Portal de Periódicos da CAPES

Paper Alert

2001; Elsevier BV; Volume: 9; Issue: 5 Linguagem: Inglês

10.1016/s0969-2126(01)00605-0

1878-4186

Chosen by Robert Liddington, Christin Frederick, Stephen D. Fuller, Sophie Jackson,

Respiratory viral infections research

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. □ Hierarchical folding of intestinal fatty acid binding protein. Syun-Ru Yeh, Ira J. Ropson, and Denis L. Rousseau (2001). Biochemistry 40, 4205–4210. Intestinal fatty acid binding protein (IFABP) is a member of the lipid binding protein family, members of which have a clam shell type of motif formed by two five-stranded β-sheets. Understanding the folding mechanism of these proteins has been hindered by the presence of an unresolved burst phase. By initiating the reaction with a sub-millisecond mixer and following its progression by Trp fluorescence, we discovered three distinct phases in the folding reaction of the W6Y mutant of IFABP from which we postulate the following sequence of events. The first phase (k1 > 10 000 s−1) involves collapse of the polypeptide chain around a hydrophobic core. During the second phase (k2 ∼ 1500 s−1), β-strands B-G, mostly located on the top half of the clam shell structure, propagate from this hydrophobic core. It is followed by the final phase (k3 ∼ 5 s−1) involving the formation of the last three β-strands on the bottom half of the clam shell and the establishment of the native hydrogen bonding network throughout the protein molecule. □ Structure of the reovirus outer capsid and dsRNA-binding protein σ3 at 1.8 Å resolution. Andrea M. Olland, Judit Jané-Valbuena, Leslie A. Schiff, Max L. Nibert and Stephen C. Harrison (2001). EMBO J. 20, 979–989. The crystal structure of the reovirus outer capsid protein σ3 reveals a tightly associated dimer, each subunit having a two-lobed structure organized around a long central helix. A fit of this model to a reconstruction of the whole virion from electron cryomicroscopy suggests that the small lobe of each σ3 subunit anchors it to the protein μ1 on the surface of the virion, while the large lobe protrudes outwards. A positively charged surface patch is implicated in binding dsRNA, an activity that inhibits the interferon response in infected cells. □ Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus. Philip M. Leonard, Sander H.J. Smits, Svetlana E. Sedelnikova, Arie B. Brinkman, Willem M. de Vos, John van der Oost, David W. Rice, and John B. Rafferty (2001). EMBO J. 20, 990–997. The LrpA protein from Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins. Its crystal structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed α/β character reminiscent of a number of RNA- and DNA-binding domains. A homodimer is formed mainly through interactions between the β-sheets of the C-terminal domain, and further interactions lead to octamer formation. The structure suggests how it binds and distorts its DNA substrate through the use of its HtH motifs. □ Structure of a multifunctional protein: mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine. Marilyn D. Yoder, Leonard M. Thomas, Jacqueline M. Tremblay, Randall L. Oliver, Lynwood R. Yarbrough, and George M. Helmkamp, Jr. (2001). J. Biol. Chem. 276, 9246–9252. Phosphatidylinositol transfer protein transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The crystal structure reveals a single β-sheet and several long α-helices defining an enclosed internal cavity in which one molecule of phospholipid is bound and suggests mechanisms for lipid exchange and binding to lipid and protein kinases. □ Crystal structure of lyme disease antigen outer surface protein C from Borrelia burgdorferi. Christoph Eicken, Vivek Sharma, Thomas Klabunde, Rick T. Owens, Dagmar S. Pikas, Magnus Höök, and James C. Sacchettini (2001). J. Biol. Chem. 276, 10010–10015. The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. The crystal structure reveals a largely α-helical protein: a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Similar results are described by Kumaran et al., EMBO J. 20, 971–978. □ Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. Betsy L. Lytle, Brian F. Volkman, William M. Westler, Matthew P. Heckman, and J. H. David Wu1 (2001). J. Mol. Biol. 307, 745–753. The type I dockerin domain is responsible for incorporating its associated glycosyl hydrolase into the bacterial cellulosome. It is a 69 residue domain with two Ca2+-binding sites with sequence similarity to the EF-hand motif. The solution structure of consists of two Ca2+-binding loop-helix motifs connected by a linker; in its non-complexed state, the dockerin fold displays a dramatic departure from that of Ca2+-bound EF-hand domains. □ Comparison of the structural and dynamical properties of Holo and Apo bovine α-lactalbumin by NMR spectroscopy. Ramani Wijesinha-Bettoni, Christopher M. Dobson, and Christina Redfield (2001). J. Mol. Biol. 307, 885–898. In the presence of 0.5 M NaCl at pH 7.1, the Ca2+-free apo form of recombinant bovine α-lactalbumin (BLA) is sufficiently stabilized in its native state to give well-resolved NMR spectra at 20°C. The 1H and 15N NMR resonances of native apo-BLA have been assigned, and the chemical-shifts compared with those of the native holo protein. Large changes observed between the two forms of BLA are mainly limited to the Ca2+-binding region of the protein. These data suggest that Na+ stabilizes the native apo state through the screening of repulsive negative charges, at the Ca2+-binding site or elsewhere, rather than by a specific interaction at the vacant Ca2+-binding site. The hydrogen exchange protection of residues in the Ca2+-binding loop and the C-helix is reduced in the apo form compared to that in the holo form. This indicates that the dynamic behavior of this region of the protein is substantially increased in the absence of the bound Ca2+. Real-time NMR experiments show that the rearrangements of the structure associated with the conversion of the holo to apo form of the protein do not involve the detectable population of partially unfolded intermediates. Rather, the conversion appears to involve local reorganizations of the structure in the vicinity of the Ca2+-binding site that are coupled to the intrinsic fluctuations in the protein structure. □ Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states. Yu-Keung Mok, Elena L. Elisseeva, Alan R. Davidson, and Julie D. Forman-Kay (2001). J. Mol. Biol. 307, 913–928. The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging β-turn connecting the 3 and 4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native β-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability. □ SOCKET: a program for identifying and analyzing coiled-coil motifs within protein structures. John Walshaw and Derek N. Woolfson (2001). J. Mol. Biol. 307, 1427–1450. The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions. Coiled coils comprise two or more α-helices that wind around each other to form “supercoils.” The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat. Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states. To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures. We used SOCKET to gather a set of unambiguous coiled-coil structures from the RCSB Protein Data Bank. Rather than searching for sequence features, the algorithm recognizes the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains. SOCKET unambiguously defines coiled-coil helix boundaries, oligomerization states, and helix orientations, and also assigns heptad registers. Structures retrieved from the Protein Data Bank included parallel and anti-parallel variants of two, three, and four-stranded coiled coils, one example of a parallel pentamer, and a small number of structures that extend the classical description of a coiled coil. We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction, and design. To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino acid profiles for the heptad repeats of different motifs. □ A mechanism for initiating RNA-dependent RNA polymerization. Sarah J. Butcher, Jonathan M. Grimes, Eugeny V. Makeyev, Dennis H. Bamford, and David I. Stuart (2001). Nature 410, 235–240. The crystal structure of the active polymerase subunit from the double-stranded RNA bacteriophage 6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. Co-crystal structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), provide a working model for the initiation of replication and transcription. □ Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex. Guozhou Xu, Maurizio Cirilli, Yihua Huang, Rebecca L. Rich, David G. Myszka, and Hao Wu (2001). Nature 410, 494–497. The p35 protein from baculoviruses prevents apoptosis by its broad-spectrum caspase inhibition. The crystal structure of p35 in complex with human caspase-8 reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase that prevents hydrolysis of the thioester intermediate, thus defining a novel mechanism of caspase inhibition. □ Structural basis for co-stimulation by the human CTLA-4/B7-2 complex. Jean-Claude D. Schwartz, Xuewu Zhang, Alexander A. Fedorov, Stanley G. Nathenson, and Steven C. Almo. Nature 410, 604–608. T-cell activity is regulated by the homodimeric T-cell surface receptors CD28 and CTLA-4. In the crystal structure of the complex between the homodimer of CTLA-4 and the receptor-binding domain of human B7-2, the unusual dimerization properties of both molecules place their respective ligand-binding sites distal to the dimer interface in each molecule, and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal, providing a model for their periodic organization within the immunological synapse and suggesting a mechanism for signaling. Similar results are reported by Stamper et al., Nature 410, 608–611. □ Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping. Robert Janowski, Maciej Kozak, Elzbieta Jankowska, Zbigniew Grzonka, Anders Grubb, Magnus Abrahamson, and Mariusz Jaskolski (2001). Nat. Struct. Biol. 8, 316–320. The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the “open interface” of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe “conformational disease” is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage, and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains. □ Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin. Thirunavukkarasu Sivaraman, Cammon B. Arrington, and Andrew D. Robertson (2001). Nat. Struct. Biol. 8, 331–333. Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins. □ Crystal structure of threonine synthase from Arabidopsis thaliana. Karine Thomazeau, Gilles Curien, Renaud Dumas, and Valérie Biou (2001). Protein Sci. 10, 638–648. Threonine synthase (TS) is a PLP-dependent enzyme that catalyzes the last reaction in the synthesis of threonine from aspartate. In plants, TS is activated allosterically by S-adenosyl-methionine (SAM), a downstream product of methionine synthesis. The crystal structure reveals a four-domain dimer with a two-stranded β-sheet arm protruding from one monomer onto the other. This domain swap could form a lever through which the allosteric effect is transmitted. The N-terminal domain (domain 1) has a unique fold and is partially disordered, whereas the structural core (domains 2 and 3) shares the functional domain of PLP enzymes of the same family. Sites for pyridoxal-phosphate and SAM binding are proposed. □ Structures of β-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery. Johan Gotthardt Olsen, Anders Kadziola, Penny von Wettstein-Knowles, Mads Siggaard-Andersen, and Sine Larsen (2001). Structure 9, 233–243. β-ketoacyl-acyl carrier protein synthase (KAS) I is required for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. Crystal structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates are described. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A model is proposed for the catalysis of KAS I. □ The structure of the fusion glycoprotein of Newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion. Lin Chen, Jeffrey J. Gorman, Jenny McKimm-Breschkin, Lynne J. Lawrence, Peter A. Tulloch, Brian J. Smith, Peter M. Colman, and Michael C. Lawrence (2001). Structure 9, 255–266. Membrane fusion within the Paramyxoviridae family of viruses is mediated by a surface glycoprotein termed the “F,” or fusion, protein. The crystal structure of the fusion protein of Newcastle disease virus (NDV-F) reveals a trimeric protein that is organized into head, neck, and stalk regions. An axial channel extends through the head and neck and is fenestrated by three large radial channels located approximately at the head-neck interface.The structure is quite different from that of influenza virus hemagglutinin, in that the central coiled coil is in the opposite orientation with respect to the viral membrane. A model of membrane fusion is proposed.