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PubMed1999

Structural roles of acetylcholinesterase variants in biology and pathology.

Apart from its catalytic function in hydrolyzing acetylcholine, acetylcholinesterase (AChE) affects cell proliferation, differentiation and responses to various insults, including stress. These responses are at least in part specific to the three C-terminal variants of AChE which are produced by alternative splicing of the single ACHE gene. 'Synaptic' AChE-S constitutes the principal multimeric enzyme in brain and muscle; soluble, monomeric 'readthrough' AChE-R appears in embryonic and tumor cells and is induced under psychological, chemical and physical stress; and glypiated dimers of erythrocytic AChE-E associate with red blood cell membranes. We postulate that the homology of AChE to the cell adhesion proteins, gliotactin, glutactin and the neurexins, which have more established functions in nervous system development, is the basis of its morphogenic functions. Competition between AChE variants and their homologs on interactions with the corresponding protein partners would inevitably modify cellular signaling. This can explain why AChE-S exerts process extension from cultured amphibian, avian and mammalian glia and neurons in a manner that is C-terminus-dependent, refractory to several active site inhibitors and, in certain cases, redundant to the function of AChE-like proteins. Structural functions of AChE variants can explain their proliferative and developmental roles in blood, bone, retinal and neuronal cells. Moreover, the association of AChE excess with amyloid plaques in the degenerating human brain and with progressive cognitive and neuromotor deficiencies observed in AChE-transgenic animal models most likely reflects the combined contributions of catalytic and structural roles.

Acetylcholinesterase↗
PubMed1997

Complete primary structure of two splice variants of collagen XII, and assignment of alpha 1(XII) collagen (COL12A1), alpha 1(IX) collagen (COL9A1), and alpha 1(XIX) collagen (COL19A1) to human chromosome 6q12-q13.

Overlapping cDNA clones that encode the full-length human alpha 1(XII) collagen polypeptides were isolated. The long variant molecule cDNA of 9750 nucleotides (nt) contains a 9189-nt open reading frame encoding 3063 amino acid residues. The short variant molecule cDNA of 6258 nt contains a 5697-nt open reading frame encoding 1899 amino acid residues. At the amino terminus of each variant is a 24-residue signal peptide that is followed by the mature polypeptides of 3039 amino acid residues with a calculated molecular mass of 330,759 Da for the long variant and 1875 amino acid residues with a calculated molecular mass of 203,163 Da for the short variant polypeptide. The human collagen XII chains are predicted to have all the structural domains described for the molecules in chicken and mouse, including, fibronectin type III repeats, von Willebrand factor A domains, and two triple-helical domains similar to those of all the other collagen family members. The amino acid residue sequence of human alpha 1(XII) collagen showed 92% identity to the mouse chain and 78% identity to the chicken chain. The sequence of three peptide fragments of collagen XII isolated from human placenta was identical to the sequence predicted from the deduced cDNA sequence and confirms that the cDNA encodes human alpha 1(XII) collagen. An isolated genomic clone was used to map the locus of the COL12A1 gene to chromosome 6q12-q13, very close to the locus of the FACIT collagen genes COL9A1 and COL19A1. RT-PCR on a variety of cDNAs demonstrates that both variant transcripts appear in human amnion, chorion, skeletal muscle, small intestine, and in cell cultures of human dermal fibroblasts, keratinocytes, and endothelial cells. Only the small variant transcript is apparent in human lung, placenta, kidney, and a squamous cell carcinoma cell line. These results confirm the previous observations showing that collagen XII is found in collagen I-containing tissues.

Alternative Splicing↗
PubMed2005

Structural characterization of the histone variant macroH2A.

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.

Acetylation↗
PubMed1992

Mass spectrometry in the integrated strategy for the structural analysis of protein variants.

A significant proportion of genetic disorders are caused by point mutations in proteins, and many variants of haemoglobin have been characterized by mass spectrometry. The strategy employs fast atom bombardment and/or tandem mass spectrometry of enzymatically digested peptides for elucidating the type and position of the mutations. Electrospray ionization mass spectrometry is useful for detecting mutations in the intact protein molecule, but the competence diminishes with increasing mass or increasing number of 13C atoms. Recent developments of DNA techniques requires us to re-evaluate the status of mass spectrometry in this field. The role of mass spectrometry in the structural analysis of protein variants is discussed, and is compared with chemical methods and DNA strategies.

Amino Acid Sequence↗
PubMed2005

Design of lambda Cro fold: solution structure of a monomeric variant of the de novo protein.

One of the classical DNA-binding proteins, bacteriophage lambda Cro, forms a homodimer with a unique fold of alpha-helices and beta-sheets. We have computationally designed an artificial sequence of 60 amino acid residues to stabilize the backbone tertiary structure of the lambda Cro dimer by simulated annealing using knowledge-based structure-sequence compatibility functions. The designed amino acid sequence has 25% identity with that of natural lambda Cro and preserves Phe58, which is important for formation of the stably folded structure of lambda Cro. The designed dimer protein and its monomeric variant, which was redesigned by the insertion of a beta-hairpin sequence at the C-terminal region to prevent dimerization, were synthesized and biochemically characterized to be well folded. The designed protein was monomeric under a wide range of protein concentrations and its solution structure was determined by NMR spectroscopy. The solved structure is similar to that of a monomeric variant of natural lambda Cro with a root-mean-square deviation of the polypeptide backbones at 2.1A and has a well-packed protein core. Thus, our knowledge-based functions provide approximate but essential relationships between amino acid sequences and protein structures, and are useful for finding novel sequences that are foldable into a given target structure.

Amino Acid Sequence↗
PubMed2005

Crystal structure of the D94S/G98E variant of rat alpha-parvalbumin. An explanation for the reduced divalent ion affinity.

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat alpha-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca(2+) affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the alpha D94S/G98E variant. The Ca(2+) coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the alpha 94/98E variant in both Na(+)- and K(+)-containing buffers. Although the Ca(2+) and Mg(2+) affinities are higher in K(+) solution, the increases are comparable to those observed for wild-type alpha. Consistent with that finding, the apparent Na(+) stoichiometry, estimated from stability studies conducted as a function of Na(+) concentration, is 1.0 +/- 0.1, identical to that of wild-type alpha. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the C(gamma) atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.

Amino Acid Substitution↗
PubMed1992

Variable and conserved structural elements of trypanosome variant surface glycoproteins.

The characterization of B cell epitopes on the trypanosome variant surface glycoprotein (VSG) rests on elucidation of variant specific amino acid sequences that may be exposed or buried as a result of the natural conformation of these molecules in the surface coat. Despite the fact that different VSGs have heterogeneous primary sequences and unique antigenic characteristics, recent high resolution X-ray crystallographic analyses of VSGs have revealed a conserved 3-dimensional structure common to these surface proteins [19]. We took advantage of this conserved structural conformation to help predict which variant subregions of VSG molecules may contain exposed or buried variant specific B cell epitopes. Using Staden data tables, we aligned the deduced amino acid sequence of Trypanosoma brucei rhodesiense LouTat 1 VSG, a molecule that has been characterized immunologically in this laboratory, with 12 other complete VSG sequences including the T. b. brucei MiTat 1.2 VSG that has been characterized in crystallographic studies. Results of this analysis predict that there are eight defined clusters of variant amino acids which may contribute to exposed B cell epitopes, and ten defined clusters of variant amino acids which may contribute to buried B cell epitopes, on all VSG molecules. Interestingly, this analysis also revealed a VSG consensus sequence in which certain conserved motifs are present in all VSGs. The shared elements of VSG sequences corresponded to known secondary structures present in MiTat 1.2, and included groups of conserved amino acids responsible for turns in subregions of the protein, for structural positioning of the variable residues on the exposed surface, and for the dimerization of VSG monomers. Overall, these observations may aid in the targeting and mapping of exposed and buried VSG specific B cell epitopes, and also may offer clues as to elements of the primary sequence that are important for the conserved 3-dimensional structure of antigenically distinct VSG molecules.

Amino Acid Sequence↗
PubMed2001

Rational Redesign of Inhibitors of Furin/kexin Processing Proteases.

Furin/kexin processing proteases catalyze the proteolysis of large protein precursors involved in many biological processes, such as zymogen activation, peptide hormone synthesis, viral protein processing and receptor maturation, making them potential targets for therapeutic agents. Herein, homology modeling and weighted evolutionary tracing were combined to investigate the interactionmechanism of furin/kex2 with eglin C mutants. The model structures showed that there were many acidic residues in the furin (kex2) binding interface, contributing to specificity for multiple basic residues of their corresponding substrates or inhibitors. Besides, some rational explanations were presented for the different inhibitor/substrate specificity of the furin/kexin members by combining the model structures with results of evolutionary tracing. Based on these analyses,an attempt was made to rationally redesign the eglin C by interface engineering with heterogeneous self-consistent ensemble optimization to improve its inhibitory specificity on furin/kex2. With the model complex structures of furin/kex2 and eglin C variants as structural templates, the P(1), P(2) and P(4) of eglin C were redesigned, respectively. The design results show that both furin and kex2 favored basic residues at P(1), P(2) and P(4) in eglin C, in good agreement with the experimental data. The detection of many specific residues in S' part of furin/kexin sequences made possible designing inhibitors with high specific binding to furin and kex2, respectively. As for furin, the best inhibitor designed was eglin C-P(2)'Glu-P(3)'Asp-P(4)'Arg (only these three positions were shown), while the best eglin C variant for kex2 designed was P(2)'Arg-P(3)'Arg-P(4)'Glu. The structures show that furin and kex2 form distinct interactions with these two eglin C variants. Herein, a strategy was proposed that combine homology modeling, evolutionary tracing and rational interface redesign to investigate enzyme-inhibitor interactions and inhibitor engineering. This computational design gives some rational guidance to further experimental inhibitor engineering.

Journal Article↗
PubMedDate not supplied

[Forensic-medical evaluation of the histologic structure of the hymen].

Variants of microscopic structure of the hymen are described and its histological classification based on the stromal component structure is offered. Hypotheses on the hymen histogenesis under different conditions are formulated. Histological analysis and histological classification of the hymen are needed for objective solution of questions arising during expert evaluation of virginity and its violation, probability or improbability of coitus without defloration.

Adolescent↗
PubMed2003

Solution structure of a circular-permuted variant of the potent HIV-inactivating protein cyanovirin-N: structural basis for protein stability and oligosaccharide interaction.

The high-resolution solution structure of a monomeric circular permuted (cp) variant of the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by NMR. Comparison with the wild-type (wt) structure revealed that the observed loss in stability of cpCV-N compared to the wt protein is due to less favorable packing of several residues at the pseudo twofold axis that are responsible for holding the two halves of the molecule together. In particular, the N and C-terminal amino acid residues exhibit conformational flexibility, resulting in fewer and less favorable contacts between them. The important hydrophobic and hydrogen-bonding network between residues W49, D89, H90, Y100 and E101 that was observed in wt CV-N is no longer present. For instance, Y100 and E101 are flexible and the tryptophan side-chain is in a different conformation compared to the wt protein. The stability loss amounts to approximately 2kcal/mol and the mobility of the protein is evident by fast amide proton exchange throughout the chain. Mutation of the single proline residue to glycine (P52G) did not substantially affect the stability of the protein, in contrast to the finding for wtCV-N. The binding of high-mannose type oligosaccharides to cpCV-N was also investigated. Similar to wtCV-N, two carbohydrate-binding sites were identified on the protein and the Man alpha1-->2Man linked moieties on the sugar were delineated as binding epitopes. Unlike in wtCV-N, the binding sites on cpCV-N are structurally similar and exhibit comparable binding affinities for the respective sugars. On the basis of the studies presented here and previous results on high-mannose binding to wtCV-N, we discuss a model for the interaction between gp120 and CV-N.

Amino Acid Sequence↗
PubMed2001

Structural comparison of monomeric variants of the chemokine MIP-1beta having differing ability to bind the receptor CCR5.

MIP-1beta, a member of the chemokine family of proteins, tightly binds the receptor CCR5 as part of its natural function in the immune response, and in doing so also blocks the ability of many strains of HIV to enter the cell. The single most important MIP-1beta residue known to contribute to its interaction with the receptor is Phe13, which when mutated reduces the ability of MIP-1beta to bind to CCR5 by more than 1000-fold. To obtain a structural understanding of the dramatic effect of the absence of Phe13 in MIP-1beta, we used multidimensional heteronuclear NMR to determine the three-dimensional structure of the MIP-1beta F13A variant. We had previously shown that, unlike the wild-type protein which has been shown to be a tight dimer, the F13A mutant is monomeric even at high concentrations [Laurence, J. S., Blanpain, C., Burgner, J. W., Parmentier, M., and LiWang, P. J. (2000) Biochemistry 39, 3401-3409], leading to significant changes in the NMR spectra of F13A and the wild-type protein. We have obtained a total of 940 structural restraints for MIP-1beta F13A, and have calculated a family of structures having a backbone rmsd from the average of 0.55 A (residues 12-67). A structural comparison of the F13A mutant with a fully active monomeric variant, P8A, shows that despite some differences in the (1)H-(15)N HSQC spectra the two are nearly identical in NOE distance restraints and in backbone conformation. A comparison of F13A with the wild-type protein shows largely the same fold, although differences exist in the N-terminal and loop regions for which the loss of the dimer in F13A can mainly account. A dynamics comparison confirms greater flexibility in F13A than in the wild-type protein in regions of dimer contact in the wild-type protein. In an analysis to determine if the large functional effect resulting from the loss of Phe13 is due to the local side chain change or due to more global structural changes, we conclude that local effects predominate. This suggests that a strategy for designing tight binding anti-CCR5 therapeutics should include a Phe-like component.

Amino Acid Substitution↗
PubMed1993

A structural motif in the variant surface glycoproteins of Trypanosoma brucei.

The variable domain of the trypanosome variant surface glycoprotein (VSG) ILTat 1.24 has been shown by X-ray crystallography to resemble closely the structures of VSG MITat 1.2, despite their low sequence similarity. Specific structural features of these VSGs, including substitution of carbohydrate for an alpha-helix, can be found in other VSG sequences. Thus antigenic variation in trypanosomes is accomplished by sequence variation, not gross structural alteration; the extensive sequence differences among VSGs may be required for another reason, such as the avoidance of recognition by helper T cells. Additionally, VSG sequences are found to define families, within a VSG superfamily, which have evolved in the trypanosome genome.

Amino Acid Sequence↗
PubMed1987

Structural features of antigenic determinants on variant surface glycoproteins from Trypanosoma brucei.

The immunochemical structure of two variant surface glycoproteins (VSGs) from Trypanosoma brucei has been studied using monoclonal and polyclonal antibodies. These two VSGs, WaTat 1.1 and WaTat 1.12 have been shown to possess cross-reactive surface-exposed antigenic determinants [Barbet et al., Nature 300, 53-57 (1982)] and similar N-terminal amino acid sequences [Olafson et al., Molec. Biochem. Parasit. 12, 287-298 (1984)]. Monoclonal and polyclonal antibodies were raised against the soluble forms of the two VSGs and against their reduced, alkylated and cyanogen bromide (CNBr) cleaved forms. None of the monoclonal antibodies which bound to the surface of living trypanosomes bound to CNBr fragments of the VSGs nor to denatured VSGs. Polyclonal antibodies raised against denatured and cleaved VSG did not bind to the surface of the living trypanosomes. These results suggest that the variable surface exposed antigenic determinants of VSG are topographically assembled structures. It was also shown that the conserved amino terminal peptides of WaTat 1.1 and WaTat 1.12 do not contain antigenic determinants.

Animals↗
PubMed2026

Genomic and structural analysis of dacB variants associated with cephalosporin resistance in Pseudomonas aeruginosa.

The rise of resistance to fourth-generation cephalosporin in Pseudomonas aeruginosa (P. aeruginosa) is a global concern. The resistance is largely driven by variants of chromosomally encoded AmpC β-lactamase, known as Pseudomonas-derived cephalosporinase (PDC), which arise from the mutations in the ampC gene. In addition, alteration in dacB, which encode the penicillin-binding protein 4 (PBP4), can lead to the overexpression of ampC, thereby contributing to β-lactam resistance. Present work analyzed 208 clinical isolates of P. aeruginosa using whole-genome sequencing (WGS) and detected multiple nonsynonymous single nucleotide polymorphisms (nsSNPs), such as Y264C, G444D, and a double mutation (A394P-T428P). All nsSNPs were predicted to be deleterious by several prediction program. Molecular dynamics (MD) simulations suggested that these substitutions destabilize PBP4, increase structural flexibility, and contribute to the resistance mechanism, which favored their selection. To determine the effective therapeutics against these mutations, molecular docking was conducted with various antibiotics. Cefoperazone exhibited the highest binding affinity (-7.3 kcal/mol) among multiple PBP4 variants. The Molecular dynamics (MD) simulations and Molecular Mechanics Poisson Boltzmann Surface Area calculations (MMPBSA) further confirmed the favorable interactions between cefoperazone and PBP4 variants. In vitro MIC analyses supported these findings, indicating that cefoperazone displayed significant activity against clinical dacB mutants of P. aeruginosa. The study offers structural insight of dacB variants leading to antibiotic resistance and emphasizes the need to prioritize specific antibiotics to address the challenges arising from protein mutations.

Pseudomonas aeruginosa↗
PubMed1997

Structure-function relations of variant and fragment nisins studied with model membrane systems.

Nisin, a 34 residue lantibiotic produced by strains of Lactococcus lactis subsp. lactis, exerts antimicrobial activity against Gram-positive bacteria at the cytoplasmic membrane. The structural aspects of nisin which facilitate membrane interaction and permeabilization have been investigated in planar lipid bilayers and liposomes with proteolytic fragments and site-directed variants. N-Terminal nisin fragments N1-12 and N1-20 had little effect on phospholipid mobility, on macroscopic electrical conductance, or on calcein release from liposomes. By contrast, the I30W nisin A variant induced a time-dependent reduction in lipid mobility, indicative of nisin-membrane surface interactions, as well as a decline in membrane capacitance, rise in conductance, and calcein release from liposomes. In these respects I30W nisin A is similar to native nisin. Charge substitutions were also engineered to generate K12L and H27K nisin A variants, both of which were similar to I30W nisin A with respect to an overall reduction in phospholipid mobility. While the K12L nisin A variant elicited a higher increase in membrane capacitance and electrical conductance than I30W nisin A, the H27K nisin A variant elicited weaker effects. These results point to a substantial role for intramembrane charged residues in controlling ion flow through nisin-doped membranes. Native nisin and variants elicit an enhanced release of calcein from liposomes composed of the negatively-charged phospholipids cardiolipin and phosphatidylserine, compared with phospholipid bearing no net charge, suggesting that an electrostatic attraction encourages the initial nisin-membrane association. The results are discussed in the context of other recently proposed models for nisin action.

Amino Acid Sequence↗
PubMed2003

Fibrinogen gamma-chain splice variant gamma' alters fibrin formation and structure.

Fibrinogen gammaA/gamma' results from alternative splicing of mRNA. This variant, which constitutes approximately 8% to 15% of plasma fibrinogen, contains FXIII and thrombin binding sites. Our objective was to investigate whether gammaA/gamma' differs in fibrin formation and structure from the more common variant gammaA/gammaA. Both variants were separated and purified by anion-exchange chromatography. Fibrin formation and clot structure of the variants and unfractionated fibrinogen were investigated by turbidity and scanning electron microscopy (SEM). Thrombin cleavage of fibrinopeptides was analyzed by high-performance liquid chromatography (HPLC). Turbidity analysis showed significantly altered polymerization rates and overall fiber thickness in gammaA/gamma' clots compared with gammaA/gammaA and unfractionated fibrinogen. This finding was consistent with a range of thrombin concentrations. HPLC demonstrated reduced rates of fibrinopeptide B (FpB) release from gammaA/gamma' fibrinogen compared with gammaA/gammaA. Delayed FpB release was associated with delayed lateral aggregation of protofibrils and significant differences were found on SEM, with gammaA/gamma' clots consisting of smaller diameter fibers and increased numbers of branch points compared with both gammaA/gammaA and unfractionated fibrinogen. These results demonstrate that the gammaA/gamma' splice variant of fibrinogen directly alters fibrin formation and structure, which may help to explain the increased thrombotic risk associated with this variant.

Alternative Splicing↗
PubMed1999

Effects of H1 histone variant overexpression on chromatin structure.

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.

3T3 Cells↗
PubMed1988

A new genetic fibrinogen variant (fibrinogen Erfurt I). Structurally characterized by an abnormal B beta-chain and present both in plasma and platelets.

An abnormal fibrinogen was discovered in the plasma of a clinically asymptomatic woman. This fibrinogen variant was analyzed by high resolution two-dimensional gel electrophoresis and its molecular abnormality established consisting in a slight decrease in molecular mass of the B beta-chains. Analysis of fibrin revealed that cleavage of fibrinopeptide B by thrombin is normal, the molecular defect being confined to the beta-portion of the B beta-chain. The same fibrinogen variant was detected in the blood platelets of the proposita. This finding supports the assumption of a common origin of plasma and platelet fibrinogen pools. Family studies revealed the presence of the abnormal fibrinogen in a brother of the proposita, thus confirming the genetic nature of the observed variant. The underlying mutant gene occurs in both carriers in heterozygous state.

Adult↗