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1.
Figure 5

Figure 5. AOA1 mutations. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

hAptx AOA1 associated single amino acid substitutions. Corresponding S. pombe Aptx variants for are shown in tabular format, and are mapped onto the S. pombe Aptx structure.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.
2.
Figure 6

Figure 6. Nicked and gap DNA binding by Aptx. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

Molecular model for Aptx interaction with a nicked or gapped 5′-adenylated substrate is shown (center). A steric clash of the wedge (α1) with the upstream region of a nick implies nicked or gapped DNA must be bent for binding and access to the 5′-adenylate lesion.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.
3.
Figure 3

Figure 3. Aptx DNA binding and deadenylation activity. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

(a) Substrate specific DNA binding by AptxFL. DNA binding to FITC-conjugated 39-mer SSB (1bp gap with 3′- and 5′-phosphates), 39-mer ssDNA and 39-mer dsDNA substrates was monitored by fluorescence anisotropy. Binding isotherms are expressed as fraction bound. Error bars reflect standard deviation of three measurements. Inset: schematics of DNA binding substrates. (b) Commassie stained gel of purified AptxFL structure-based mutants. (c) DNA binding activity of Aptx 5′ adenylated strand binding Znf and FPK motif mutants. Binding to the SSB substrate (as in panel a) shown. (d) DNA deadenylase activity of structure-specific Aptx mutants. Measured release of α-P32-AMP (relative to WT) from an abortively ligated nicked (form II) φX174 is shown. Error bars are standard deviation of two measurements.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.
4.
Figure 4

Figure 4. Aptx adenylate access and catalytic mechanism. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

(a) Aptx DNA end binding and adenylate rotation is mediated by the HIT domain wedge and pivot (FPK) elements that extract and align 5′-adenylate into the HIT-Znf active site. Unbiased σ-A weighted Fo-Fc positive difference density map (green, contoured at 2.4σ) calculated before building the DNA model is displayed. Phe65 and Lys67 of the pivot directly bind the sugar-phosphate backbone of the incoming 5′-strand, and AMP. (b) DNA 5′-phosphate binding pocket. An electrostatic surface potential with electropositive surface (blue) and electronegative surface (red) is displayed with a orange surface representation for the AMP. The DNA 5′-phosphate binding pocket is indicated as a dotted line. (c) The AMP binding pocket is displayed with bound AMP. Blue electron density is finalσ-A weighted 2Fo-Fc map, contoured at 1.4 σ. (d) The Aptx active site catalytic residues. Canonical HIT motif residues are marked (green "HIT"). Residues His138 and His168 are conserved in the Aprataxins, and assemble to from the Aptx HIT-Znf composite active site. (e) Structure based mechanism for 2-step direct reversal of DNA 5′-adenylation.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.
5.
Figure 1

Figure 1. X-ray crystal structure of the Aptx–DNA–AMP–Zn quaternary complex. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

(a) To restore ligatable DNA 5′-phosphates, Aptx reverses DNA 5′-adenylation from abortive ligation reactions created when DNA ligases engage nicks with 3′ (red circles) or 5′ (yellow circles) nick distorting lesions. (b) Overall Aptx–DNA–AMP–Zn complex architecture. Orthogonal views of the fused Aptx HIT-Znf assembly with the HIT domain (purple) and Znf (gold) collaborating to assemble the structure specific DNA end (green) binding surface. A DNA pseudo-duplex axis is formed by pairing of 3-bases across a crystallographic 2-fold axis. Symmetry related DNA is shown in grey. (c) DNA protein complex formation in the crystal. σ-A weighted Fo-Fc positive difference density map calculated before building the DNA model is displayed for symmetry related molecules (red and blue density, contoured at 2.4σ). (d) Domain architectures of S. pombe and human Aprataxin. S. pombe Aptx lacks an N-terminal FHA domain, but retains the HIT-Znf catalytic core. "T" marks site of tryptic proteolysis delineating a minimal conserved catalytic core encompassing residues 30-232 of S. pombe Aptx.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.
6.
Figure 2

Figure 2. Aptx structure-specific DNA binding. From: Structure of an Aprataxin–DNA complex with insights into AOA1 Neurodegenerative Disease.

(a) The Aptx HIT and Znf surfaces assemble an electropositive DNA binding platform. AMP (orange) binds in a recessed hydrophobic pocket. A dotted line demarcates surface boundaries of the HIT and Znf domains. (b) Five conserved regions (DB1-5) mediate Aptx DNA binding contacts. DNA base stacking from DB1 (turquoise), DB3 (red) probe and interrogate the dsDNA end base stack for end recognition. DB2 (blue) and DB4 (grey) bind the incoming 5′ strand, whereas DB5 engages the complementary "non-adenylated" strand. (c) Surface representation of the DNA base-stacking wedge. The AMP product is sandwiched between the wedge, 5′ strand, and the HIT-Znf active site. (d) The Aptx C2HE zinc finger is structurally related to C2H2 zinc fingers. Aptx E221 replaces the second histidine of a canonical C2H2. (e) A model-phased Zn anomalous difference Fourier map (purple, displayed at 14σ) reveals the position of the single bound Zn. Blue electron density is the final σ-A weighted 2Fo-Fc map is contoured at 1.4 σ. (f) The Aptx C2HE Znf domain engages the DNA minor groove. Left: the Aptxββα-fold and α7 bind the phosphate backbone in a novel minor groove DNA binding mode. Right: canonical major groove interaction by the Zif268 C2H2 domains.

Percy Tumbale, et al. Nat Struct Mol Biol. ;18(11):1189-1195.

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