scholarly journals Structure and enzymatic mechanism of a moonlighting dUTPase

2013 ◽  
Vol 69 (12) ◽  
pp. 2298-2308 ◽  
Author(s):  
Ibolya Leveles ◽  
Veronika Németh ◽  
Judit E. Szabó ◽  
Veronika Harmat ◽  
Kinga Nyíri ◽  
...  
Keyword(s):  
Kinetic Data ◽  
Metal Ion ◽  
Three Dimensional ◽  
Genome Integrity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Enzymatic Mechanism ◽  
Kinetics Studies ◽  
Inner Channel

Genome integrity requires well controlled cellular pools of nucleotides. dUTPases are responsible for regulating cellular dUTP levels and providing dUMP for dTTP biosynthesis. InStaphylococcus, phage dUTPases are also suggested to be involved in a moonlighting function regulating the expression of pathogenicity-island genes. Staphylococcal phage trimeric dUTPase sequences include a specific insertion that is not found in other organisms. Here, a 2.1 Å resolution three-dimensional structure of a φ11 phage dUTPase trimer with complete localization of the phage-specific insert, which folds into a small β-pleated mini-domain reaching out from the dUTPase core surface, is presented. The insert mini-domains jointly coordinate a single Mg2+ion per trimer at the entrance to the threefold inner channel. Structural results provide an explanation for the role of Asp95, which is suggested to have functional significance in the moonlighting activity, as the metal-ion-coordinating moiety potentially involved in correct positioning of the insert. Enzyme-kinetics studies of wild-type and mutant constructs show that the insert has no major role in dUTP binding or cleavage and provide a description of the elementary steps (fast binding of substrate and release of products). In conclusion, the structural and kinetic data allow insights into both the phage-specific characteristics and the generally conserved traits of φ11 phage dUTPase.

scholarly journals Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

2000 ◽  
Vol 74 (23) ◽  
pp. 11388-11393 ◽  
Author(s):  
Peggy P. Li ◽  
Akira Nakanishi ◽  
Mary A. Tran ◽  
Adler M. Salazar ◽  
Robert C. Liddington ◽  
...  
Keyword(s):  
Life Cycle ◽  
Disulfide Bonds ◽  
Three Dimensional ◽  
Simian Virus 40 ◽  
Experimental System ◽  
Simian Virus ◽  
Dimensional Structure ◽  
Wild Type

ABSTRACT We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants—C9A, C49A, C87A, C104A, C207S, C254A, and C267L—retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca2+-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca2+ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

scholarly journals Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

2015 ◽  
Vol 71 (11) ◽  
pp. 2354-2361 ◽  
Author(s):  
Vaheh Oganesyan ◽  
Yariv Mazor ◽  
Chunning Yang ◽  
Kimberly E. Cook ◽  
Robert M. Woods ◽  
...  
Keyword(s):  
Functional Role ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Replacement ◽  
Wild Type ◽  
Direct Role ◽  
Three Dimensional Structure ◽  
Human Igg1 ◽  
Structural Insights

The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc `LLGG' motif at positions 234–237, and particularly of Leu235, via a `lock-and-key' mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

scholarly journals The Mitochondrial Genome Integrity Gene, MGI1, of Kluyveromyces lactis Encodes the β-Subunit of F1-ATPase

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1445-1454 ◽  
Author(s):  
Xin Jie Chen ◽  
G Desmond Clark-Walker
Keyword(s):  
Mitochondrial Genome ◽  
Kluyveromyces Lactis ◽  
Three Dimensional ◽  
Genome Integrity ◽  
Dimensional Structure ◽  
Deletion Mutants ◽  
Β Subunit ◽  
Gain Of Function ◽  
F1 Atpase ◽  
Γ Subunit

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

scholarly journals Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 267-286 ◽  
Author(s):  
J D Fackenthal ◽  
J A Hutchens ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Variable Region ◽  
Internal Variable ◽  
Microtubule Assembly ◽  
Dimensional Structure ◽  
Wild Type ◽  
Beta Tubulin ◽  
Assembly Kinetics ◽  
Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

scholarly journals Analysis of structural similarities between brain Thy-1 antigen and immunoglobulin domains. Evidence for an evolutionary relationship and a hypothesis for its functional significance

1981 ◽  
Vol 195 (1) ◽  
pp. 31-40 ◽  
Author(s):  
F E Cohen ◽  
J Novotný ◽  
M J E Sternberg ◽  
D G Campbell ◽  
A F Williams
Keyword(s):  
Functional Significance ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Three Dimensional ◽  
Evolutionary Relationship ◽  
Dimensional Structure ◽  
Sequence Homologies ◽  
Beta Structure ◽  
Beta 2 Microglobulin

The Thy-1 membrane glycoprotein from rat brain is shown to have structural and sequence homologies with immunoglobulin (Ig) domains on the basis of the following evidence. 1. The two disulphide bonds of Thy-1 are both consistent with the Ig-fold. 2. The molecule contains extensive beta-structure as shown by the c.d. spectrum. 3. Secondary structure prediction locates beta-strands along the sequence in a manner consistent with the Ig-fold. 4. On the basis of rules derived from known beta-sheet structures, a three-dimensional structure with the Ig-fold is predicted as favourable for Thy-1. 5. Sequences in the proposed beta-strands of Thy-1 and known beta-strands of Ig domains show significant sequence homology. This homology is statistically more significant than for the comparison of proposed beta-strand sequences of beta 2-microglobulin with Ig domains. An hypothesis is presented for the possible functional significance of an evolutionary relationship between Thy-1 and Ig. It is suggested that both Thy-1 and Ig evolved from primitive molecules, with an Ig fold, which mediated cell--cell interactions. The present-day role of Thy-1 may be similar to that of the primitive domain.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Solvent Accessibility of Residues Undergoing Pathogenic Variations in Humans: From Protein Structures to Protein Sequences

2021 ◽  
Vol 7 ◽  
Author(s):  
Castrense Savojardo ◽  
Matteo Manfredi ◽  
Pier Luigi Martelli ◽  
Rita Casadio
Keyword(s):  
Solvent Accessibility ◽  
Protein Structures ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Large Data ◽  
Human Protein ◽  
Dimensional Structure ◽  
Wild Type ◽  
Solvent Exposure ◽  
Data Set

Solvent accessibility (SASA) is a key feature of proteins for determining their folding and stability. SASA is computed from protein structures with different algorithms, and from protein sequences with machine-learning based approaches trained on solved structures. Here we ask the question as to which extent solvent exposure of residues can be associated to the pathogenicity of the variation. By this, SASA of the wild-type residue acquires a role in the context of functional annotation of protein single-residue variations (SRVs). By mapping variations on a curated database of human protein structures, we found that residues targeted by disease related SRVs are less accessible to solvent than residues involved in polymorphisms. The disease association is not evenly distributed among the different residue types: SRVs targeting glycine, tryptophan, tyrosine, and cysteine are more frequently disease associated than others. For all residues, the proportion of disease related SRVs largely increases when the wild-type residue is buried and decreases when it is exposed. The extent of the increase depends on the residue type. With the aid of an in house developed predictor, based on a deep learning procedure and performing at the state-of-the-art, we are able to confirm the above tendency by analyzing a large data set of residues subjected to variations and occurring in some 12,494 human protein sequences still lacking three-dimensional structure (derived from HUMSAVAR). Our data support the notion that surface accessible area is a distinguished property of residues that undergo variation and that pathogenicity is more frequently associated to the buried property than to the exposed one.

3D Structure of Striations in the North Pacific

2021 ◽  
Author(s):  
YU ZHANG ◽  
YU PING GUAN ◽  
RUI XIN HUANG
Keyword(s):  
North Pacific ◽  
3D Structure ◽  
Three Dimensional ◽  
Vertical Plane ◽  
Dimensional Structure ◽  
The North ◽  
Layer Analysis ◽  
Fluid Potential ◽  
The North Pacific

AbstractOcean striations are composed of alternating quasi-zonal band-like flows; this kind of organized structure of currents be found in all world’s oceans and seas. Previous studies have mainly been focused on the mechanisms of their generation and propagation. This study uses the spatial high-pass filtering to obtain the three-dimensional structure of ocean striations in the North Pacific in both the z-coordinate and σ-coordinate based on 10-yr averaged SODA3 data. First, we identify an ideal-fluid potential density domain where the striations are undisturbed by the surface forcing and boundary effects. Second, using the isopycnal layer analysis, we show that on isopycnal surfaces the orientations of striations nearly follow the potential vorticity (PV) contours, while in the meridional-vertical plane the central positions of striations are generally aligned with the latitude of zero gradient of the relative PV. Our analysis provides a simple dynamical interpretation and better understanding for the role of ocean striations.

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