Crystal structure of a dUTPase

Cedergren-Zeppezauer, Eila; Larsson, Gunilla; Nyman, Per Olof; Dauter, Zbigniew; Wilson, K.S.

doi:10.1038/355740a0

Cited by 136 publications

(116 citation statements)

References 15 publications

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“…Quantitative evaluation of the spectra by the k2d program (60) resulted in estimates for secondary structural content (␣, 8 Ϯ 1%; ␤, 45 Ϯ 3%; other, 47 Ϯ 2%), revealing an overwhelming majority of ␤-structure over ␣-helices in the folded protein. These values are very close to those estimated from the spectrum of the E. coli enzyme (␣, 11%; ␤, 42%; other, 47%) (33), which were also confirmed by the crystal structure (26).…”

Section: Table I Mass Spectrometric Analysis Of a Total Tryptic Digessupporting

confidence: 76%

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54°C, 23°C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg 2؉ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

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Section: Table I Mass Spectrometric Analysis Of a Total Tryptic Digessupporting

confidence: 76%

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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“…A. Rogers, G. Larsson & E O. Nyman, unpublished work). The E. coli dUTPase was first shown to be a trimer in the crystal (Cedergren-Zeppezauer et al, 1992). This is proven by gel-filtration experiments (this work) to be true in solution also.…”

Section: Introductionmentioning

confidence: 71%

The Refined Structure of dUTPase from Escherichia coli

Dauter¹,

Wilson²,

Larsson³

et al. 1998

Acta Cryst D

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Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 A MIRAS phases differed from the final set by 40 c on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a 'jelly-roll' type, containing mostly/3-sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its fl-sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the fl-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.

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“…Moreover, our domainlevel searches have failed to reveal similarity of the inner domain to any known atomic structures, although the missing terminal segments might conceal relationships. We do, however, find fragmentary similarity for portions of the outer domain with known structures.and dUTP pyrophosphatase 28 has elements in common with both barrels of the outer domain. In each case, the superimposable fraction is limited.…”

Section: Structure Of Gp120mentioning

confidence: 81%

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Kwong

Wyatt

Robinson

et al. 1998

Nature

2,742

108

3,142

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The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.The human immunodeficiency viruses HIV-1 and HIV-2 and the related simian immunodeficiency viruses (SIV) cause the destruction of CD4 + lymphocytes in their respective hosts, resulting in the development of acquired immunodeficiency syndromeCorrespondence and requests for materials should be addressed to W. A.H. (wayne@convex.hhmi.columbia.edu).Coordinates have been deposited in the Brookhaven Protein Data Bank (accession code 1gc1) and maybe obtained from the authors. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (AIDS) 1,2 . The entry of HIV into host cells is mediated by the viral envelope glycoproteins, which are organized into oligomeric, probably trimeric spikes displayed on the surface of the virion. These envelope complexes are anchored in the viral membrane by the gp41 transmembrane envelope glycoprotein. The surface of the spike is composed primarily of the exterior envelope glycoprotein, gp120, associated by non-covalent interactions with each subunit of the trimeric gp41 glycoprotein complex 3,4 . Comparison of the gp120 sequences of different primate immunodeficiency viruses identified five variable regions (V1-V5) (ref. 5 ). The first four variable regions form surface-exposed loops that contain disulphide bonds at their bases 6 . The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gp120 regions are extensively glycosylated 6 . The variability and glycosylation of the gp120 surface probably modulate the immunogenicity and antigenicity of the gp120 glycoprotein, which is the main target for neutralizing antibodies elicited during natural infection 7 .Entry of primate immunodeficiency viruses into the host cell involves the binding of the gp120 envelope glycoprotein to the CD4 glycoprotein, which serves as the primary receptor. The gp120 glycoprotein binds to the most amino-terminal of the four immunoglobulin-like domains of CD4. S...

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Crystal structure of a dUTPase

Cited by 136 publications

References 15 publications

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

The Refined Structure of dUTPase from Escherichia coli

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

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