices, which are believed to number 10–12. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Zouni A, Witt H-T, Kern J, Fromme P, Krauss N, Saenger W, Orth P: Nature 2001, 409:739-743. •• Significance: Oxygenic photosynthesis provides the atmosphere with oxygen and is the principal energy converter on earth. Structural information from electron crystallography was previously obtained for photosystem II (PSII) fragments without water oxidising activity (at 8 Å) and intact PSII, but at low resolution (15–30 Å). The X-ray structure of PSII at 3.8 Å resolution shows the spatial arrangement of protein subunits and cofactors. The presence of the manganese cluster will stimulate specific investigations on the mechanism of water oxidation. Findings: PSII from the thermophilic cyanobacterium Synechococcus elongatus was isolated as a homodimer, as confirmed also by electron microscopy. PSII consists of at least 17 protein subunits, of which 14 are located within the membrane. Porphyrin molecules could be modelled into the electron density and the proteins are represented by Cα traces. The membrane integral part comprises 36 transmembrane α helices; 22 of these, assigned to D1, D2, CP43 and CP47 proteins, are arranged with a local pseudo-twofold rotation
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symmetry. As expected, the arrangement of the α helices from the D1 and D2 proteins resembles that of the reaction centre from photosynthetic bacteria and of the C-terminal helices of PsbA and PsbB in PSI. This supports the hypothesis that all photosynthetic reaction centres have evolved from one common ancestor. Seven transmembrane helices could not be assigned in the present structure and the extrinsic 12 kDa subunit could not be located because of unassigned secondary structure elements. The arrangement of the cofactors is presented. The interatomic distances between the four manganese ions of the manganese cluster are close to values derived from spectroscopic data.
Carbohydrates and glycoconjugates Selected by Sabine L Flitsch and Philip AS Lowden* Edinburgh University, Edinburgh, UK *University of Exeter, Exeter, UK
Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Persson K, Ly HD, Dieckelmann M, Wakarchuk WW, Withers SG, Strynadka NCY: Nat Struct Biol 2000, 8:166-175. •• Significance: The first three-dimensional structure of a retaining, nucleotide-sugar-dependent glycosyltransferase and the first of any glycosyltransferase in complex with both donor and acceptor sugars. Findings: LgtC is an α-1,4-galactosyltransferase that transfers galactose from uridine diphosphogalactose (UDP-Gal) to the terminal lactose of the cell-surface lipooligosaccharides of Neisseria meningitidis with retention of anomeric configuration. This paper reports the 2.0 Å crystal structures of LgtC complexed with Mn2+ and UDP 2-deoxy-2-fluoro-galactose (an inactive glycosyl donor analogue), with and without 4′-deoxylactose (an inactive glycosyl acceptor analogue). The overall fold of the enzyme resembles some of the inverting glycosyltransferase structures already known, despite low sequence homology. By analogy with retaining glycosidases, a catalytic nucleophile was expected to be found. The structure suggested either the 6′-hydroxyl of lactose or the sidechain oxygen of Gln189 as this nucleophile. However, studies with alternative substrates or with Gln189 mutants provided no support for either of these cases. The catalytic mechanism therefore remains obscure, with the intriguing possibility that the reaction may proceed by a rare SNi mechanism, in which nucleophile and leaving group act on the same side of the substrate. Direct isolation and sequencing of specific protein-binding glycosaminoglycans. Keiser N, Venkataraman G, Shriver Z, Sasisekharan R: Nat Med 2001, 7:123-128. •• Significance: Heparin/heparan-sulfate-like glycosaminoglycans (HLGAG) play important roles in the regulation of protein stability and function. Identification of the precise glycosaminoglycan sequences involved in these events has been very difficult. This paper demonstrates for the first time how surface noncovalent affinity mass spectrometry can be used to rapidly isolate and directly sequence the biologically relevant HLGAG oligosaccharides. Findings: A recently reported method of using matrix-assisted laser desorption ionisation mass spectrometry to analyse picomole amounts of HLGAG oligosaccharides was combined with immobilisation of an HLGAG-binding protein on a surface to perform affinity isolation and sequencing of tissue-derived HLGAG oligosaccharides on a mass spectrometer surface.
This method of analysis was first tested to study the binding of known ligands to antithrombin III and was able to distinguish between specific and nonspecific binding interactions, even within a mixture of saccharides. The studies were then extended to find and identify oligosaccharide sequences that bind to the fibroblast growth factor FGF-2 by combining the method with other sequencing techniques, such as selective HLGAG hydrolysis. Structural basis for catalysis and inhibition of N-glycan processing class I α1,2-mannosidases. Vallee F, Karaveg K, Herscovics A, Moremen KW, Howell PL: J Biol Chem 2000, 275:41287-41298. • Significance: Endoplasmic reticulum class I α1,2-mannosidase is a key enzyme in the maturation of N-linked oligosaccharides and plays an important role in the degradation of misfolded glycoproteins by the proteasome. It is therefore a target of inhibitor design for the treatment of genetic diseases that are characterised by rapid degradation of misfolded glycoproteins. This paper provides a first structure of the enzyme with and without bound inhibitors, and provides a basis for further inhibitor design. Findings: Crystal structures of the catalytic domain of the α1,2-mannosidase in the presence and absence of two inhibitors, kifunesine and 1-deoxymannojirimycin, were determined. The data show that the specificity of the inhibitors is dictated by the coordination of the essential calcium at the catalytic site to the O-2′ and O-3′ hydroxyl groups, and resulting stabilisation of the sixmembered rings in the 1C4 conformation. Furthermore, the distance of any putative catalytic carboxyl-sidechain to the substrate-binding site suggests that the enzyme operates by a novel mechanism in which the active site carboxyl groups do not directly react with the substrate.
Biophysical methods Selected by Steve Matthews Imperial College of Science, Technology and Medicine, London, UK
Reaction-induced infrared difference spectroscopy for the study of protein reaction mechanisms. Zscherp C, Barth A: Biochemistry 2001, 40:1875-1883. • Significance: This paper gives an up-to-date illustration of the state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterisation of enzyme function on the level of single bonds of proteins, cofactors or substrates. Findings: Protein structure and function can be studied by various biochemical and biophysical approaches. High-resolution structural information can be gained from X-ray crystallography, electron cryomicroscopy and NMR spectroscopy. Infrared spectroscopy provides the ability to study structural information with a high time resolution, which enables molecular mechanisms of protein reactions to be elucidated. The number of methods used to initiate protein reactions has constantly increased over the past decade. Light-driven reactions, redox reactions, molecule–protein interaction and protein folding can be studied by infrared spectroscopy and are discussed in detail.
Proteins Selected by Martin Noble University of Oxford, Oxford, UK
Mechanism based design of a protein kinase inhibitor. Parang K, Till JH, Ablooglu AJ, Kohanski RA, Hubbard SR, Cole PA: Nat Struct Biol 2001, 8:37-41.
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• Significance: Specific inhibition of protein kinases could significantly advance both research into cell biology and the development of novel treatments for proliferative diseases. This paper presents a major advance in a strategy for specifically inhibiting particular members of the large family of protein kinases, achieved through structure-based design. Findings: These authors have synthesised a molecule that mimics all of the constituent elements of the transition state of phospho transfer from ATP to peptide substrate. In designing this molecule, the authors assumed a transition-state structure along a dissociative phospho-transfer pathway. The resulting compound has a Ki of 370 nM and is competitive with both nucleotide and peptide substrates of the kinase reaction. Crystallographic analysis of the resulting compound in complex with the target kinase (the insulin receptor tyrosine kinase) showed success in all of the major design goals. The structure of Sky1p reveals a novel mechanism for constitutive activity. Nolen B, Yun CY, Wong CF, McCammon A, Fu XD, Ghosh G: Nat Struct Biol 2001, 8:176-183. • Significance: The active conformations observed in the family of protein kinases are all closely related. Maintenance of this conformation is achieved in either a regulated or a constitutive fashion through a relatively small repertoire of interactions. This paper describes an extension to this repertoire that acts as a paradigm for a subfamily of kinases, with substrate preference for an arginine C-terminal to the phosphorylatable serine. Findings: The authors have determined a 2.1 Å resolution structure of a trimmed form of Sky1p from Saccharomyces cerevisiae. Crystallisation resulted from excision of the divergent N-terminal part of the structure and of a spacer region that characterises the SR family of protein kinases, of which Sky1p is an example. The constitutive activity of Sky1p could be understood in terms of noncore sequences of Sky1p that stabilise the active conformations of both the αC helix and the kinase activation segment. Design of three-dimensional domain-swapped dimers and fibrous oligomers. Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D: Proc Natl Acad Sci USA 2001, 98:1404-1409. • Significance: New avenues of application are opened up by the possibility of designing protein oligomers that extend in three dimensions. One mechanism by which this might be achieved is through the phenomenon of domain swapping, whereby oligomeric states are stabilised by an exchange of secondary structural elements between constituent monomers. This paper describes the design and characterisation of a domain-swapped protein structure for this purpose. Findings: The authors describe the structure and properties of two forms of designed three-helical bundles: one with an updown-up topology and the other with an up-down-down topology. Whereas the former gave rise to a domain-swapped, but globular, dimeric protein, the latter produced fibrils visible by electron microscopy.
Catalysis and regulation Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA
Novel catalytic mechanism of nucleophilic substitution by asparagine residue involving cyanoalanine intermediate revealed by mass spectrometric monitoring of an enzyme reaction. Ichiyama S, Kurihara T, Li Y-F, Kogure Y, Tsunasawa S, Esaki N: J Biol Chem 2000, 275:40804-40809.
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• Significance: This is a remarkable example of how the chemical course of an enzyme mechanism can change to surmount an active site mutation that would otherwise appear to preclude catalysis. Findings: The normal mechanism of L-2-haloacid dehalogenase from Pseudomonas sp. YL involves an SN2 displacement of chloride by the sidechain of Asp10 to form an ester intermediate that is subsequently hydrolysed to give the 2-hydroxyacid. Surprisingly, the mutant in which Asp10 was converted to asparagine (D10N) retained a low level of catalytic activity that was not due to deamidation of Asn10. Mass spectrometry was used to monitor the dehalogenation of L-2-chloropropionate by the D10N mutant. The data were consistent with SN2 displacement of chloride by the Asn10 sidechain to form an imidate from which lactic acid was eliminated to produce a β-cyanoalanine residue at position 10. The active site nitrile was partially hydrolysed to regenerate the amide sidechain of Asn10, presumably by the same catalytic machinery normally responsible for hydrolysing the ester intermediate. Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase. Mizuguchi H, Hayashi H, Okada K, Miyahara I, Hirotsu K, Kagamiyama H: Biochemistry 2001, 40:353-360. • Significance: The fact that individual protein residues play multiple roles in structure and function often complicates the interpretation of site-directed mutagenesis experiments. This study describes a way to address this issue, using an aminotransferase as an example. Findings: Aspartate aminotransferase is a well-studied transaminase that utilises a pyridoxal phosphate cofactor that is bound to the sidechain of Lys258 via a Schiff base in the resting form of the enzyme. Substrate binding increases the pKa of the Schiff base nitrogen by 2.0 pH units and this is critical in facilitating the transaldimination process that yields an imine with the α-amine of the donor amino acid, the first intermediate in transamination. Two active site guanidinium groups, contributed by Arg292 and Arg386, were clearly important in this process; however, the precise way in which these two residues increased the pKa of the Schiff base was not clear. Using a series of site-directed mutants, the manner by which these two arginine residues increase the pKa by a combination of electrostatic effects, domain motion and Schiff base conformational strain was elucidated. Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis. Fedorov A, Shi W, Kicska G, Fedorov E, Tyler PC, Furneaux RH, Hanson JC, Gainsford GJ, Larese JZ, Schramm VL, Almo SC: Biochemistry 2001, 40:853-860. •• Significance: A transition-state mimic designed from the experimental transition-state structure was shown by X-ray crystallography to mimic many of its steric and electronic properties, including ground-state destabilisation and enzyme movement. Findings: Purine nucleoside phosphorylase catalyses the displacement of purine nucleoside bases by phosphate to yield the free base and the β-anomer of ribose-1-phosphate. Kinetic isotope effect studies supported an oxocarbenium-like transition state. Immucillin-H was designed to mimic this species as a protonated pyrrolidine ring with a C–C bond to a 9-deazahypoxanthine ring, which itself is protonated on N7. Comparing the X-ray crystal structures of free and enzyme-
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bound immucillin-H revealed that the inhibitor conformation was distorted upon binding. Specifically, the pucker of the pyrrolidine ring was altered significantly and the angle between the hypoxanthine and pyrrolidine rings was flattened. These distortions of the inhibitor mirror the steps necessary for the normal substrate to reach the transition state. Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity. Polekhina G, Board PG, Blackburn AC, Parker MW: Biochemistry 2001, 40:1567-1576. • Significance: The crystal structure of this enzyme shows both intriguing similarities and differences with other glutathione S-transferases and provides the first step toward explaining the diversity of chemical reactions observed for this remarkable enzyme.
Findings: Human maleylacetoacetate isomerase catalyses two physiologically significant glutathione-dependent reactions: a cis-trans olefin isomerisation that is the penultimate step of phenylalanine and tyrosine degradation, and the hydrolysis of 1,1-dichloroacetate to glyoxalate. X-ray crystallography showed that the structure of the human enzyme was similar to other glutathione S-transferases and that the position of bound glutathione was consistent with its mechanistic roles proposed for both cis-trans isomerisation and dichloroacetate hydrolysis. Although the sequence of the human isomerase/glutathione transferase is very similar to that of the bacterial enzyme tetrachlorohydroquinone dehalogenase, this similarity apparently does not extend to the catalytic role of a conserved cysteine residue (Cys16) that is essential for the bacterial enzyme, but not for the human protein.
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 2001, 11:131–138 Contents (chosen by) 131 Protein–nucleic acid interactions (Convery et al.) 132 Folding and binding (Kortemme et al.) 132 Theory and simulation (Fiser) 132 Nucleic acids (Parkinson) 133 Sequences and topology (Copley) 133 Engineering and design (Gilardi) 135 Lipids (Newman) 135 Membrane proteins (Sinning) 136 136 136 137 • ••
Carbohydrates and glycoconjugates (Flitsch and Lowden) Biophysical methods (Matthews) Proteins (Noble) Catalysis and regulation (Stewart) of special interest of outstanding interest
analogue reveals stabilisation of the extrahelical target base and provides a basis for the general mechanism of AdoMetdependent DNA N-methylation. To catalyse the transfer of the methyl group from the cofactor to the DNA, the target nucleoside is rotated out of the DNA helix. This extrahelical conformation is stabilised by compression of the DNA helix at the target base pair position and relocation of the partner thymine base. Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Carter AP, Clemons WM Jr, Brodersen DE, Morgan-Warren RJ, Hartsch T, Wimberly BT, Ramakrishnan V: Science 2001, 291:498-501. • Significance: The 3.2 Å crystal structure of initiation factor IF1 bound to the small ribosomal subunit reveals initiation-factor-induced conformational changes in the ribosomal subunit. Findings: The structure of the complex reveals how localised changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit. Binding of IF1 occludes the ribosomal A site (blocking tRNA binding to this site) and ‘flips out’ functionally important 16S rRNA bases, burying them in pockets in the initiation factor. Binding of IF1 also causes longrange changes in the conformation of helix 44 of 16S rRNA and leads to movement of the domains of the 30S subunit with respect to each other.
Protein–nucleic acid interactions Selected by Máire Convery*, Caitríona Dennis† and Siân Rowsell‡ *GlaxoSmithKline, Stevenage, UK †University of Leeds, Leeds, UK ‡AstraZeneca, Macclesfield, UK
The structure of Pariacoto virus reveals a dodecahedral cage of duplex RNA. Tang L, Johnson KN, Ball LA, Lin T, Yeager M, Johnson JE: Nat Struct Biol 2001, 8:77-83. • Significance: The structure of Pariacoto virus reveals extensive interactions between the viral RNA genome and the protein capsid. Findings: The crystal structure of Pariacoto virus has been determined at 3.0 Å resolution and a three-dimensional cryo-EM reconstruction has been determined to better than 23 Å resolution; both were obtained from the same sample. The structures reveal a dodecahedral cage of 30 RNA duplexes inside the icosahedral virus capsid. The ordered portion of the viral RNA associates closely with the capsid and, through its interactions with the protein shell, acquires icosahedral symmetry. The protein capsid shows remarkable complementarity to the RNA, both in shape and in electrostatic environment. Structure of the N6-adenine DNA methyltransferase M••TaqI in complex with DNA and a cofactor analog. Goedecke K, Pignot M, Goody RS, Scheidig AJ, Weinhold E: Nat Struct Biol 2001, 8:121-125. • Significance: The co-crystal structure of M•TaqI in complex with its DNA substrate reveals a novel method of stabilisation of the ‘flipped-out’ base. Findings: The 2.0 Å crystal structure of the N6-adenine DNA methyltransferase M•TaqI in complex with DNA and a cofactor
Arrangement of RNA and protein in the spliceosomal U1 small nuclear ribonucleoprotein particle. Stark H, Dube P, Luhrmann R, Kastner B: Nature 2001, 409:539-542. •• Significance: The smallest structure determined to date by single-particle electron cryomicroscopy. Findings: The U1 small nuclear ribonucleoprotein (snRNP) is the first particle of the spliceosome to assemble on the mRNA. It contains elements common to all the snRNPs, along with protein and RNA unique to the U1 particle. This reconstruction at 10 Å shows that the seven Sm proteins, common to all the snRNPs, form a central doughnut-shaped core with an outer diameter of 80 Å. The other three proteins and the RNA make extensive contacts with the core and with each other. Architecture of nucleotide excision repair complexes: DNA is wrapped by UrvB before and after damage recognition. Verhoeven EE, Wyman C, Moolenaar GF, Hoeijmakers JHJ, Goosen N: EMBO J 2001, 20:601-611. • Significance: An insight into the role UvrA, UvrB and UvrA2B complexes with DNA play in damage recognition and repair in Escherichia coli. Findings: This study was carried out using atomic force microscopy (AFM) methods. The lengths of DNA arms on either side of the protein bound to the DNA at a defined damage site were measured. The amount of DNA involved in the protein–DNA complex could therefore be ascertained. It was shown that, in both the pre-incision UvrB–DNA complex and UvrA2B–DNA complexes on undamaged DNA, the DNA is wrapped around UvrB. The initial presence of UvrA imposes asymmetry on the complex and is postulated as being important for damage recognition.
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Folding and binding
Theory and simulation
Selected by Tanja Kortemme, Sehat Nauli and Alex Watters University of Washington, Seattle, Washington, USA
Selected by Andras Fiser Rockefeller University, New York, New York, USA
Design of three-dimensional domain-swapped dimers and fibrous oligomers. Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D: Proc Natl Acad Sci USA 2001, 98:1404-1409. • Significance: The authors rationally designed proteins that form domain-swapped dimers and fibrillar aggregates. Findings: The domain-swapped dimers and aggregates were designed using an α-helical bundle structure as the basic motif. Carefully placed charged residues determine the topology of the monomers by their electrostatic interactions. X-ray diffraction shows that the domain-swapped dimer is formed and a combination of electron microscopy with other biophysical studies shows that the fibrillar aggregates are formed as designed. Biomaterial design using synthetic fibrous proteins is one exciting possibility suggested by the authors.
Automated discovery of structural signatures of protein fold and function. Turcotte M, Muggleton SH, Sternberg JE: J Mol Biol 2001, 306:591-605. • Significance: An automatic approach is presented to search systematically for protein fold signatures. Findings: The authors applied Inductive Logic Programming, a machine learning technique that derives rules from observations and encoded principles. The paper shows that, applying this approach for protein fold recognition, fold signatures do exist and superfamilies can be automatically and systematically discovered. The derived new rules were partly tested by presenting them in a form of a questionnaire to the world expert Murzin, who identified virtually all of them. The approach aims to replace the need for human expertise, used so far in constructing protein family databases such as SCOP.
Barstar is electrostatically optimized for tight binding to barnase. Lee L-P, Tidor B: Nat Struct Biol 2001, 8:73-76. • Significance: The importance of electrostatic interactions for protein stability and binding is unclear, as the replacement of polar with hydrophobic sidechains can be energetically favourable. Here, the authors present evidence that the optimisation of electrostatic interactions may have been used in naturally occurring proteins to achieve tight binding. Findings: Lee and Tidor developed an algorithm to design a charge distribution on a protein ligand that optimises the electrostatic contribution to binding to its receptor. For a complex between a protein inhibitor, barstar, and its receptor, barnase, the computed charge distribution is very similar to the actual one. Furthermore, mutation of interface sidechains on barstar with each of the naturally occurring amino acids is not predicted to increase binding affinity, suggesting that this interface is electrostatically optimized.
Protein structural alignments and functional genomics. Irving JA, Whisstock JC, Lesk AM: Proteins 2001, 42:378-382. • Significance: A new approach is introduced to locate functional sites in proteins using pairwise structural alignments alone. Findings: The strong conservation of active site conformations suggests a method for identifying active sites. This depends on the relationship between size and goodness-of-fit of aligned substructures in homologous proteins. This relationship was well established before, but not analysed in quantitative detail. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins.
Tailoring in vitro evolution for protein affinity or stability. Jermutus L, Honegger A, Schwesinger F, Hanes J, Pluckthun A: Proc Natl Acad Sci USA 2001, 98:75-80. • Significance: Due to limited knowledge of structure/function relationships, rational design of proteins with specific physical properties has not been consistently achieved. Directed evolution using display techniques that specifically optimise certain properties, such as protein stabilities and affinities, may provide a convenient method to overcome current limitations in rational design. Findings: Using single-chain Fv antibody fragments (scFvs), the authors were able to use a series of PCR mutagenesis and directional selection rounds to generate variants with either higher overall stability or greater affinity for a specific antigen, fluorescein. scFv phage libraries bound to biotin-tagged fluorescein were incubated with saturating levels of untagged fluorescein for increasing lengths of time, such that only individual scFvs with low dissociation rates would remain bound to the biotin-labelled fluorescein. These phage were then recovered by binding to streptavidin-coated magnetic beads. This gave a variant with a 30-fold decrease in the dissociation rate. Stability selections were selected in increased reducing environments to produce stable scFvs without disulfide bonds. When disulfide bonds were reintroduced by returning the selected proteins to an oxidising environment, the selected proteins had a stability twice that of the starting protein.
A Gaussian statistical mechanics model for the equilibrium thermodynamics of barnase folding. Crippen MG: J Mol Biol 2001, 306:565-573. • Significance: A new statistical mechanical model is presented that is able to reproduce the experimental free energy of a real protein during unfolding. Findings: It was possible to adjust a potential function to give the correct free energies for the isothermal denaturation of the protein barnase. By extending a statistical mechanical model with the empirical estimate of the partial molar heat capacity of denatured proteins as a function of amino acid composition, it could quantitatively account for experimental observations during barnase denaturation, such as melting temperature, width of thermal transition and cooperativity. The model described in this paper makes an attempt to bridge experimental observations of a real protein and theory, at least in the case of one protein and at least for the equilibrium thermodynamics.
Nucleic acids Selected by Gary Parkinson The Institute of Cancer Research, Sutton, UK
Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection. van Buuren BN, Overmars FJ, Ippel JH, Altona C, Wijmenga SS: J Mol Biol 2000, 304:371-383. • Significance: The authors report the first high-resolution NMR solution structure of a DNA three-way junction (3H). Three-way junctions are a recurring motif in nucleic acid structures; such DNA motifs are associated with recombination involving phage, with replication and with site-specific integration of viral DNA
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into the host genome. Using this structure and previous data, the authors developed rules for the predictive stacking preferences of a three-way junction, of any sequence, with two unpaired bases (3HS2). Findings: Arms A and B are co-axially stacked. Arm C is in an antiparallel orientation with respect to the A–B-stacked helix. The interhelix angle of 53(±11)° is in agreement with previous FRET studies on three-way junctions. Both the A–B-stacked helices and arm C show an overall B-DNA geometry. Only small deviations from this standard helical geometry are observed, such as a reduced twist in the A–B-stacked helix at the junction with arm C. The two unpaired bases are stacked onto arm C reminiscent of a type I hairpin fold. The authors propose two rules, the ‘pyrimidine rule’ and the ‘loop rule’, that, when combined, can reproduce experimentally known conformational preferences for 3H2S structures.
• Significance: An interesting approach to the evolution of gene context, using a local alignment algorithm to detect conserved gene order in different organisms. Findings: The authors’ claims of the value of genome context for function prediction are relatively modest. Predictions for proteins of unknown function or major clarifications of function were found for 4% of the orthologous groups analysed. As is pointed out, however, the true value of such approaches is that the information provided is completely complementary to homology-based methods, helping place proteins within systems and complexes. Some of the examples found include new components of the translation machinery and proteins having replication- and repair-related functions.
Sequences and topology
Protein design of an HIV-1 entry inhibitor. Root MJ, Kay MS, Kim PS: Science 2001, 291:884-888. •• Significance: The design of a small protein may serve as the basis for a new class of antiviral agents displaying potent inhibitory activity against diverse HIV-1 variants. Findings: Infection by HIV-1 requires fusion of the viral and cellular membranes via the formation of a trimer of hairpin structures in gp41. The core of gp41 is a bundle of six α helices. The authors designed a protein, denoted 5-Helix, in which five of the six helices of the core of gp41 are connected with short peptide linkers. The resulting protein is shown not only to be well folded, soluble and stable under physiological conditions, but also to potently inhibit HIV-1 membrane fusion (nanomolar IC50), as measured by viral infectivity and cell–cell fusion assays. It also inhibits infection by viruses pseudo-typed with a variety of HIV-1 envelope proteins with similar potency. Finally, the authors suggest that, as a potent broad-spectrum inhibitor of viral entry, 5-Helix may serve as the basis for development of a new class of therapeutic agents against HIV-1.
Selected by Richard Copley EMBL, Heidelberg, Germany
KH domain: one motif, two folds. Grishin NV: Nucleic Acids Res 2001, 29:638-643. •• Significance: A rare example of homologous sequences adopting folds with different topologies. Findings: The KH domain was originally detected in various proteins using sequence-profile-based methods. With the availability of 3D structures, Grishin has noticed that these original sequence definitions fall into two distinct fold topologies. The differences between the two types of fold are not minor — the central β sheet undergoes major rearrangement, in a manner reminiscent of the two forms of serpin. On the other hand, the sequence evidence that the two types of folds are indeed homologous is persuasive. Grishin goes on to discuss evolutionary scenarios that could have led to this situation. It will be interesting to see how many such results are uncovered in the coming years and whether they can be drawn into a consistent theoretical framework. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Krogh A, Larsson B, von Heijne G, Sonnhammer EL: J Mol Biol 2001, 305:567-580. • Significance: A method to predict 97–98% of transmembrane helices from single sequences. Findings: Transmembrane helix prediction is important not only for understanding the function of proteins, but also from a practical point of view in determining domain boundaries and guiding procedures that aim to express the soluble portions of proteins. The method presented here is particularly accurate, although (in common with other approaches) it can occasionally confuse signal peptides with transmembrane helices (particularly for some bacterial proteins). Depending on the organism, between 20 and 30% of genes are predicted to encode transmembrane-domain-containing proteins and proteins with both the N and C termini on the cytoplasmic side of the membrane are preferred. Organisms with large genomes do not seem to be relatively enriched in transmembrane-domain-containing proteins compared with organisms encoding fewer genes. Genome alignment, evolution of prokaryotic genome organization and prediction of gene function using genomic context. Wolf YI, Rogozin IB, Kondrashov AS, Koonin EV: Genome Res 2001, 11:356-372.
Engineering and design Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK
Thermostabilization of a chimeric enzyme by residue substitution: four amino acid residues in loop regions are responsible for the thermostability of Thermus thermophilus isopropylmalate dehydrogenase. Numatu K, Hayashi-Iwasaki Y, Kawaguchi J, Sakurai M, Moriyama H, Tanaka N, Oshima T: Biochem Biophys Acta 2001, 1545:174-183. • Significance: Thermostability of proteins is a key factor in the development of enzymes for industry. The authors investigated the effect on thermostability of single amino acid changes within a thermophilic–mesophilic chimeric enzyme that restore the thermophilic sequence. By following the temperature at which 50% irreversible thermal denaturation (Th) occurs, they were able to quantify the effect of different changes. Findings: The chimeric enzyme had 22 amino acid changes relative to the thermophilic enzyme in a domain comprising 20% of the entire protein and a Th of 63ºC compared with 83ºC. The authors found four amino acid reversions that led to a significant increase in Th. The changes resulted in tightening hydrophobic packing by removing cavities (e.g. A115L: 6ºC increase in Th), neutralising a surface charge (e.g. S62R: 6ºC increase in Th and kcat increases from 121 to 149 s—1) and placing glycine in a position requiring strained torsion angles (N125G: 3ºC increase in Th and kcat increases from 121 to 133 s–1). All effective changes were found to be in loops. Investigations on double mutations showed that the effects are additive. Changes that were predicted to result in similar
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effects failed to increase thermostability, probably as a result of steric effects. Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent proteins through zinc-site engineering. Jensen KK, Martini L, Schwartz TW: Biochemistry 2001, 40:938-945. • Significance: The authors exploited metal-binding site design principles to stabilise the interaction of two protein fluorescence partners. Close proximity of the fluorophores buried within the partners allowed enhanced fluorescence resonance energy transfer (FRET) signal. The authors suggest that such a system could be adapted to the study of protein–protein interactions in vivo. Findings: Partial zinc-binding sites were incorporated into cyan fluorescent protein (CFP) with complementary sites in yellow fluorescent protein (YFP), such that a stable heterodimer will form by binding two zinc ions in symmetrical sites. A zinc-dependent FRET signal was observed for heterodimers of CFP and YFP (excitation of CFP at 433 nm leads to emission at 475 nm, which, in turn, excites YFP, leading to emission at 533 nm). For a bidendate zinc-binding site design, a complete YFP signal was observed in 10–2.5 M ZnCl2. The authors investigate both bidendate and tridendate zinc-binding site designs; the apparent enhancement in the tridendate constructs was attributed to an increased availability of potential metal ligands, rather than tridendate metal binding. An approximately 100-fold increase in dimer formation was observed for zinc-binding mutants compared with autodimerisation in the wildtype. By designing proteins with only a single potential zinc-binding site, an enhancement in the FRET signal was observed (signal ratio I530:I475 increased from 4.8 to 8.1). This was attributed to a reduction in the propensity of nonproductive homodimer formation. A limitation of the constructs was a low zinc affinity (10–3 M), which would prevent the use of the current system in vivo. Structure of a genetically engineered molecular motor. Kliche W, Fujita-Becker S, Kollmar M, Manstein DJ, Kull FJ: EMBO J 2001, 20:40-46. •• Significance: Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. This work shows the possibility of engineering single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Findings: An active molecular motor was created by fusing a domain of myosin with an artificial lever arm composed of α-actinin. The artificial lever arm showed a structure similar to that of human α-actinin, with both repeats folded into a triple helix and connected by a continuous helical linker. Interestingly, the engineered linker connecting the myosin converter domain to α-actinin was also found to form an α helix. As the bending of helix 1 in the first α-actinin repeat dictates the position of the end of the lever arm, changing the helical register in the motor–lever linker region allowed accurate repositioning of the C-terminal end of the lever arm. Previous studies had shown a linear relationship between lever arm length and in vitro sliding velocity in artificial motors with lever arms composed of one or two α-actinin repeats. As two α-actinin repeats are about 13 nm long, it is argued that removing a single α-actinin repeat would shorten the lever arm by 6.5 nm. If the lever were coupled to an identical rotational displacement, the predicted step size would be about 55% of the dual repeat construct. This is
in agreement with a previously measured step size ratio, showing the possibility of having molecular motors with different properties. The authors also investigated the disruption of a salt bridge that appears to interfere with the formation of myosin’s catalytically active ‘closed’ conformation. Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation. Isalan M, Patel SD, Balasubramanian S, Choo Y: Biochemistry 2001, 40:830-836. • Significance: Telomeres are highly conserved DNA repeat sequences associated with proteins that are found at the end of chromosomes in nearly all eukaryotes. This paper shows the successful design of a Zn-finger protein with high specificity for binding to the G-quadruplex structure formed by the human telomere sequence (5′-GTTAGG-3′)n for potential therapeutic applications. Findings: A phage display library containing three Zn-finger modules was designed to maximise the chance of finding roteins that bind the single-stranded human telomeric G-quadruplex. This led to the isolation of four clones with a significant degree of sequence similarity. The library was screened with 5′biotin—(5′-GTTAGG-3′)5 (Biotin-Htelo) and the structure of the single-stranded G-quadruplex was tested using a DMS protection assay in order to confirm the specificity for the three-dimensional conformation adopted by the human G-quadruplex. The analysis of the binding properties of the selected Zn-finger, using analogues of a Biotin-Htelo oligonucleotide containing adenine or inosine in place of the critical guanine bases and of a double-stranded Htelo oligonucleotide, showed that the selected clone (Gq1) had high sequence and/or structure specificity for the human G-quadruplex, with an apparent Kd of approximately 26 nM. Gq1, cloned and overexpressed as a glutathione-S-transferase fusion protein, was tested for electromobility shift and DMS protection of the DNA–protein complex. The Kd of 34 nM for the Gq1–Htelo complex, obtained by analysis of the mobility shift in nondenaturing gel, was in good agreement with the one measured by the biotin assay. Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase. Gutierrez A, Yoshimura T, Fuchikami Y, Esaki N: Eur J Biochem 2000, 267:7218-7223. • Significance: The activity of an important family of enzymes could be modified towards different substrates. The method of inserting a short glycine loop may have wider applications to other enzymes. Findings: A loop of one, three or five glycines was inserted between the two domains of the D-amino acid aminotransferase homodimer. The activity of the mutant enzymes towards amino acid substrates of different sizes was found to have a different pattern compared with the wildtype. In particular, the five-glycine insertion allowed the protein to turn over small-chain amino acids more efficiently. Also, the one glycine mutant could use larger bulkier amino acids as substrates. The specific activity of the protein could be tuned by the insertion of a small glycine loop of different lengths. Critical role of the residue six at position 87 in H2O2-dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3. Li Q-S, Ogawa J, Shimizu S: Biochem Biophys Res Commun 2000, 280:1258-1261.
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•• Significance: The driving of the cytochrome P450 catalytic cycle using hydrogen peroxide presents a feasible and inexpensive alternative to the usual cofactor, NADPH. Findings: The group replaced a single phenylalanine in the active site of cytochrome P450BM-3 with either glycine or alanine. These single point mutations allowed the enzyme to hydroxylate substrates using hydrogen peroxide at around 40% of the wildtype enzyme’s rate when utilising NADPH. The mutations also improved the enzyme’s stability in hydrogen peroxide. These studies provide another important step forward for the commercial use of P450s. Engineering of a functional human NADH-dependent cytochrome P450 system. Dohr O, Paine MJI, Friedberg T, Roberts GCK, Wolf CR: Proc Natl Acad Sci USA 2001, 98:81-86. •• Significance: The residue responsible for distinguishing an enzyme’s normal cofactor, NADPH, has been identified and replaced so that NADH can be utilised instead. Findings: By replacing Trp676 of human cytochrome P450 NADPH-dependent oxido-reductase (CPR) with an alanine, the enzyme was engineered to use NADH instead of NADPH as the source for its reducing equivalents. The wildtype CPR has essentially no affinity for NADH and exhibits very little reducing activity in its presence. The W676A mutant was able to utilise NADH and exhibited around 90% of the reducing (NADPHdependent) activity of the wildtype CPR. These findings open up the possibility for the application of this approach to other oxido-reductases, aiding our understanding of how they distinguish between different cofactors.
Lipids Selected by Richard Newman EMBL-European Bioinformatics Institute, Cambridge, UK
Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Peters C, Bayer MJ, Buehler S, Andersen JS, Mann M, Mayer A: Nature 2001, 409:581-588. •• Significance: The mechanism of membrane fusion, without which all membrane traffic within a cell would grind to a halt, was thought to be mediated solely by SNAREs (proteins that inhabit all fusion-competent membranes of a cell). The authors describe a situation in which SNAREs do not cause fusion directly, but instead enable an ion-permeable membrane protein, the vacuolar H+ATPase, to form a bridge between membranes, allowing fusion to proceed. The important implication of these observations is that the mechanism of membrane fusion is explained in terms of the radial expansion of a proteolipid-lined protein channel. Findings: The authors show that two yeast vacuoles fuse by forming a head-to-head dimer of the V0 part of the vacuolar proton pump, which is found in most intracellular membranes and vesicles. This pump contains two multisubunit sectors — V1, which hydrolyses ATP, and V0, a complex of six so-called proteolipids — together with associated subunits, through which proteins are transported. The dimerisation of V0 is proposed to allow the two proteolipid hexamers to form a closed-off channel from one vacuole into the other, suggesting a mechanism for fusion. Calcium-bound calmodulin is believed to cause the proteolipids to separate within the plane of the membrane, so that lipids invade the space between them and the aqueous channel in the centre opens. The separating proteolipids expose surfaces between them that have a hydrophobic portion sandwiched between two hydrophilic
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strips. Such surfaces mimic lipid bilayers and allow lipids to invade the proteolipid ring without leaving their bilayer configuration. This model is attractive because it explains fusion without invoking temporary lipid intermediates.
Membrane proteins Selected by Irmgard Sinning EMBL, Heidelberg, Germany
Projection structure of a ClC-type chloride channel at 6.5 Å resolution. Mindell JA, Maduke M, Miller C, Grigorieff N: Nature 2001, 409:219-223. •• Significance: Chloride channels are present in virtually all eukaryotic cells. They serve a wide range of functions, such as controlling electrical excitability in skeletal muscle and maintaining systemic blood pressure. Two fundamentally different models for the channel architecture were proposed previously: a double-barrelled homodimer with two pores per dimer and a conventional barrel-stave architecture with a single pore on the homodimer twofold axis. The projection structure from twodimensional crystals of a prokaryotic ClC-channel homologue (EriC) allows a first glimpse at this dimeric channel complex with water-filled pores and can serve as a prototype, at least for the muscle subfamily. Findings: EriC was crystallised by reconstituting the purified channel into phospholipid membranes. The crystals have P22121 symmetry with a unit cell of 140 × 80 Å. Two EriC channels per unit cell are related by crystallographic twofold axes, confirming the homodimeric nature of the complex. EriC shows two pairs of two low-density troughs, each pair separated by a density peak of about 7 Å across. The authors interpret their data such that each pair of ‘density wells’ represents a single pore that is kinked roughly halfway through the membrane. The intracellular and extracellular openings are offset when viewed normal to the membrane plane. The model with two pores in the dimeric channel explains the double-barrelled gating behaviour of eukaryotic ClC channels. The distance of about 25–40 Å between the two pores would be far enough for them to be electrostatically independent. Although the authors count 12 peaks in all, the projection structure does not allow the assignment of transmembrane helices, which are believed to number 10–12. Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Zouni A, Witt H-T, Kern J, Fromme P, Krauss N, Saenger W, Orth P: Nature 2001, 409:739-743. •• Significance: Oxygenic photosynthesis provides the atmosphere with oxygen and is the principal energy converter on earth. Structural information from electron crystallography was previously obtained for photosystem II (PSII) fragments without water oxidising activity (at 8 Å) and intact PSII, but at low resolution (15–30 Å). The X-ray structure of PSII at 3.8 Å resolution shows the spatial arrangement of protein subunits and cofactors. The presence of the manganese cluster will stimulate specific investigations on the mechanism of water oxidation. Findings: PSII from the thermophilic cyanobacterium Synechococcus elongatus was isolated as a homodimer, as confirmed also by electron microscopy. PSII consists of at least 17 protein subunits, of which 14 are located within the membrane. Porphyrin molecules could be modelled into the electron density and the proteins are represented by Cα traces. The membrane integral part comprises 36 transmembrane α helices; 22 of these, assigned to D1, D2, CP43 and CP47 proteins, are arranged with a local pseudo-twofold rotation
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symmetry. As expected, the arrangement of the α helices from the D1 and D2 proteins resembles that of the reaction centre from photosynthetic bacteria and of the C-terminal helices of PsbA and PsbB in PSI. This supports the hypothesis that all photosynthetic reaction centres have evolved from one common ancestor. Seven transmembrane helices could not be assigned in the present structure and the extrinsic 12 kDa subunit could not be located because of unassigned secondary structure elements. The arrangement of the cofactors is presented. The interatomic distances between the four manganese ions of the manganese cluster are close to values derived from spectroscopic data.
Carbohydrates and glycoconjugates Selected by Sabine L Flitsch and Philip AS Lowden* Edinburgh University, Edinburgh, UK *University of Exeter, Exeter, UK
Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Persson K, Ly HD, Dieckelmann M, Wakarchuk WW, Withers SG, Strynadka NCY: Nat Struct Biol 2000, 8:166-175. •• Significance: The first three-dimensional structure of a retaining, nucleotide-sugar-dependent glycosyltransferase and the first of any glycosyltransferase in complex with both donor and acceptor sugars. Findings: LgtC is an α-1,4-galactosyltransferase that transfers galactose from uridine diphosphogalactose (UDP-Gal) to the terminal lactose of the cell-surface lipooligosaccharides of Neisseria meningitidis with retention of anomeric configuration. This paper reports the 2.0 Å crystal structures of LgtC complexed with Mn2+ and UDP 2-deoxy-2-fluoro-galactose (an inactive glycosyl donor analogue), with and without 4′-deoxylactose (an inactive glycosyl acceptor analogue). The overall fold of the enzyme resembles some of the inverting glycosyltransferase structures already known, despite low sequence homology. By analogy with retaining glycosidases, a catalytic nucleophile was expected to be found. The structure suggested either the 6′-hydroxyl of lactose or the sidechain oxygen of Gln189 as this nucleophile. However, studies with alternative substrates or with Gln189 mutants provided no support for either of these cases. The catalytic mechanism therefore remains obscure, with the intriguing possibility that the reaction may proceed by a rare SNi mechanism, in which nucleophile and leaving group act on the same side of the substrate. Direct isolation and sequencing of specific protein-binding glycosaminoglycans. Keiser N, Venkataraman G, Shriver Z, Sasisekharan R: Nat Med 2001, 7:123-128. •• Significance: Heparin/heparan-sulfate-like glycosaminoglycans (HLGAG) play important roles in the regulation of protein stability and function. Identification of the precise glycosaminoglycan sequences involved in these events has been very difficult. This paper demonstrates for the first time how surface noncovalent affinity mass spectrometry can be used to rapidly isolate and directly sequence the biologically relevant HLGAG oligosaccharides. Findings: A recently reported method of using matrix-assisted laser desorption ionisation mass spectrometry to analyse picomole amounts of HLGAG oligosaccharides was combined with immobilisation of an HLGAG-binding protein on a surface to perform affinity isolation and sequencing of tissue-derived HLGAG oligosaccharides on a mass spectrometer surface.
This method of analysis was first tested to study the binding of known ligands to antithrombin III and was able to distinguish between specific and nonspecific binding interactions, even within a mixture of saccharides. The studies were then extended to find and identify oligosaccharide sequences that bind to the fibroblast growth factor FGF-2 by combining the method with other sequencing techniques, such as selective HLGAG hydrolysis. Structural basis for catalysis and inhibition of N-glycan processing class I α1,2-mannosidases. Vallee F, Karaveg K, Herscovics A, Moremen KW, Howell PL: J Biol Chem 2000, 275:41287-41298. • Significance: Endoplasmic reticulum class I α1,2-mannosidase is a key enzyme in the maturation of N-linked oligosaccharides and plays an important role in the degradation of misfolded glycoproteins by the proteasome. It is therefore a target of inhibitor design for the treatment of genetic diseases that are characterised by rapid degradation of misfolded glycoproteins. This paper provides a first structure of the enzyme with and without bound inhibitors, and provides a basis for further inhibitor design. Findings: Crystal structures of the catalytic domain of the α1,2-mannosidase in the presence and absence of two inhibitors, kifunesine and 1-deoxymannojirimycin, were determined. The data show that the specificity of the inhibitors is dictated by the coordination of the essential calcium at the catalytic site to the O-2′ and O-3′ hydroxyl groups, and resulting stabilisation of the sixmembered rings in the 1C4 conformation. Furthermore, the distance of any putative catalytic carboxyl-sidechain to the substrate-binding site suggests that the enzyme operates by a novel mechanism in which the active site carboxyl groups do not directly react with the substrate.
Biophysical methods Selected by Steve Matthews Imperial College of Science, Technology and Medicine, London, UK
Reaction-induced infrared difference spectroscopy for the study of protein reaction mechanisms. Zscherp C, Barth A: Biochemistry 2001, 40:1875-1883. • Significance: This paper gives an up-to-date illustration of the state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterisation of enzyme function on the level of single bonds of proteins, cofactors or substrates. Findings: Protein structure and function can be studied by various biochemical and biophysical approaches. High-resolution structural information can be gained from X-ray crystallography, electron cryomicroscopy and NMR spectroscopy. Infrared spectroscopy provides the ability to study structural information with a high time resolution, which enables molecular mechanisms of protein reactions to be elucidated. The number of methods used to initiate protein reactions has constantly increased over the past decade. Light-driven reactions, redox reactions, molecule–protein interaction and protein folding can be studied by infrared spectroscopy and are discussed in detail.
Proteins Selected by Martin Noble University of Oxford, Oxford, UK
Mechanism based design of a protein kinase inhibitor. Parang K, Till JH, Ablooglu AJ, Kohanski RA, Hubbard SR, Cole PA: Nat Struct Biol 2001, 8:37-41.
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• Significance: Specific inhibition of protein kinases could significantly advance both research into cell biology and the development of novel treatments for proliferative diseases. This paper presents a major advance in a strategy for specifically inhibiting particular members of the large family of protein kinases, achieved through structure-based design. Findings: These authors have synthesised a molecule that mimics all of the constituent elements of the transition state of phospho transfer from ATP to peptide substrate. In designing this molecule, the authors assumed a transition-state structure along a dissociative phospho-transfer pathway. The resulting compound has a Ki of 370 nM and is competitive with both nucleotide and peptide substrates of the kinase reaction. Crystallographic analysis of the resulting compound in complex with the target kinase (the insulin receptor tyrosine kinase) showed success in all of the major design goals. The structure of Sky1p reveals a novel mechanism for constitutive activity. Nolen B, Yun CY, Wong CF, McCammon A, Fu XD, Ghosh G: Nat Struct Biol 2001, 8:176-183. • Significance: The active conformations observed in the family of protein kinases are all closely related. Maintenance of this conformation is achieved in either a regulated or a constitutive fashion through a relatively small repertoire of interactions. This paper describes an extension to this repertoire that acts as a paradigm for a subfamily of kinases, with substrate preference for an arginine C-terminal to the phosphorylatable serine. Findings: The authors have determined a 2.1 Å resolution structure of a trimmed form of Sky1p from Saccharomyces cerevisiae. Crystallisation resulted from excision of the divergent N-terminal part of the structure and of a spacer region that characterises the SR family of protein kinases, of which Sky1p is an example. The constitutive activity of Sky1p could be understood in terms of noncore sequences of Sky1p that stabilise the active conformations of both the αC helix and the kinase activation segment. Design of three-dimensional domain-swapped dimers and fibrous oligomers. Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D: Proc Natl Acad Sci USA 2001, 98:1404-1409. • Significance: New avenues of application are opened up by the possibility of designing protein oligomers that extend in three dimensions. One mechanism by which this might be achieved is through the phenomenon of domain swapping, whereby oligomeric states are stabilised by an exchange of secondary structural elements between constituent monomers. This paper describes the design and characterisation of a domain-swapped protein structure for this purpose. Findings: The authors describe the structure and properties of two forms of designed three-helical bundles: one with an updown-up topology and the other with an up-down-down topology. Whereas the former gave rise to a domain-swapped, but globular, dimeric protein, the latter produced fibrils visible by electron microscopy.
Catalysis and regulation Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA
Novel catalytic mechanism of nucleophilic substitution by asparagine residue involving cyanoalanine intermediate revealed by mass spectrometric monitoring of an enzyme reaction. Ichiyama S, Kurihara T, Li Y-F, Kogure Y, Tsunasawa S, Esaki N: J Biol Chem 2000, 275:40804-40809.
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• Significance: This is a remarkable example of how the chemical course of an enzyme mechanism can change to surmount an active site mutation that would otherwise appear to preclude catalysis. Findings: The normal mechanism of L-2-haloacid dehalogenase from Pseudomonas sp. YL involves an SN2 displacement of chloride by the sidechain of Asp10 to form an ester intermediate that is subsequently hydrolysed to give the 2-hydroxyacid. Surprisingly, the mutant in which Asp10 was converted to asparagine (D10N) retained a low level of catalytic activity that was not due to deamidation of Asn10. Mass spectrometry was used to monitor the dehalogenation of L-2-chloropropionate by the D10N mutant. The data were consistent with SN2 displacement of chloride by the Asn10 sidechain to form an imidate from which lactic acid was eliminated to produce a β-cyanoalanine residue at position 10. The active site nitrile was partially hydrolysed to regenerate the amide sidechain of Asn10, presumably by the same catalytic machinery normally responsible for hydrolysing the ester intermediate. Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase. Mizuguchi H, Hayashi H, Okada K, Miyahara I, Hirotsu K, Kagamiyama H: Biochemistry 2001, 40:353-360. • Significance: The fact that individual protein residues play multiple roles in structure and function often complicates the interpretation of site-directed mutagenesis experiments. This study describes a way to address this issue, using an aminotransferase as an example. Findings: Aspartate aminotransferase is a well-studied transaminase that utilises a pyridoxal phosphate cofactor that is bound to the sidechain of Lys258 via a Schiff base in the resting form of the enzyme. Substrate binding increases the pKa of the Schiff base nitrogen by 2.0 pH units and this is critical in facilitating the transaldimination process that yields an imine with the α-amine of the donor amino acid, the first intermediate in transamination. Two active site guanidinium groups, contributed by Arg292 and Arg386, were clearly important in this process; however, the precise way in which these two residues increased the pKa of the Schiff base was not clear. Using a series of site-directed mutants, the manner by which these two arginine residues increase the pKa by a combination of electrostatic effects, domain motion and Schiff base conformational strain was elucidated. Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis. Fedorov A, Shi W, Kicska G, Fedorov E, Tyler PC, Furneaux RH, Hanson JC, Gainsford GJ, Larese JZ, Schramm VL, Almo SC: Biochemistry 2001, 40:853-860. •• Significance: A transition-state mimic designed from the experimental transition-state structure was shown by X-ray crystallography to mimic many of its steric and electronic properties, including ground-state destabilisation and enzyme movement. Findings: Purine nucleoside phosphorylase catalyses the displacement of purine nucleoside bases by phosphate to yield the free base and the β-anomer of ribose-1-phosphate. Kinetic isotope effect studies supported an oxocarbenium-like transition state. Immucillin-H was designed to mimic this species as a protonated pyrrolidine ring with a C–C bond to a 9-deazahypoxanthine ring, which itself is protonated on N7. Comparing the X-ray crystal structures of free and enzyme-
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bound immucillin-H revealed that the inhibitor conformation was distorted upon binding. Specifically, the pucker of the pyrrolidine ring was altered significantly and the angle between the hypoxanthine and pyrrolidine rings was flattened. These distortions of the inhibitor mirror the steps necessary for the normal substrate to reach the transition state. Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity. Polekhina G, Board PG, Blackburn AC, Parker MW: Biochemistry 2001, 40:1567-1576. • Significance: The crystal structure of this enzyme shows both intriguing similarities and differences with other glutathione S-transferases and provides the first step toward explaining the diversity of chemical reactions observed for this remarkable enzyme.
Findings: Human maleylacetoacetate isomerase catalyses two physiologically significant glutathione-dependent reactions: a cis-trans olefin isomerisation that is the penultimate step of phenylalanine and tyrosine degradation, and the hydrolysis of 1,1-dichloroacetate to glyoxalate. X-ray crystallography showed that the structure of the human enzyme was similar to other glutathione S-transferases and that the position of bound glutathione was consistent with its mechanistic roles proposed for both cis-trans isomerisation and dichloroacetate hydrolysis. Although the sequence of the human isomerase/glutathione transferase is very similar to that of the bacterial enzyme tetrachlorohydroquinone dehalogenase, this similarity apparently does not extend to the catalytic role of a conserved cysteine residue (Cys16) that is essential for the bacterial enzyme, but not for the human protein.