Showing papers on "Structural biology published in 1999"
TL;DR: It is shown that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity.
Abstract: Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular v-SNARE and a target membrane z-SNARE is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase Rab5. Here we show that Rab5-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated proteins seem to act upstream of EEA1, implying that Rab5 effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that EEA1 mediates endosome docking and, together with SNAREs, leads to membrane fusion.
836 citations
TL;DR: In vitro experiments suggest that Hsp90 contains two different binding sites for non-native proteins, which allow it to combine the properties of a promiscuous chaperone with those of a dedicated folding-helper protein.
664 citations
TL;DR: The human immunodeficiency virus (HIV) genome encodes a total of three structural proteins, two envelope proteins, three enzymes, and six accessory proteins as mentioned in this paper, which have been provided high-resolution three-dimensional structural information over the past ten years.
431 citations
TL;DR: The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods.
Abstract: A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.
213 citations
TL;DR: It is shown that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI +] elements, the first mutation of its type among these newly described elements of inheritance.
Abstract: The yeast [PSI+] element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases. The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be. Sup35, the [PSI+] protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP. Repeat expansions in PrP produce spontaneous prion diseases. Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI+] elements, the first mutation of its type among these newly described elements of inheritance. In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides. Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.
202 citations
111 citations
TL;DR: The structure of signal peptidase suggests how it releases functional proteins and how the GTP affinities of the signal recognition particle and its receptor are coupled to the targeting of ribosomes to translocational membrane pores.
109 citations
TL;DR: This work presents new insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells through detailed analysis of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), Hla-E, HFE, ZAG and MIC-A.
108 citations
104 citations
TL;DR: The host defense functions of the C-reactive protein using transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice and a complement-independent host protective effect was also demonstrated.
Abstract: Human C-reactive protein is a Ca2+-binding acute phase-protein with binding specificity for phosphocholine. Recent crystallographic and mutagenesis studies have provided a solid understanding of the structural biology of the protein, while experiments using transgenic mice have confirmed its host-defense function. The protein consists of five identical protomers in cyclic symmetry. On one face of each protomer there is a binding site for phosphocholine consisting of two Ca2+ ions that ligate the phosphate group and a hydrophobic pocket that accommodates the methyl groups of phosphocholine. On the opposite face is a deep cleft formed by parts of the N and C termini and bordered by an alpha-helix. Mutational studies indicate that the C1q-binding site of the molecule is located at the open end of this cleft with Asp112 and Tyr175 representing contact residues. Using human C-reactive protein transgenic mice, we investigated the host defense functions of the protein. Transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice. This was attributable to an up to 400-fold reduction in bacteremia mediated mainly by the interaction of C-reactive protein with complement. A complement-independent host protective effect was also demonstrated.
102 citations
TL;DR: The method selects only on the basis of structure and stability will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.
Abstract: The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive the folding and stab...
TL;DR: Analysis of membrane proteins as “inside‐out” proteins is an unfounded notion, suggesting that packing of α‐helices in membranes is better understood by maximization of van der Waal's forces, rather than by a general segregation of hydrophobicities driven by lipid exclusion.
Abstract: One of the central paradigms of structural biology is that membrane proteins are "inside-out" proteins, in that they have a core of polar residues surrounded by apolar residues This is the reverse of the characteristics found in water-soluble proteins We have decided to test this paradigm, now that sufficient numbers of transmembrane alpha-helical structures are accessible to statistical analysis We have analyzed the correlation between accessibility and hydrophobicity of both individual residues and complete helices Our analyses reveal that hydrophobicity of residues in a transmembrane helical bundle does not correlate with any preferred location and that the hydrophilic vector of a helix is a poor indicator of the solvent exposed face of a helix Neither polar nor hydrophobic residues show any bias for the exterior or the interior of a transmembrane domain As a control, analysis of water-soluble helical bundles performed in a similar manner has yielded clear correlations between hydrophobicity and accessibility We therefore conclude that, based on the data set used, membrane proteins as "inside-out" proteins is an unfounded notion, suggesting that packing of alpha-helices in membranes is better understood by maximization of van der Waal's forces, rather than by a general segregation of hydrophobicities driven by lipid exclusion
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TL;DR: The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats, which account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.
TL;DR: It is shown that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA.
Abstract: The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.
TL;DR: The structural and biochemical basis of antigen recognition by the T‐cell receptor (TCR)‐CD3 signaling complex has been illuminated greatly over the past few years, and functional experiments are bridging the gap and providing preliminary working models of the multimeric assemblies.
Abstract: The structural and biochemical basis of antigen recognition by the T-cell receptor (TCR)-CD3 signaling complex has been illuminated greatly over the past few years. Structural biology has contributed enormously to this understanding through the determination of crystal structures of many of the individual components of this complex, and some of the complexes. A number of general principles can be derived for the structure of the alpha beta TCR and its interaction with peptide-major histocompatibility complex (pMHC) in class I systems, as well as interaction of the CD8 co-receptor with MHC. Large buried surface areas within the protein-protein interfaces, and varying degrees of shape complementarity appear critical for modulating the stability of the multicomponent, low-affinity macromolecular complexes consisting of TCR, pMHC, CD8 or CD4, and CD3 gamma, delta, epsilon and zeta. Significant structural alterations in TCR and pMHC, upon complex formation, hint at an as yet unclear role for conformational change in both recognition and activation. Subtle chemical alterations in key peptide residues which contact the TCR can have dramatic agonist or antagonist effects on receptor activation, which correlate only loosely with the TCR/pMHC complex affinity, implying an ability of the signaling complex to "sense" fine differences in the interface. The stoichiometry of an activated TCR signaling complex is still an unresolved issue, as is the structure and disposition of the CD3 components. However, functional experiments are bridging this gap and providing us with preliminary working models of the multimeric assemblies.
TL;DR: It is shown that the apical domain binds aggregation-sensitive polypeptides but cannot significantly assist their refolding in vitro and fails to replace the groEL gene or to complement defects of groEL mutants in vivo, suggesting sequestration of aggregation-prone intermediates in a folding cage is an important element of the chaperonin mechanism.
TL;DR: Hydrogen exchange measured by electrospray (ES) mass spectrometry (MS) is used to probe the solution dynamics of amyloidogenic proteins.
Abstract: Publisher Summary Evidence has suggested that the formation of amyloid fibrils may involve the structural rearrangement of native monomeric protein through partially folded intermediates. For example, the kinetics of amyloid fibril formation in vitro is most favorable for wild-type transthyretin under solution conditions that destabilize the native state tetramer of the protein. Furthermore, naturally occurring variants of human lysozyme, transthyretin, and immunoglobulin light chain VL domain have been shown to destabilize the native states of these proteins. Destabilization allows these proteins to unfold more readily, generating partially folded states that are proposed as amyloidogenic intermediates. These aggregation-prone intermediates are difficult to study by classical structural biology techniques as they are too dynamic for X-ray crystallography and are not amenable to nuclear magnetic resonance (NMR) measurements directly because the concentration necessary for their study promotes aggregation. In this article, hydrogen exchange measured by electrospray (ES) mass spectrometry (MS) is used to probe the solution dynamics of amyloidogenic proteins.
TL;DR: The proposed system allows to examine structural changes and molecular complex of proteins at work, which will become a great advantage for the next generation in structural biology after static structures of proteins are all determined.
Abstract: Single particle analysis is a straightforward method for structural studies of protein and biological macromolecules developed in image analysis on electron microscopy [1]. It is a predominant structural probe for proteins which decline to crystallize and exceed the limit in molecular weight for NMR analysis. Furthermore, it allows to examine structural changes and molecular complex of proteins at work, which will become a great advantage for the next generation in structural biology after static structures of proteins are all determined. Our system, named BESPA (Backplane for Electron microscopic Single Particle Analysis) will be packaged soon featuring new algorithms and techniques together with practically useful programs. The single particle analysis has also raised topics for a computational science both in practical algorithm and in establishment of theoretical background.
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TL;DR: A model is proposed based on the hypothesis that a thermodynamically irreversible step is involved in protein folding and it is shown that, under certain conditions, the transitory secondary structure formed during the earlier step of folding could interact with chaperone.
Abstract: It is generally accepted that prion infection is due solely to a protein i.e. the protein-only hypothesis. The essential constituent of infectious prions is the scrapie prion protein (PrPSc) which is chemically indistinguishable from the normal, cellular protein (PrPC) but exhibits distinct secondary and tertiary structure. This very unusual feature seems to be in contradiction with a major paradigm of present structural biology stated by Anfinsen: a protein folds to the most stable conformation, this means only one structure. In order to reconcile the results obtained on prions with the biophysics of protein folding, a model is proposed. It is based on the hypothesis that a thermodynamically irreversible step is involved in protein folding. The model is then extended to chaperone-assisted protein folding. It is shown that, under certain conditions, the transitory secondary structure formed during the earlier step of folding could interact with chaperone. Analysis shows that chaperone may help the protein to find correct conformation. On the other hand, analysis reveals the possibility that more than one structure may form from a single polypeptide chain. Under these conditions, the behaviour of chaperones resembles the characteristics of prion diseases.
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