PII: S0969-2126(01)00614-1

Abstract

A selection of interesting papers that were published in the month before our press date in major journals most likely to report significant results in structural biology, protein and RNA folding. □ Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold. Sergei V. Pletnev, Wei Zhang, Suchetana Mukhopadhyay, Bonnie R. Fisher, Raquel Hernandez, Dennis T. Brown, Timothy S. Baker, Michael G. Rossmann, and Richard J. Kuhn (2001). Cell 105, 127–136. This paper together with Ferlenghi et al. and Lescar et al. (described here) present a step forward in our understanding of enveloped virus structure. The best-characterized viral fusion proteins (Type I) follow the paradigm of the influenza hemagglutinin. They are trimeric proteins that contain fusion peptides near their N-terminae, adopt a helical coiled-coil conformation after triggering for fusion as presaged by strong helical coiled-coil tendency, and are activated for fusion by proteolytic cleavage of the fusion protein precursor. The systems described in these three papers, alphaviruses and the flaviviruses, are examples of type II fusion proteins. The proteins bear internal fusion peptides, show no helix-forming tendency, and are activated by the cleavage of the precursor of a companion protein rather than the fusion protein itself. These three papers show that an architectural similarity exists between viruses that utilize type II fusion proteins: the presence of a fusion protein shell. Pletnev et al. (2001) use cEM and difference imaging to characterize mutant Sindbis viruses from which defined glycosylation sites have been removed. Sindbis, like the other alphaviruses, contains 240 copies of each envelope protein arrayed as 80 trimeric spikes in a T = 4 arrangement. Two of the glycosylation sites are present on E1, the viral fusion protein, and the other two are on E2, the receptor binding subunit of the spike complex. The precursor of E2 is the companion protein to E1, whose cleavage is required to activate the spike complex for fusion. Difference imaging shows that the E1 sites lie at the base of the spike in a skirt-like layer of protein that surrounds the spike and covers the membranes. The E2 sites are on the projecting regions of the spikes. Similar analysis of corresponding mutants in another alphavirus, Ross River virus, leads to a similar localization. The authors point out that the arrangement of the fusion protein, E1, resembles the arrangement seen by Ferlenghi et al. (2001) in a flavivirus particle and suggest that both type II fusion proteins follow a similar mechanism. □ The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Julien Lescar, Alain Roussel, Michelle W. Wien, Jorge Navaza, Stephen D. Fuller, Giesela Wengler, Gert Wengler, and Felix A. Rey (2001). Cell 105, 137–48. This paper combines the structure of the fusion protein with a cEM reconstruction of the virion to define the details of a fusion protein shell. The authors show the trace of the polypeptide chain of the SFV fusion glycoprotein, E1, derived from their 3.5 Å electron density map. E1 is unexpectedly similar to the flavivirus envelope protein TBE-E, with three structural domains disposed in the same primary sequence arrangement. The similarity of the E1 to the flavivirus E extends to its arrangement on the viral surface as seen from the fitting of the protein to a 9 Å resolution map of SFV [Mancini et al. (2000) Mol. Cell 5, 255–266] showing that most of E1 forms the flat skirt region between the projecting spikes. The fusion peptide of E1 is buried against the projecting spikes formed by E2. This suggests a mechanism by which E2 rearrangement can control the oligomerization of E1 and fusion. □ The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel. Montserrat Barcéna, Theresa Ruiz, Luis Enrique Donate, Susan E. Brown, Nicholas E. Dixon, Michael Radermacher, and José Marı́a Carazo (2001). EMBO J. 20, 1462–1468. Escherichia coli DnaB is the best characterized member of the replicative helicase family. The authors define the structure of this 480 kDa protein complex of a DnaB hexamer with six copies of its loading partner, DnaC, by cEM and three-dimensional reconstructions using Radon transforms to 26 Å resolution. The volume that is generated by combining 8000 particle images reveals the elaborate interactions among DnaC and DnaB dimers that lead to a variation of symmetry of the complex and modulation of its helicase activity. □ Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion. Jean Lepault, Isabelle Petitpas, Inge Erk, Jorge Navaza, Dominique Bigot, Michel Dona, Patrice Vachette, Jean Cohen, and Félix A. Rey (2001). EMBO J. 20, 1485–1497. The structural protein VP6 of rotavirus forms the middle layer in the triple-layered viral capsid. The crystal structure of VP6 was determined and fitted into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal β-barrel domain and a proximal α-helical domain, which interact with the outer and inner layer of the virion, respectively. The atomic model of the middle layer derived from the fit shows that quasiequivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer. □ Structural polymorphism of the major capsid protein of rotavirus. Jean Lepault, Isabelle Petitpas, Inge Erk, Jorge Navaza, Dominiqe Bigot, Michel Dona, Patrice Vachette, Jean Cohen, and Felix A. Rey (2001). EMBO J. 20, 1498–1507. Rotaviruses contain a triple-layered icosahedral capsid. The authors show that the major capsid protein, VP6, self-assembles into spherical or helical particles primarily as a function of pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca2+ and Zn2+). The structures of two types of helical tubes were determined by cEM and helical image reconstruction to resolutions of 20 Å and 25 Å , respectively. The closeness of the match between the molecular envelope of VP6 and the atomic model determined by X-ray crystallography [Mathiu et al. (2001). EMBO J. 20, 1485–97] shows that the fold of the protein is unchanged in the different assemblies. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. The authors propose a simple model to account for VP6 assembly in the charge of a single residue controls the assembly process. The 3-fold symmetry of the VP6 trimer is lost when only one or two monomers are charged. The switch between the assembly of spheres, large tubes and the small tubes would reflect the presence of three, two, or one neutral subunit, respectively. The system is a nice example of the influence of charge state on the formation of different contacts that leads to flexibility in macromolecular assembly. □ The hairpin structure of the 6F11F22F2 fragment from human fibronectin enhances gelatin binding. Andrew R. Pickford, Steven P. Smith, David Staunton, Jonathan Boyd, and Iain D. Campbell (2001). EMBO J. 20, 1519–1529. The solution structure of the 6F11F22F2 fragment from the gelatin binding region of fibronectin reveals an extensive hydrophobic interface between the noncontiguous 6F1 and 2F2 modules. The buried surface area between 6F1 and 2F2 (870 Å2) is the largest intermodule interface seen in fibronectin to date. The hairpin topology of 6F11F22F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. □ Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus. Ikuko Hayashi, Takuji Oyama, and Kosuke Morikawa (2001). EMBO J. 20, 1819–1828. Proper placement of the bacterial cell division site requires the site-specific inactivation of other potential division sites. In Escherichia coli, selection of the correct mid-cell site is mediated by the MinC, MinD, and MinE proteins. MinD is a membrane-associated ATPase that works as an activator of MinC. Crystal structures with a substrate analog, AMPPCP, and with the product ADP reveal general similarities to H-Ras p21. Mutagenesis suggests that the residues around the ATP binding site are required for the direct interaction with MinC, and that ATP binding and hydrolysis play a role as a Ras-like molecular switch to control the mechanisms of MinCDE-dependent bacterial cell division. □ The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR. Daan M.F. van Aalten, Concetta C. DiRusso, and Jens Knudsen (2001). EMBO J. 20, 2041–2050. FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold. The structures of the FadR-operator and FadR-myristoyl-CoA binary complexes reveal a novel winged helix-turn-helix protein-DNA interaction and that binding of acyl-CoA results in dramatic conformational changes throughout the protein. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 Å in separation of the DNA recognition helices and the loss of DNA binding, thus revealing the molecular basis of acyl-CoA-responsive regulation. □ Cryo-electron microscopic localization of protein L7/L12 within the Escherichia coli 70 S ribosome by difference mapping and Nanogold labeling. Luisa Montesano-Roditis, Dohn G. Glitz, Robert R. Traut, and Phoebe L. Stewart (2001). J. Biol. Chem. 276, 14117–14123. The landmark crystallographic results on the ribosome structure have left a number of areas of the structure undefined. Among these is the location of the protein L7 (also called L12 or L7/L12) that is known to be flexible under some conditions and is not visualized in the 2.4 Å resolution structure from Ban et al. [(2000). Science 289, 905–920]. This paper uses a combination of cEM with gold labeling and difference imaging to localize the L7. Difference imaging of ribosome cores from which L7 had been removed or added by reconstitution shows a density near the L11 shoulder. The gold labeling of the N-terminal region of L7 allows visualization of four incompletely occupied sites. One of these matches that seen in the later 5.5 Å resolution structure determined by X-ray crystallography by Yuspov et al. [(2001). Science 292, 883–96]. The other three are at interfacial regions of the ribosome. The combination of difference imaging and labeling allows the visualization of sites that are variably occupied in the structure. The authors interpretation of their finding of multiple sites for the C-terminal domain of L7 suggests that the conformation of this protein may change during the steps of elongation and translocation. □ Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui. Rishi Matadeen, Petr Sergiev, Andrej Leonov, Tillmann Pape, Eli van der Sluis, Florian Mueller, Monika Osswald, Klaus von Knoblauch, Richard Brimacombe, Alexey Bogdanov, Marin van Heel, and Olga Dontsova (2001). J. Mol. Biol. 307, 1341–1349. cEM was combined with difference imaging to localize 17 bp insertions within regions of the Escherichia coli 23 S rRNA that differ from that of Haloarcula marismortui, for which a high resolution structure has been determined. cEM and image reconstruction of 50S subunits to 25 Å resolution for two insertions revealed extra density that matched the locations expected for the insertions. Another insertion was found slightly displaced from its expected position. A fourth gave rise to no observable difference. The combination of engineered insertions and difference imaging provides a useful tool for correlating the structural features of ribosomes from extremophiles with those of E. coli, for which more extensive biochemical characterization is available. □ Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax. W. Versées, K. Decanniere, R. Pellé, J. Depoorter, E. Brosens, D.W. Parkin, and J. Steyaert (2001). J. Mol. Biol. 307, pp. 1363–1379. (doi:10.1006/jmbi.2001.4548) Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and have no equivalent in mammalian cells. The enzyme is a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). The crystal structure of the complex with the substrate analog 3-deaza-adenosine shows that T. vivax IAG-NH is a homodimer, with each subunit consisting of 10 β-strands, 12 α-helices, and three small 310-helices. Six strands of the central β-sheet form a Rossmann-like fold. Comparison with the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the substrate specificity. □ Locating the thapsigargin binding site on Ca2+-Atpase by cryoelectron microscopy. Howard S. Young, Chen Xu, Peijun Zhang, and David L. Stokes (2001). J. Mol. Biol. 308, 231–240. The binding site of thapsigargin (TG), a potent inhibitor of sarcoplasmic reticulum and endoplasmic reticulum Ca2+-ATPases, was determined by cEM and helical reconstruction. Enzymatic studies had shown that the TG-bound enzyme is locked in a dead-end complex that resembles the E2 state of this P-type ATPase. The similarity of structure of the TG inhibitted enzyme to the native enzyme support the E2-like nature of the Ca2+ ATPase in the tubular crystals. Difference maps revealed a consistent difference on the lumenal side of the membrane that the authors conclude marks the thapsigargin binding site. Modeling the atomic structure for Ca2+-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments. The authors propose indirect interactions to explain the effects on TG affinity as well as TG-induced changes in ATP affinity. Hence, TG inhibition represents a specific example of the structural coupling that generally characterizes Ca2+-ATPase and other P-type ATPases. □ Structural changes in GroEL effected by binding a denatured protein substrate. Scott Falke, Mark T. Fisher, and Edward P. Gogol (2001). J. Mol. Biol. 308, 569–577. In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in nonnative conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring. □ The fast folding pathway in human lysozyme and its blockage by appropriate mutagenesis: A sequential stopped-flow fluorescence study. Katrien Noyelle, Marcel Joniau, and Herman Van Dael (2001). J. Mol. Biol. 308, 807–819. In this work we were able to show that human lysozyme refolds along two parallel pathways: a fast path followed by 13% of the molecules that leads directly from a collapsed state to the native protein and a slow one for the remaining molecules that involves a partially unfolded intermediate state. However, in the refolding process of LYLA1, a chimera of human lysozyme which possesses the Ca2+ binding loop and helix C of bovine α-lactalbumin, the direct pathway is no longer accessible. This indicates that these structural elements, which are located in the interface region between the α- and β-domain of the protein, and their interaction with the environment play an important role in the fast folding of the molecules. These results also shed some light on the conservation of folding patterns among structurally homologous proteins. In recent years it was often stated that structurally homologous proteins with high sequence identity follow the same folding pattern. Human lysozyme and LYLA1 have a sequence identity of 87%. However, we have shown that their folding patterns are different. Therefore, a high degree of sequence identity for two proteins belonging to the same family is not a guarantee for an identical folding pattern. □ Calculation of ensembles of structures representing the unfolded state of an SH3 domain. Wing-Yiu Choy and Julie D. Forman-Kay (2001). J. Mol. Biol. 308, 1011–1032. The N-terminal SH3 domain of drk (drkN SH3 domain) exists in equilibrium between a folded (Fexch) and an unfolded (Uexch) form under nondenaturing conditions. In order to further our previous descriptions of the Uexch state, we have developed a protocol for calculating ensembles of structures, based on experimental spectroscopic data, which broadly represent the unfolded state. A large number of unfolding trajectories were generated, starting from the folded state structure of the protein, in order to provide a reasonable sampling of the conformational space accessible to this sequence. Unfolded state ensembles have been “calculated” using a newly developed program, ENSEMBLE, which optimizes the population weights assigned to each structure based on experimental properties of the Uexch state. Pseudoenergy terms for nuclear Overhauser effects, J-coupling constants, 13C chemical shifts, translational diffusion coefficients, and tryptophan ring burial based on NMR and fluorescence data have been implemented. The population weight assignment procedure was performed for different starting ensembles. Small numbers of structures (<60) dominate the final ensembles compared to the total number in the starting ensembles, suggesting that the drkN SH3 domain Uexch state can be described by a limited number of lower-energy conformations. The calculated Uexch state ensembles are much more compact than a “random coil” chain, with significant native-like residual structure observed. In particular, a sizable population of conformers having the n-src loop and distal β-hairpin structures exist in the calculated Uexch state ensembles, and Trp36 is involved in a large number of interactions, both native and nonnative. □ Interaction of coxsackievirus A21 with its cellular receptor, ICAM-1. Chuan Xiao, Carol M. Bator, Valorie D. Bowman, Elizabeth Rieder, Yongning He, Benoı̂t Hébert, Jordi Bella, Timothy S. Baker, Eckard Wimmer, Richard J. Kuhn, and Michael G. Rossmann (2001). J. Virol. 75, 2444–2451. Coxsackievirus A21 (CAV21) uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of human rhinoviruses (HRV). Cryo-electron microscopy (cEM) and image reconstruction of CAV21 at 25 Å resolution reveals a structure that is consistent with the highly homologous of poliovirus 1. CAV21 shares a canyon-like depression around each of the 12 5-fold vertices with the other enteroviruses and HRVs. A cEM reconstruction of CAV21 complexed with ICAM-1 reveals all five domains of the extracellular component of ICAM-1 for the first time; allowing the greater visibility of the molecule in the complex could reflect the use of a greater number of particles and FEG illumination for the cEM reconstruction. The improved quality of the map allows the known structures of the amino-terminal domains D1 and D2 to be fitted into the density of the complex. The position of the ICAM-1 binding site within the canyon of CAV21 overlaps the site of receptor recognition utilized by other enteroviruses. Originally, this use of this site was proposed as a way of avoiding immune surveillance. Recent work suggests that it overlaps the antibody recognition site. This suggests that receptor interactions within this common region may trigger viral destabilization after attachment. The attachment of receptors to similar sites in CAV21, HRV and poliovirus may represent a similar sequential step in a similar process. □ Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Ilaria Ferlenghi, Mairi Clarke, Twan Rutten, Stephen C. Harrison, Felix A. Rey, and Stephen D. Fuller (2001). Mol. Cell 7, 593–602. The tick-borne encephalitis flavivirus contains two transmembrane proteins: E, a type II fusion protein, and M, the companion protein that must be cleaved to acti-vate E for fusion. Coexpression of these proteins leads to secretion of recombinant subviral particles (RSPs). In the most common form of these RSPs, analyzed at a 19 Å resolution by cEM and icosahedral image reconstruction, 60 copies of E pack as dimers in a T = 1 icosahedral surface lattice. Fitting the high-resolution structure of a soluble E fragment [Rey et al (1995). Nature 375, 291–298) into the RSP density defines interaction sites between E dimers, positions M relative to E, and allows assignment of transmembrane regions of E and M. Lateral interactions among the glycoproteins stabilize this capsidless particle. The structure suggests a picture for trimer association under fusion-inducing conditions. The position of M on the three-fold axis and hence of its precursor would inhibit E trimerization. This would explain the requirement for M precursor cleavage in fusion activation. □ Three-dimensional structure of the anaphase-promoting complex. Christian Gieffers, Prakash Dube, J. Robin Harris, Holger Stark, and Jan-Michael Peters (2001). Mol. Cell 7, 907–913. The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits and a total molecular mass near 850 kDa. cEM and three-dimensional reconstruction by angular reconstitution from 13,000 molecules was used to the determine the structure of human APC to 24 Å resolution. The APC is a complex structure of 140 Å × 140 Å × 135 Å which displays no symmetry. The complex forms an outer protein wall surrounding a large central cavity of 100 Å diameter and 80 Å depth. The authors suggest that this cavity represents a reaction chamber in which the ubiquitination reactions occur that control progression through mitosis and G1. The authors also suggest that this organization may be common to other protein machines such as the 26S proteasome and chaperone complexes. □ Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis. Peter B. Rupert and Adrian R. Ferré-D'amaré (2001). Nature 410, 780–786. The hairpin ribozyme catalyses sequence-specific cleavage of RNA. This paper describes the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions. The active site results from the docking of two irregular helices: stems A and B. One strand of stem A harbors the scissile bond.The ribozyme aligns the 2′-OH nucleophile and the 5′-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. □ Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Maria A. Schumacher, Andre F. Rivard, Hans Peter Bächinger, and John P. Adelman (2001). Nature 410, 1120–1124. Small-conductance Ca2+-activated K+ channels (SK channels) are heteromeric membrane channels comprising pore-forming α-subunits and the Ca2+ binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM binding domain (CaMBD), which is located in an intracellular region of the α-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. The crystal structure of the SK channel CaMBD/Ca2+/CaM complex reveals an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three α-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. A gating mechanism is proposed. □ Translocation pathway of protein substrates in ClpAP protease. Takashi Ishikawa, Fabienne Beuron, Martin Kessel, Sue Wickner, Michael R. Maurizi, and Alasdair C. Steven (2001). Proc. Natl. Acad. Sci. USA 98, 4328–4333. The ClpAP protease is an ATP-dependent protease involved in intracellular protein degredation. It is a protein machine that combines a chaperone-like ATPase that recognizes and unfolds protein substrates with the proteinase component to which they are delivered for digestion. The complex comprises hexameric rings of the ClpA ATPase that stack axially on either face of the ClpP proteinase consisting of two apposed heptameric rings. The authors have used cEM to characterize the interactions of ClpAP with a model protein substrate, RepA. Difference imaging between averages of micrographs reveals the position of the substrate in projection and allows the authors to trace the path of the substrate from its binding at near axial sites near one end of the substrate through its translation by ∼150 Å into the digestion chamber inside ClpP. Translocation appears to proceed without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. The density may represent intermediate points on the translocation pathway. This work shows how the use of cEM in projection can be used to characterize a dynamic process. □ Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution. Steven L. Kazmirski, Kam-Bo Wong, Stefan M.V. Freund, Yee-Joo Tan, Alan R. Fersht, and Valerie Daggett (2001). Proc. Natl. Acad. Sci. USA 98, 4349–4354. Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution. □ Tryptophan zippers: Stable, monomeric β-hairpins. Andrea G. Cochran, Nicholas J. Skelton, and Melissa A. Starovasnik (2001). Proc. Natl. Acad. Sci. USA 98, 5578–5583. A structural motif, the tryptophan zipper (trpzip), greatly stabilizes the β-hairpin conformation in short peptides. Peptides (12 or 16 aa in length) with four different turn sequences are monomeric and fold cooperatively in water, as has been observed previously for some hairpin peptides. However, the folding free energies of the trpzips exceed substantially those of all previously reported β-hairpins and even those of some larger designed proteins. NMR structures of three of the trpzip peptides reveal exceptionally well-defined β-hairpin conformations stabilized by cross-strand pairs of indole rings. The trpzips are the smallest peptides to adopt an unique tertiary fold without requiring metal binding, unusual amino acids, or disulfide crosslinks. □ Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues. F. Rousseau, J.W.H. Schymkowitz, H.R. Wilkinson, and L.S. Itzhaki (2001). Proc. Natl. Acad. Sci. USA 98, 5596–5601. p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer-dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein. □ Hepatitis C virus IRES RNA-induced changes in the conformation of the 40S ribosomal subunit. Christian M. Spahn, Jeffery S. Kieft, Robert A. Grassucci, Pawel A. Penczek, Kaihong Zhou, Jennifer A. Doudna, and Joachim Frank (2001). Science 291, 1959–1962. This paper presents a structural characterization of an IRES (internal ribosome entry site) with the 40S ribosome by cEM and three-dimensional reconstruction. The hepatitis C virus IRES was used for the study although other viral and cellular IRES's should interact with the 40S similarly. Difference imaging between two new ∼20 Å resolution maps defines the IRES density and the IRES/40S complex. The 40S structure combined 18,801 particles to achieve and improved 21.9 Å resolution. The IRES/40S complex combined 20,939 particles to define the structure to 19.8 Å. The authors used comparison with a reconstruction of a complex of 40S and a truncated IRES to define the IRES orientation in the complex. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center. □ Virus maturation involving large subunit rotations and local refolding. James F. Conway, W.R. Wikoff, N. Cheng, R.L. Duda, R.W. Hendrix, J.E. Johnson, and A.C. Steven (2001). Science 292, 744–748. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. This paper combines cEM with simulation to model the conformational changes which occur in the maturation of the precursor, Prohead-II, to produce the Head II structure. Rigid-body rotations of ∼40 degrees result in a switch of the interactions of the precursor. This motion combined with the refolding of two motifs stabilize the capsid by increasing the surface area buried at interfaces. The change also brings the cross-linking residues close together. The authors suggest that DNA packaging may trigger the rearrangement through electrostatic interaction. □ Crystal structure of Streptococcus mutans pyrophosphatase: A new fold for an old mechanism. Michael C. Merckel, Igor P. Fabrichniy, Anu Salminen, Nisse Kalkkinen, Alexander A. Baykový, Reijo Lahti, and Adrian Goldman (2001). Structure 9, 289–297. Streptococcus mutans pyrophosphatase (Sm-PPase) is a member of a widely dispersed sequence family (family II) of inorganic pyrophosphatases. The first family II PPase structure, described here, reveals an N-terminal α/β domain and a C-terminal mixed β sheet domain that bears no resemblance to family I PPase, even though the arrangement of active site ligands and the residues that bind them shows significant similarity. This is a remarkable example of convergent evolution. The large change in C-terminal conformation suggests that domain closure might be the mechanism by which Sm-PPase achieves specificity for pyrophosphate over other polyphosphates.