1. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins 
Ben E. Clifton, Colin J. Jackson 
Cell Chemical Biology 
Volume 23, Issue 2, Pages (February 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 236-245DOI: (10. 1016/j. chembiol. 2015
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3. Figure 1 Reconstruction of Ancestral Amino Acid-Binding Proteins
(A) Maximum-likelihood phylogeny of the AABP family used for ancestral reconstruction. Tips corresponding to representative AABPs that have been characterized previously are annotated with the source organism and the amino acids bound by the protein. The scale bar represents the mean number of substitutions per site. bm, Bacillus megaterium; cg, Corynebacterium glutamicum; cj, Campylobacter jejuni; ec, Escherichia coli; ef, Enterococcus faecalis; sp, Streptococcus pneumoniae; Cyi, l-cystine; Orn, l-ornithine.
(B) Expanded view of (A) showing the four ancestral nodes characterized in this work and bootstrap values from 100 replicates on major branches.
(C) Posterior probability (PP) distributions of the reconstructed ancestral sequences.
(D) Posterior probability distributions for individual positions near the amino acid-binding sites of ancestral AABPs. Residues are numbered according to the equivalent position in AncQR.
See also Figure S1.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Binding Specificity of Ancestral and Extant AABPs
Association constants (Ka) for AABP-amino acid interactions determined by ITC at 25°C. Data represent the mean ± SD from at least three titrations. See also Figure S2, Tables S1 and S2.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3 Crystal Structures of AncQR
(A and B) Binding sites of the AncQR-Arg (A) and AncQR-Gln (B) complexes. Electron density for the ligands and ordered water molecules is shown by omit Fo − Fc maps contoured at +3σ. Phe58 is positioned on top of the ligands and is omitted for clarity. The flexible loop Lys145-Glu151 is shown in red.
(C) Existence of multiple low-energy conformational substates in the binding site of the AncQR-Arg complex. Residues 123 and 145–151 were modeled in the conformation unique to the AncQR-Arg structure (gray) with occupancy of 0.5. The Fo – Fc electron density map resulting from refinement of this model is contoured at ±3σ and matches the alternative conformation observed in the AncQR-Gln structure (orange, with an associated water molecule in blue).
(D) Global conformational differences between the AncQR-Arg (gray) and AncQR-Gln (orange) structures. Backbone atoms of the large domain of each protein (residues 7–95 and 193–232) were superimposed, revealing a rigid body displacement of the small domain, which corresponds to a 5.3° rotation of the small domain about the axis shown by the blue arrow. The two hinge strands (residues 94–97 and 190–195) connecting the two domains are shown in cartoon representation.
See Supplemental Experimental Procedures for further details. See also Figure S3 and Table S3.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
Contrasting Enthalpic and Entropic Modes of Binding in the AABP Family
Thermodynamic parameters for AABP-amino acid interactions were determined by ITC at 25°C. ΔHobs values represent the mean ± SD from at least three titrations. TΔS and ΔG values were calculated from mean ΔHobs and Ka values, and errors in these quantities were propagated.
(A) Distribution of ΔHobs and ΔG values for AABP-amino acid interactions showing enthalpic binding for ligands of AncQR, AncQ, seLAOBP, and ecGlnBP, and entropic binding for ligands of AncCE, AncE, and ecDEBP.
(B) Thermodynamic signatures for interactions between AncQR and l-arginine, AncQR and l-glutamine, and ecGlnBP and l-glutamine.
(C) Comparison of AncQR-Gln (orange) and ecGlnBP-Gln (green; PDB: 1WDN) complexes shows that water molecules (blue) in the AncQR-Gln complex are displaced through mutations to binding site residues.
See also Figure S4.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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