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PubMed1992

Sequence-structure matching in globular proteins: application to supersecondary and tertiary structure determination.

A methodology designed to address the inverse globular protein-folding problem (the identification of which sequences are compatible with a given three-dimensional structure) is described. By using a library of protein finger-prints, defined by the side chain interaction pattern, it is possible to match each structure to its own sequence in an exhaustive data base search. It is shown that this is a permissive requirement for the validation of the methodology. To pass the more rigorous test of identifying proteins that are not close sequence homologs, but that have similar structure, the method has been extended to include insertions and deletions in the sequence, which is compared to the fingerprint. This allows for the identification of sequences having little or no sequence homology to the fingerprint. Examples include plastocyanin/azurin/pseudoazurin, the globin family, different families of proteases and cytochromes, including cytochromes c' and b-562, actinidin/papain, and lysozyme/alpha-lactalbumin. Turning to supersecondary structure prediction, we find that alpha/beta/alpha fragments possess sufficient specificity to identify their own and related sequences. By threading a beta-hairpin through a sequence, it is possible to predict the location of such hairpins and turns with remarkable fidelity. Thus, the method greatly extends existing techniques for the prediction of both global structural homology and local supersecondary structure.

Amino Acid Sequence↗

PubMed1992

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.

Amino Acid Sequence↗

PubMed1990

Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved alpha-1-proteinase inhibitor.

The crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin, has been determined to a resolution of 2.8 A by the isomorphous replacement method and preliminary refinement. The structure closely resembles that of the cleaved form of alpha-1-proteinase inhibitor, with some important exceptions. The disposition of the new carboxyl chain terminus liberated by proteolysis is different with respect to the central beta-sheet A in the structures of these two molecules. In alpha-1-proteinase inhibitor, the new chain terminus inserts in beta-sheet A to add a middle strand to the sheet. In plakalbumin, this strand remains free near the site at which the cleavage occurs. A structural basis for this difference in behavior is proposed from the structures and sequences of these two molecules and other members of the serpin family. The structures and positions of the putative signal peptide of ovalbumin, the several post-translational modifications, and the relationship of the intron-exon patterns of plakalbumin and alpha-1-proteinase inhibitor to their protein structures are also described.

Amino Acid Sequence↗

PubMed1989

Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam.

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.

Animals↗

PubMed1990

Internal transcribed spacer 1 of the yeast precursor ribosomal RNA. Higher order structure and common structural motifs.

The higher order structure of the first internal transcribed spacer between the 18S and the 5.8S rRNA sequences in the Saccharomyces cerevisiae precursor ribosomal RNA has been investigated. Sites of potential base pairing in the RNA region have been determined by using a combination of enzymatic and chemical structure sensitive probes. Data generated have been used to evaluate secondary structure models predicted by minimum free energy calculations. Several alternative suboptimal structures were also evaluated. The derived model contains several stable hairpins. Theoretical secondary structural models for the corresponding RNA region from S. carlsbergensis, S. pombe, N. crassa, X. laevis, and mung bean have also been derived from identical calculations and assumptions. Certain structural motifs appear to be conserved despite extensive divergence in the base sequence. The yeast model should be a useful prototype for investigation of structure and function of precursor ribosomal RNA molecules.

Alkylating Agents↗

PubMed1992

Cap structure of U3 small nucleolar RNA in animal and plant cells is different. gamma-Monomethyl phosphate cap structure in plant RNA.

U3 small nucleolar RNA (snoRNA) is an abundant small RNA involved in the processing of pre-ribosomal RNA of eukaryotic cells. U3 snoRNA has been previously characterized from several sources, including human, rat, mouse, frog, fruit fly, dinoflagellates, slime mold, and yeast; in all these organisms, U3 snoRNA contains trimethylguanosine cap structure. In all instances where investigated, the trimethylguanosine-capped snRNAs including U3 snoRNA, are synthesized by RNA polymerase II. However, in higher plants, the U3 snoRNA is synthesized by RNA polymerase III and contains a cap structure different from trimethylguanosine (Kiss, T., and Solymosy, F. (1990) Nucleic Acids Res. 18, 1941-1949; Marshallsay, C., Kiss, T., and Filipowicz, W. (1990) Nucleic Acids Res. 18, 3451-3458; Kiss, T., Marshallsay, C., and Filipowicz, W. (1991) Cell 65, 517-526). In this study, we present evidence that cowpea and, most likely, tomato plant U3 snoRNA contains a methyl-pppA cap structure. These data show that the same U3 snoRNA contains different cap structures in different species and suggest that the kind of cap structure that an uridylic acid-rich small nuclear RNA contains is dependent on the RNA polymerase responsible for its synthesis. In vitro synthesized plant U3 snoRNA, with pppA or pppG as its 5' end, was converted to methyl-pppA/G cap structure in vitro when incubated with extracts prepared from wheat germ or HeLa cells. These data show that the capping machinery is conserved in organisms as evolutionarily distant as plants and mammals. Nucleotides 1-45 of tomato U3 snoRNA, which are capable of forming a stem-loop structure, are sufficient to direct the methyl cap formation in vitro.

Animals↗

PubMed1990

[Stratum-structure of the spinal nerves in the understanding of body structure].

The main methodology employed in the progressive gross anatomy of today's Japan is typological anatomy (typologische Anatomie) as introduced by Seiho Nishi (1943; 1950; 1961). This typological methodology is sufficient to suggest the existence of a certain constancy between muscle phylogeny and innervation in spite of the classic theory of nerve-muscle specificity having been discredited. On the basis of findings obtained from the many recent Japanese studies including the author's study on the cervical plexus (Kida, 1987), it has been noted that the stratum-structure of the spinal nerves may be an important key for the phylogenetic understanding of animal body structure (Kida, 1988). 1) The stratum-structure of the spinal nerves was initially observed only in the limb plexus by Fürbringer (Schwalbe, 1981). At that time, it was accepted as the longitudinal strata corresponding to the portion occupied by nerve fibers to the flexors or the extensors (Bolk, 1902). This theory was effective for the systematic understanding of limb structure. 2) The stratum-structure of the spinal nerves was at first considered to be related to the concept of nerve-muscle specificity. After the review of Straus (1946) on the concept of nerve-muscle specificity, this concept was disproved and has since been generally regarded as a theory of the past, therefore stratum-structure is now treated independently of that concept. 3) However, recent Japanese studies have modified and progressed beyond the concept of the stratum-structure of the spinal nerves.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗

PubMed1989

Predicted structures of apolipoprotein II mRNA constrained by nuclease and dimethyl sulfate reactivity: stable secondary structures occur predominantly in local domains via intraexonic base pairing.

Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation.

Animals↗

PubMed1988

Nature and biosynthesis of structural matrix protein in melanosomes: melanosomal structural protein as differentiation antigen for neoplastic melanocytes.

Two major classes of protein, i.e., structural matrix and enzymic, exist in the melanosome. In normal melanocytes, synthesis of these two components is under a strict genetic control with regulatory steps associated with transcription and translation coded with appropriate pigment genes. In abnormal neoplastic melanocytes, they become markedly aberrant. The aberrant melanogenesis can be typically manifested by an abnormality in fine structure of the melanosome. The fine structural heterogeneity of the melanosome can often be diagnostic to certain forms of malignant melanoma and dysplastic melanocytic nevus. This study brings about the importance of the structural matrix protein for the expression of fine structural heterogeneity in the melanosome by developing the 2 monoclonal antibodies, MoAb HMSA-1 and HMSA-2. The 2 MoAbs recognized the cytoplasmic antigen on paraffin embedded sections, which under immunoelectron microscopy and cell fractionation studies, were found to be localization the melanosome and smooth ER, but not Golgi complex and coated vesicles as seen in the tyrosinase studies. It is indicated (a) that the sites of the synthesis for the melanosomal protein and tyrosinase are different, (b) that the melanosomal structural protein is accumulated in the dilated vacuole of smooth ER from which the stage I melanosome is synthesized, (c) that the synthesis of melanosomal protein becomes markedly aberrant and directly reflects the abnormal fine structure of the melanosome and (d) the heterogeneity in synthesis of melanosomal protein as expressed by MoAb HMSA-1 and HMSA-2 is a useful adjunct for immunohistopathological differentiation of malignant melanoma cells from benign nevoid cells and normal melanocytes.

Antigens, Differentiation↗

PubMedDate not supplied

[Investigation of the structure of magnesium and lithium salts of T2 phage DNA by the method of x-ray diffraction. The possible mechanisms of the participation of cations in the structural transformation of double-stranded DNA].

The secondary structure of DNA is known to be largely determined by the kind of counterion bound to it. We have used the X-ray diffraction method to study the structure of magnesium and lithium salts of T2 phage DNA in oriented fibres. The structural behaviour of this glucosylated DNA in the form of magnesium and lithium salts was shown to be identical to the behaviour of the same salts of "normal" calf thymus DNA throughout the studied range of relative humidities (44-95%). However these two DNAs in the form of sodium salt are known to behave quite differently. One can presume that Mg2+ and Li+ influence the structural behaviour of double-stranded DNA so effectively as to be able to "ignore" the fact that T2 phage DNA contains glucoside residues. The results of this work and the already known facts concerning the structure of DNA in the form of various cation salts (in solution and in "solid" fibres) indicate that the structural behaviour of double-stranded DNA is mainly determined by the cation located in the region of the narrow groove of the double helix. If cations are graded according to the efficiency of their influence on the structural behaviour of DNA in fibres, the scale will coincide with that of their DNA-binding strength in water solution, that is: Mg2+ greater than Li+ greater than Na+ greater than K+ greater than Rb+. A qualitative consideration of electrostatic interaction between the cations and the negatively charged DNA strands leads one to suppose that this interaction must obstruct the transition of individual DNA molecules from the B-form to the A-form. Aggregation of self-aggregation of DNA molecules is presumed necessary to enable them to adopt the A-conformation.

Cations, Monovalent↗

PubMedDate not supplied

The crystal structure of alpha-bungarotoxin at 2.5 A resolution: relation to solution structure and binding to acetylcholine receptor.

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.

Animals↗

PubMed2026

Dynamic Protein Structure Paradox: An Integrative Framework for Endpoint-Conditioned Evidentiary Sufficiency in Structure-to-Function Claims.

Accurate coordinates for a represented protein state do not, by themselves, establish activity or any other condition-specific function. This article defines the Dynamic Protein Structure Paradox (DPSP) as the apparent conflict between structural accuracy and functional underdetermination and develops it as an integrative evidentiary assessment framework rather than a new theory or paradigm. The underlying problem has been longstanding, since structural genomics, function annotation, allostery, and disorder research each established that fold does not determine function and that function does not determine fold. DPSP consolidates those results into one endpoint-conditioned rule. Once a measurable endpoint is defined, it assesses four coupled dimensions: relevant-state completeness, context completeness, ensemble or kinetic dependence, and chemical dependence. A rubric rates each dimension as adequate, uncertain, or missing, and a materiality test determines which gaps influence the stated decision. The outcome is one of three mutually exclusive modes of utilization: geometry-led, conditional, or function-measured. The deliverable is a concise evidence statement delineating what the structure supports, which decisive variable remains unmeasured, and what corroboration is necessary. DPSP complements, rather than replaces, existing structural, ensemble, and computational approaches. The framework remains unvalidated, its thresholds are provisional, and the studies necessary to confirm or refute it are specified.

Proteins↗

PubMed1987

Molecular dynamics simulations of cyclosporin A: the crystal structure and dynamic modelling of a structure in apolar solution based on NMR data.

The conformation of the immunosuppressive drug cyclosporin A (CPA), both in apolar solution and in crystalline state, has been studied by computer simulation techniques. Three molecular dynamics (MD) simulations have been performed: one modelling the crystal structure and two modelling the structure in apolar solution, using a restrained MD approach in which data from nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy are taken into account. The simulation of the crystalline state (MDC) concerns a system of 4 unit cells containing 16 cyclosporin A molecules and 22 water molecules, which is simulated using crystalline periodic boundary conditions. The simulations modelling the apolar solvent conformation (MDS) concern one isolated cyclosporin A molecule. In these simulations an extra term in the interatomic potential function is used, which forces the molecule to satisfy a set of 57 atom-atom distance constraints originating from nuclear Overhauser effects (NOEs) obtained from NMR spectroscopy and one distance constraint deduced from IR spectroscopy. From a comparison of the results of the crystal simulation to those of the X-ray experiment in terms of structure, atomic fluctuations, hydrogen bond pattern, etc., it is concluded that the force field that is used yields an adequate representation of crystalline cyclosporin A. Secondly, it is shown that the dynamic modelling technique that is used to obtain a structure in a polar solution from NMR distance information works well. Starting from initial conformations which have a root mean square difference of 0.14 nm both distance restrained MD simulations converge to the same final solution structure. A comparison of the crystal structure of cyclosporin A and the one in apolar solution shows that there are significant differences. The overall difference in atomic positions is 0.09 nm for the C alpha atoms and 0.17 nm for all atoms. In apolar solution, the molecule is slightly more bent and the side chains of 1 MeBmt and 10 MeLeu adopt a different conformation.

Computer Simulation↗

PubMed1992

Design, synthesis and solution structure of a renin inhibitor. Structural constraints from NOE, and homonuclear and heteronuclear coupling constants combined with distance geometry calculations.

A macrocyclic renin inhibitor was designed using molecular modeling and a model of human renin. The synthesized molecular displayed poor binding affinity. To investigate the reasons for the observed inactivity, the structure of the compound has been studied by NMR spectroscopy and distance geometry. Structural constraints for distance geometry calculations were derived from nuclear Overhauser effects and homonuclear and heteronuclear three bond coupling constants. Homonuclear coupling constants were measured directly from the resolution-enhanced proton spectra and heteronuclear coupling constants were measured from the natural abundance 15N- and 13C-edited TOCSY experiments. One phi angle was determined uniquely by this method and two were reduced to two possible values each. By using a statistical analysis of 400 structures generated with distance geometry, two families of structures were found to be consistent with the NMR data. The solution structures so derived were different from the originally designed structure, including an internal hydrogen bond. This provides a possible explanation for the lack of effectiveness of this compound.

Humans↗

PubMed1989

Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine.

The three-dimensional structure of the native unliganded form of the Leu/Ile/Val-binding protein (Mr = 36,700), an essential component of the high-affinity active transport system for the branched aliphatic amino acids in Escherichia coli, has been determined and further refined to a crystallographic R-factor of 0.17 at 2.4 A resolution. The entire structure consists of 2710 non-hydrogen atoms from the complete sequence of 344 residues and 121 ordered water molecules. Bond lengths and angle distances in the refined model have root-mean-square deviations from ideal values of 0.05 A and 0.10 A, respectively. The overall shape of the protein is a prolate ellipsoid with dimensions of 35 A x 40 A x 70 A. The protein consists of two distinct globular domains linked by three short peptide segments which, though widely separated in the sequence, are proximal in the tertiary structure and form the base of the deep cleft between the two domains. Although each domain is built from polypeptide segments located in both the amino (N) and the carboxy (C) terminal halves, both domains exhibit very similar supersecondary structures, consisting of a central beta-sheet of seven strands flanked on either side by two or three helices. The two domains are far apart from each other, leaving the cleft wide open by about 18 A. The cleft has a depth of about 15 A and a base of about 14 A x 16 A. Refining independently the structure of native Leu/Ile/Val-binding protein crystals soaked in a solution containing L-leucine at 2.8 A resolution (R-factor = 0.15), we have been able to locate and characterize an initial, major portion of the substrate-binding site of the Leu/Ile/Val-binding protein. The binding of the L-leucine substrate does not alter the native crystal structure, and the L-leucine is lodged in a crevice on the wall of the N-domain, which is in the inter-domain cleft. The L-leucine is held in place primarily by hydrogen-bonding of its alpha-ammonium and alpha-carboxylate groups with main-chain peptide units and hydroxyl side-chain groups; there are no salt-linkages. The charges on the leucine zwitterion are stabilized by hydrogen-bond dipoles. The side-chain of the L-leucine substrate lies in a depression lined with non-polar residues, including Leu77, which confers specificity to the site by stacking with the side-chain of the leucine substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Solution structures of two zinc-finger domains from SWI5 obtained using two-dimensional 1H nuclear magnetic resonance spectroscopy. A zinc-finger structure with a third strand of beta-sheet.

This paper describes the detailed three-dimensional structures of two zinc-finger domains from the yeast transcription factor SWI5, calculated using the results of the n.m.r. experiments described in the accompanying paper. The structure of finger 2 is essentially similar to those previously obtained by others for isolated, synthetic single zinc-finger domains in solution, and for the three zinc-finger peptide Zif268 in its crystalline complex with DNA. The N-terminal half of the sequence forms a two-stranded, irregular beta-sheet containing both of the metal-binding cysteine residues, while the remainder of the structure forms a helix. Approximately the first half of this helix is alpha-helical, whereas the C-terminal portion, including the two metal-binding histidine residues, is 3(10) helical. Four invariant hydrophobic residues form a core to the structure. In contrast to all previously described structures of zinc-finger domains, finger 1 has an additional strand in the beta-sheet, formed by residues N-terminal to the formal start of the finger motif. This additional strand plays a role in stabilising the folded form of finger 1, since a two-finger peptide lacking the N-terminal residues showed folded structure in finger 2 but not in finger 1.

Amino Acid Sequence↗

PubMedDate not supplied

Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects.

2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic 'loop-helix-beta-sheet' structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a 'horse saddle' topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.

Amino Acid Sequence↗

PubMed1988

Evidence from cassette mutagenesis for a structure-function motif in a protein of unknown structure.

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant Km for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the Km for adenosine triphosphate, the apparent Km for tRNA, or the rate constant kcat.

Amino Acyl-tRNA Synthetases↗