Solution Structure and Peptide Binding of the PTB Domain from the AIDA1 Postsynaptic Signaling Scaffolding Protein

<div><p>AIDA1 links persistent chemical signaling events occurring at the neuronal synapse with global changes in gene expression. Consistent with its role as a scaffolding protein, AIDA1 is composed of several protein-protein interaction domains. Here we report the NMR structure of the carboxy terminally located phosphotyrosine binding domain (PTB) that is common to all AIDA1 splice variants. A comprehensive survey of peptides identified a consensus sequence around an NxxY motif that is shared by a number of related neuronal signaling proteins. Using peptide arrays and fluorescence based assays, we determined that the AIDA1 PTB domain binds amyloid protein precursor (APP) in a similar manner to the X11/Mint PTB domain, albeit at reduced affinity (∼10 µM) that may allow AIDA1 to effectively sample APP, as well as other protein partners in a variety of cellular contexts.</p></div

Amino acid preferences of the AIDA1 PTB domain for APP determined from a peptide array.

<p>A list of peptides on the array are provided in supplementary material. <i>(a)</i> The array probed with anti 6xHis mAb only. Positive control 6xHis peptides are identified by a <b>+</b>. <i>(b)</i> The array probed with 6xHis-AIDA1 PTB domain. <i>(c)</i> Sliding window peptide scan of 12-mers spanning aa. 672–697 of APP. Peptides are duplicated on the array; for example, at A3 and A18. Since peptide content per spot can vary, if a signal was observed at the exposure presented it was deemed to be interaction. <i>(d)</i> Results of a window scan across the APP C-terminal sequence and an exhaustive positional scan. Grey boxes indicate binding was observed, regardless of signal intensity.</p

Affnities of APP-derived peptides for two solubility enhanced mutants of the AIDA1 PTB domain. ND: not done.

<p>Affnities of APP-derived peptides for two solubility enhanced mutants of the AIDA1 PTB domain. ND: not done.</p

<i>(a)</i> Sequence alignment of the AIDA1 PTB domain against the APP binding proteins, Dab1 [25], X11 [17] and Fe65 [20].

<p>Five aromatic amino acids selected for alanine substitution in AIDA1 PTB domain are boxed. <i>(b)</i> Backbone atom superposition of top15 structures according to lowest refinement energy. <i>(c)</i> Strip plots of a ¹³C-edited NOESY spectrum at the Cβ chemical shift of each alanine substituted in the PTB5M mutant. A asterisk denotes a resonance not associated with that strip. <i>(d)</i> A ribbon representation of the PTB5M model highlighting the positions of the alanine substitutions. Y6A is not shown in the figure as the first 14 amino acids are unstructured and were excluded from the structure calculation.</p

Solubilities and thermal denaturation midpoints of the AIDA PTB domain and alanine substitution mutants.

<p>Solubilities and thermal denaturation midpoints of the AIDA PTB domain and alanine substitution mutants.</p

Structural similarity of the AIDA1 PTB domain to related PTB domains that also bind APP.

<p>Structural similarity of the AIDA1 PTB domain to related PTB domains that also bind APP.</p

The Solution Structures of Two Prophage Homologues of the Bacteriophage λ Ea8.5 Protein Reveal a Newly Discovered Hybrid Homeodomain/Zinc-Finger Fold

A cluster of genes in the <i>exoxis</i> region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in <i>Escherichia coli</i>. The most indispensible gene in this region is <i>ea8.5</i>. Here, we report the nuclear magnetic resonance structures of two <i>ea8.5</i> orthologs from enteropathogenic <i>E. coli</i> and <i>Pseudomonas putida</i> prophages. Both proteins are characterized by a fused homeodomain/zinc-finger fold that escaped detection by primary sequence search methods. While these folds are both associated with a nucleic acid binding function, the amino acid composition suggests otherwise, leading to the possibility that Ea8.5 associates with other viral and host proteins

Proteins and substrates used in reconstitution of long patch base excision repair Recombinant proteins were purified as described in ‘Materials and Methods’ and separated on a 8–20% gradient SDS-PAGE gel, and stained with Coomassie Blue

<p><b>Copyright information:</b></p><p>Taken from "The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase β in long patch base excision repair"</p><p></p><p>Nucleic Acids Research 2007;35(8):2596-2608.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885638.</p><p>© 2007 The Author(s)</p> Lane 1: molecular weight markers; lane 2: APE 1 (2 μg); lane 3: Pol β (2 μg); lane 4: Fen 1 (2 μg); lane 5: Lig I (2 μg); lane 6: 9-1-1 complex (6 μg). Schematic representation of the P-5′-labeled oligonucleotide substrates used in the study: a 100 bp duplex oligonucleotide containing a THF moiety at the position 43 was used for repair reactions, the ends of the substrate were either free (unblocked substrate) or blocked with a biotin at each end (blocked substrate); a 100 bp duplex oligonucleotide with a 1 nucleotide gap at the same position was used for the Pol β assay; with a nick for the Lig I assay and with a 10 nucleotide flap for the Fen 1 reactio

Titration of FITC-labeled APP peptides with a solubility enhanced mutant (Y70A) of the AIDA1 PTB domain.

<p>Binding was monitored by fluorescence anisotropy. Legend: APP17, a short X11-like binding site; APP32, a longer Fe65-like binding site; APP17{pY}, a short X11-like phosphopeptide. The peptide sequences are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065605#pone-0065605-t004" target="_blank">Table 4</a>.</p

The 9-1-1 complex specifically stimulates the endonuclease activity of APE 1

<p><b>Copyright information:</b></p><p>Taken from "The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase β in long patch base excision repair"</p><p></p><p>Nucleic Acids Research 2007;35(8):2596-2608.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1885638.</p><p>© 2007 The Author(s)</p> The APE 1 incision assay was performed as described in ‘Materials and Methods’. Reactions were stopped by adding an equal volume of formamide-dye solution and products were analyzed on a 10% denaturing polyacrylamide gel. An APE 1 reaction mixture (10 μl) contained (besides all components described in ‘Materials and Methods’) P-5′-labeled 100 bp duplex oligonucleotide (50 fmol see B), APE 1 (2 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex. As A but with the blocked substrate (50 fmol). Quantification of the stimulation of APE 1 endonuclease cleavage by the 9-1-1 complex on the substrate with free ends (closed circles) and with the ends blocked with biotin (open circles). The values represent the mean of three independent experiments. The error bars correspond to the standard error of the mean