The two proteins of the erythropoietin receptor are structurally similar

The structure of the erythropoietin receptor has been identified in this laboratory as two proteins of 100 and 85 kDa by cross-linking 125I-erythropoietin (125I-EP) to the surface of erythroid cells purified from the spleens of mice infected with the anemia strain of Friend virus. This study investigates the relatedness of these two proteins and the possibility that these proteins are subunits of the functional receptor for EP. Other workers have claimed that the 100- and 85-kDa proteins are bridged by disulfide bonds. This most likely is an artifact due to the insolubility of the cross-linked membrane. Proteolytic digestion by the method of Cleveland (Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106) resulted in identical fragments from the 100- and 85-kDa proteins, which strongly suggests that the primary amino acid sequence of these two proteins is similar if not identical. Increasing the number of protease inhibitors during the preparation of membranes and the binding and cross-linking steps increased the ratio of 100-kDa protein labeled compared to the 85-kDa protein. Together these results suggest that the 85-kDa protein is derived by proteolytic cleavage of the 100-kDa receptor for EP. It is not clear whether the 100-kDa protein can bind EP in the absence of the 85-kDa protein.     

Cis-trans isomerism of the peptide bond

The molecular orbital method PCILO is applied to eight. N-monsubstituted amides. Experimentally known geometric properties are reasonably predicted by minimization of total energy with respect to molecular geometry. The same procedure shows that molecular deformations during rotation around the peptide bond significantly lower calculated barriers. Experimental heats of activation and the free-energy changes associated with cis–trans isomerism are in good agreement with those calculated, which include qualitative estimates of configurational entropy contributions to the isomerism energies. Both the calculations and revised infrared data indicate that N-phenylurethane, which has been used as a model for the cis peptide bond, should be predominantly trans. However the variations in rotational barriers and cis–trans isomerism energies among the N-monosubstituted amides provide no reason to suppose that the cis peptide bond should be excluded from stable protein conformations.     

Gaps in structurally similar proteins: towards improvement of multiple sequence alignment

An algorithm was developed to locally optimize gaps from the FSSP database. Over 2 million gaps were identified from all versus all FSSP structure comparisons, and datasets of non-identical gaps and flanking regions comprising between 90,000 and 135,000 sequence fragments were extracted for statistical analysis. Relative to background frequencies, gaps were enriched in residue types with small side chains and high turn propensity (D, G, N, P, S), and were depleted in residue types with hydrophobic side chains (C, F, I, L, V, W, Y). In contrast, regions flanking a gap exhibited opposite trends in amino acid frequencies, i.e., enrichment in hydrophobic residues and a high degree of secondary structure. Log-odds scores of residue type as a function of position in or around a gap were derived from the statistics. Three simple experiments demonstrated that these scores contained significant predictive information. First, regions where gaps were observed in single sequences taken from HOMSTRAD structure-based multiple sequence alignments generally scored higher than regions where gaps were not observed. Second, given the correct pairwise-aligned cores, the actual positions of gaps could be reproduced from sequence more accurately using the structurally-derived statistics than by using random pairwise alignments. Finally, revision of the Clustal-W residue-specific gap opening parameters with this new information improved the agreement of Clustal-W alignments with the structure-based alignments. At least three applications for these results are envisioned: improvement of gap penalties in pairwise (or multiple) sequence alignment, prediction of regions of single sequences likely (or unlikely) to contain indels, and more accurate placement of gaps in automated pairwise structure alignment.     

Multiresolution analysis uncovers hidden conservation of properties in structurally and functionally similar proteins

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests.     

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.     

Folding mechanism of three structurally similar β-sheet proteins

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly β-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, ΔG) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences. Proteins 33:107–118, 1998. © 1998 Wiley-Liss, Inc.     

Protein homologous cores and loops: important clues to evolutionary relationships between structurally similar proteins

Background  

To discover remote evolutionary relationships and functional similarities between proteins, biologists rely on comparative sequence analysis, and when structures are available, on structural alignments and various measures of structural similarity. The measures/scores that have most commonly been used for this purpose include: alignment length, percent sequence identity, superposition RMSD and their different combinations. More recently, we have introduced the "Homologous core structure overlap score" (HCS) and the "Loop Hausdorff Measure" (LHM). Along with these we also consider the "gapped structural alignment score" (GSAS), which was introduced earlier by other researchers.     

Persistently conserved positions in structurally similar, sequence dissimilar proteins: Roles in preserving protein fold and function

Many protein pairs that share the same fold do not have any detectable sequence similarity, providing a valuable source of information for studying sequence-structure relationship. In this study, we use a stringent data set of structurally similar, sequence-dissimilar protein pairs to characterize residues that may play a role in the determination of protein structure and/or function. For each protein in the database, we identify amino-acid positions that show residue conservation within both close and distant family members. These positions are termed "persistently conserved". We then proceed to determine the "mutually" persistently conserved (MPC) positions: those structurally aligned positions in a protein pair that are persistently conserved in both pair mates. Because of their intra- and interfamily conservation, these positions are good candidates for determining protein fold and function. We find that 45% of the persistently conserved positions are mutually conserved. A significant fraction of them are located in critical positions for secondary structure determination, they are mostly buried, and many of them form spatial clusters within their protein structures. A substitution matrix based on the subset of MPC positions shows two distinct characteristics: (i) it is different from other available matrices, even those that are derived from structural alignments; (ii) its relative entropy is high, emphasizing the special residue restrictions imposed on these positions. Such a substitution matrix should be valuable for protein design experiments.     

Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations

Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the ‘CN network’ hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the ‘CN network’ hypothesis has been revisited and the present study suggested that ‘CN network’ should be accounted in terms of ‘structural contacts’ and ‘structural strengths’ in order to precisely describe order of structural stabilities of TFTs.     

Olfactory discrimination of structurally similar alcohols by cockroaches

The capability of the cockroach Periplaneta americana to discriminate odors of structurally similar aliphatic alcohols was studied by using an operant conditioning paradigm. Cockroaches were trained to discriminate three odors: one odor associated with sucrose solution (reward) and two odors associated with NaCl solution (non-reward). After training, their odor preferences were tested by counting the number of visits to each odor source. We tested the capability of cockroaches to discriminate (1) three normal aliphatic alcohols with different numbers of carbon (1-pentanol, 1-hexanol and 1-octanol), (2) three C6 aliphatic alcohols (1-hexanol, 2-hexanol and trans-2-hexen-1-ol), (3) binary mixtures of two of these three alcohols and their components, and (4) 1-hexanol solution of three different concentrations (1, 10 and 100 micro g micro l(-1)). Cockroaches exhibited higher preferences for the odors associated with reward in these tests, and we therefore conclude that cockroaches can discriminate these odors. However, discrimination of 1-hexanol and trans-2-hexen-1-ol and their binary mixture was imperfect, in that some statistical tests suggested significant level of discrimination but other tests did not. In addition, the cockroaches learned to associate a 1-hexanol solution of the highest or lowest concentration with sucrose reward but failed to learn to associate 1-hexanol of an intermediate concentration with reward.     

Comparison of conformational characteristics in structurally similar protein pairs.

Although it is known that three-dimensional structure is well conserved during the evolutionary development of proteins, there have been few studies that consider other parameters apart from divergence of the main-chain coordinates. In this study, we align the structures of 90 pairs of homologous proteins having sequence identities ranging from 5 to 100%. Their structures are compared as a function of sequence identity, including not only consideration of C alpha coordinates but also accessibility, Ooi numbers, secondary structure, and side-chain angles. We discuss how these properties change as the sequences become less similar. This will be of practical use in homology modeling, especially for modeling very distantly related or analogous proteins. We also consider how the average size and number of insertions and deletions vary as sequences diverge. This study presents further quantitative evidence that structure is remarkably well conserved in detail, as well as at the topological level, even when the sequences do not show similarity that is significant statistically.     

Synapsin I is structurally similar to ATP-utilizing enzymes.

Synapsins are abundant synaptic vesicle proteins with an essential regulatory function in the nerve terminal. We determined the crystal structure of a fragment (synC) consisting of residues 110-420 of bovine synapsin I; synC coincides with the large middle domain (C-domain), the most conserved domain of synapsins. SynC molecules are folded into compact domains and form closely associated dimers. SynC monomers are strikingly similar in structure to a family of ATP-utilizing enzymes, which includes glutathione synthetase and D-alanine:D-alanine ligase. SynC binds ATP in a Ca2+-dependent manner. The crystal structure of synC in complex with ATPgammaS and Ca2+ explains the preference of synC for Ca2+ over Mg2+. Our results suggest that synapsins may also be ATP-utilizing enzymes.     

Detection in the structure of influenza viral proteins of sequences similar to vasoactive intestinal peptide

The paper presents the results on the influenza virus proteins (only HA, M and NS) contained in the amino acid sequence regions similar to that of the VIP. These data may be important as there is similarity in pathological reactions between VIP and influenza virus.     

Different thermodynamic binding mechanisms and peptide fine specificities associated with a panel of structurally similar high-affinity T cell receptors

To understand the mechanisms that govern T cell receptor (TCR)-peptide MHC (pMHC) binding and the role that different regions of the TCR play in affinity and antigen specificity, we have studied the TCR from T cell clone 2C. High-affinity mutants of the 2C TCR that bind QL9-L(d) as a strong agonist were generated previously by site-directed mutagenesis of complementarity determining regions (CDRs) 1beta, 2alpha, 3alpha, or 3beta. We performed isothermal titration calorimetry to assess whether they use similar thermodynamic mechanisms to achieve high affinity for QL9-L(d). Four of the five TCRs examined bound to QL9-L(d) in an enthalpically driven, entropically unfavorable manner. In contrast, the high-affinity CDR1beta mutant resembled the wild-type 2C TCR interaction, with favorable entropy. To assess fine specificity, we measured the binding and kinetics of these mutants for both QL9-L(d) and a single amino acid peptide variant of QL9, called QL9-Y5-L(d). While 2C and most of the mutants had equal or higher affinity for the Y5 variant than for QL9, mutant CDR1beta exhibited 8-fold lower affinity for Y5 compared to QL9. To examine possible structural correlates of the thermodynamic and fine specificity signatures of the TCRs, the structure of unliganded QL9-L(d) was solved and compared to structures of the 2C TCR/QL9-L(d) complex and three high-affinity TCR/QL9-L(d) complexes. Our findings show that the QL9-L(d) complex does not undergo major conformational changes upon binding. Thus, subtle changes in individual CDRs account for the diverse thermodynamic and kinetic binding mechanisms and for the different peptide fine specificities.     

Dog and human acid beta-D-galactosidases are structurally similar.

The purification of dog liver acid beta-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid beta-galactosidase cross-reacted with beta-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid beta-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid beta-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid beta-galactosidase deficiency) is an excellent model for the same disease in man.     

A novel approach to fold recognition using sequence-derived properties from sets of structurally similar local fragments of proteins

Comparative modeling methods can consistently produce reliable structural models for protein sequences with more than 25% sequence identity to proteins with known structure. However, there is a good chance that also sequences with lower sequence identity have their structural components represented in structural databases. To this end, we present a novel fragment-based method using sets of structurally similar local fragments of proteins. The approach differs from other fragment-based methods that use only single backbone fragments. Instead, we use a library of groups containing sets of sequence fragments with geometrically similar local structures and extract sequence related properties to assign these specific geometrical conformations to target sequences. We test the ability of the approach to recognize correct SCOP folds for 273 sequences from the 49 most popular folds. 49% of these sequences have the correct fold as their top prediction, while 82% have the correct fold in one of the top five predictions. Moreover, the approach shows no performance reduction on a subset of sequence targets with less than 10% sequence identity to any protein used to build the library.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.