Molecular Determinants of S100B Oligomer Formation

Background: S100B is a dimeric protein that can form tetramers, hexamers and higher order oligomers. These forms have been suggested to play a role in RAGE activation. Methodology/Principal Findings: Oligomerization was found to require a low molecular weight trigger/cofactor and could not be detected for highly pure dimer, irrespective of handling. Imidazol was identified as a substance that can serve this role. Oligomerization is dependent on both the imidazol concentration and pH, with optima around 90 mM imidazol and pH 7, respectively. No oligomerization was observed above pH 8, thus the protonated form of imidazol is the active species in promoting assembly of dimers to higher species. However, disulfide bonds are not involved and the process is independent of redox potential. The process was also found to be independent of whether Ca 2+ is bound to the protein or not. Tetramers that are purified from dimers and imidazol by gel filtration are kinetically stable, but dissociate into dimers upon heating. Dimers do not revert to tetramer and higher oligomer unless imidazol is again added. Both tetramers and hexamers bind the target peptide from p53 with retained stoichiometry of one peptide per S100B monomer, and with high affinity (lgK = 7.360.2 and 7.260.2, respectively in 10 mM BisTris, 5 mM CaCl 2, pH 7.0), which is less than one order of magnitude reduced compared to dimer under the same buffer conditions. Conclusion/Significance: S100B oligomerization requires protonated imidazol as a trigger/cofactor. Oligomers ar

Functional Assessment of Population and Tumor-Associated APE1 Protein Variants

<div><p>Apurinic/apyrimidinic endonuclease 1 (APE1) is the predominant AP site repair enzyme in mammals. APE1 also maintains 3′–5′ exonuclease and 3′-repair activities, and regulates transcription factor DNA binding through its REF-1 function. Since complete or severe APE1 deficiency leads to embryonic lethality and cell death, it has been hypothesized that APE1 protein variants with slightly impaired function will contribute to disease etiology. Our data indicate that except for the endometrial cancer-associated APE1 variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit normal thermodynamic stability of protein folding; abasic endonuclease, 3′–5′ exonuclease and REF-1 activities; coordination during the early steps of base excision repair; and intracellular distribution when expressed exogenously in HeLa cells. The R237C mutant displayed reduced AP-DNA complex stability, 3′–5′ exonuclease activity and 3′-damage processing. Re-sequencing of the exonic regions of <i>APE1</i> uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results indicate that <i>APE1</i> missense mutations are seemingly rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele.</p></div

Structural and functional insights into RAGE activation by multimeric S100B

Nervous system development and plasticity require regulation of cell proliferation, survival, neurite outgrowth and synapse formation by specific extracellular factors. The EF-hand protein S100B is highly expressed in human brain. In the extracellular space, it promotes neurite extension and neuron survival via the receptor RAGE (receptor for advanced glycation end products). The X-ray structure of human Ca2+-loaded S100B was determined at 1.9 Å resolution. The structure revealed an octameric architecture of four homodimeric units arranged as two tetramers in a tight array. The presence of multimeric forms in human brain extracts was confirmed by size-exclusion experiments. Recombinant tetrameric, hexameric and octameric S100B were purified from Escherichia coli and characterised. Binding studies show that tetrameric S100B binds RAGE with higher affinity than dimeric S100B. Analytical ultracentrifugation studies imply that S100B tetramer binds two RAGE molecules via the V-domain. In line with these experiments, S100B tetramer caused stronger activation of cell growth than S100B dimer and promoted cell survival. The structural and the binding data suggest that tetrameric S100B triggers RAGE activation by receptor dimerisation

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in motif 2 to stabilize the leaving uracilate anion. The integrated approach has important implications in studying enzyme evolution and protein structure and function

DNA damage recognition and repair by 3-methyladenine DNA glycosylase I (TAG)

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3-methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix–hairpin–helix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA