Computational design and experimental verification of a symmetric protein homodimer

Homodimers are the most common type of protein assembly in nature and have distinct features compared with heterodimers and higher order oligomers. Understanding homodimer interactions at the atomic level is critical both for elucidating their biological mechanisms of action and for accurate modeling of complexes of unknown structure. Computation-based design of novel protein–protein interfaces can serve as a bottom-up method to further our understanding of protein interactions. Previous studies have demonstrated that the de novo design of homodimers can be achieved to atomic-level accuracy by β-strand assembly or through metal-mediated interactions. Here, we report the design and experimental characterization of a α-helix–mediated homodimer with C2 symmetry based on a monomeric Drosophila engrailed homeodomain scaffold. A solution NMR structure shows that the homodimer exhibits parallel helical packing similar to the design model. Because the mutations leading to dimer formation resulted in poor thermostability of the system, design success was facilitated by the introduction of independent thermostabilizing mutations into the scaffold. This two-step design approach, function and stabilization, is likely to be generally applicable, especially if the desired scaffold is of low thermostability

Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

<div><p>Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (<em>Ipomoea batatas</em> cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (K_A) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense.</p> </div

Determination of tetrameric IPO by gel-filtration chromatography.

<p>The HiLoad™ 16/60 Superdex™ 200 column was pre-equilibrated with a running buffer containing 27 mM Tris-HCl (pH 7.0) and 2 M NaCl with and without 0.2 M Me-Glc or 1 M glucose at a flow rate of 0.6 ml/min. The elution profiles were monitored at 280 nm. (<b>A</b>) Peak 3 represents the IPO protein dissolved in the running buffer without carbohydrates and eluted at 108.2 ml. Peak 2 represents the IPO protein dissolved in the running buffer with 0.2 M Me-Glc and eluted at 80.5 ml. Peak 1 represents the IPO protein dissolved in running buffer with 1 M glucose and eluted at 78.8 ml. The retarded results from peaks 1 and 2 show that the IPO protein could bind to the dextran of the Superdex 200 column. The retarded phenomenon of IPO could be complemented by 1 M glucose. Peak 3 was found with an estimated molecular mass of 63.2 kDa corresponding to a tetramer with 69.2 kDa. (<b>B</b>) To determine the role of the N terminus of IPO protein in tetramerization, a truncated IPO by removing residues 1 to 10 was prepared. The proteins were dissolved in the running buffer with 1 M glucose. Peak 1 represents the native IPO and was eluted at 78.8 ml. Peak 2 represents the truncated IPO and was eluted at 89.6 ml. Peak 2 was calculated with a molecular mass of 22.0 kDa corresponding to a truncated monomer with 16.3 kDa. (<b>C</b>) The standard markers from BioRad containing thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa) were used to calculate the equation of linear regression. The X-axis represents the elution volume and Y-axis the log value of molecular mass from the standard markers. The equation is y = -0.0423x+5.1333 and R² = 0.9847.</p

ITC binding assay of wildtype IPO (tetremeric IPO) with carbohydrates.

<p>For the methylated carbohydrates, the thermal changes were detected with 1 mM wildtype IPO which was titrated by 25 mM Me-Man (<b>A</b>), 25 mM Me-Glc (<b>B</b>), 25 mM Me-Gal (<b>C</b>). For the non-methylated carbohydrates, the thermal changes were detected with 3 mM IPO which was titrated by 75 mM Man (<b>D</b>), 75 mM Glc (<b>E</b>) and 75 mM Gal (<b>F</b>). The upper panel of figures was presented by 18 injections and 2 µl/per injection. The interval of injection time is 180 sec. The 18 experimental data were almost fitted for a 1∶1 binding model (one-site of fitting) with Microcal Origin 7.0 software (the bottom panel). In each bottom panel, X-axis indicates the molar ratio of protein-carbohydrate and Y-axis indicates the thermal change in each injection.</p

Hydrogen bond networks between carbohydrate binding pocket of chain A and N terminus of chain B.

<p>The carbohydrate binding pocket of chain A is shown in blue, and the N terminus of chain B is shown in magenta. The monosaccharide Me-Gal is orange. Four hydrogen bonds are formed: 2 by the atom OD1 of Asn19 (chain A) and the atoms ND1 and N of His 8 (chain B); 1 by the atom N of Asn19 (chain A) and the atom O of Leu5 (chain B); and 1 by the atom ND2 of Asn139 (chain A) and the atom O of His8 (chain B).</p

Electron density of carbohydrates from the structures of IPO–Me-Glc and IPO–Me-Man, IPO–Me-Gal.

<p>These maps are contoured at 1.0 σ 2fofc electron density. The residues interacting with carbohydrates are highlighted. The carbohydrates Me-Glc only in chain A (<b>A</b>), Me-Glc with cadmium ion in chain B–D (<b>B</b>); Me-Man (<b>C</b>) and Me-Gal (<b>D</b>) form the hydrogen-bonding interactions (the yellow dashed lines) with the residues Gly21, Tyr97, Gly141, Trp142, Tyr143 and Asp145 of IPO.</p

Packing diagram of apo ipomoelin (IPO) (A) and in complex with (B) methyl α-D-mannopyranoside (Me-Man), (C) methyl α-D-glucopyranoside (Me-Glc) and (D) methyl α-D-galactopyranoside (Me-Gal).

<p>The resolved IPO structures are in green and the molecules in pink or in light green were generated by symmetric operations. (<b>A</b>) The packing diagram of apo IPO with 5 molecules in green. Four of 5 molecules form a tetramer and the 5th molecule can form another tetramer by the red one, the yellow one and the blue one in the center. The molecules in red, yellow and blue are generated by symmetric operations (-X, Y, -Z), (X, -Y, -Z), and (-X-1, -Y, Z). (<b>B</b>) The resolved IPO–Me-Man complex in green is 2 molecules in an asymmetric unit. However, the other 2 molecules in red are generated by the symmetric operation (X, -Y, -Z) to form a tetramer in the center. (<b>C</b>) The resolved IPO–Me-Glc complex is a tetrameric form, and (<b>D</b>) the IPO–Me-Gal complex is also a tetramer. All packing diagrams reveal its tetrameric nature.</p

ITC binding assay ofΔN10IPO (monomeric IPO) with methylated carbohydrates.

<p>The thermal changes were detected by 0.5 mM ΔN10IPO with 12.5 mM Me-Man (<b>A</b>), 0.75 mM ΔN10IPO with 20 mM Me-Glc (<b>B</b>) and 1 mM ΔN10IPO with 25 mM Me-Gal (<b>C</b>). The upper panel of figures was presented by 18 injections and 2 µl/per injection. The interval of injection time is 180 sec. The 18 experimental data were almost fitted for a 1∶1 binding model (one-site of fitting) with Microcal Origin 7.0 software (the bottom panel). In each bottom panel, X-axis indicates the molar ratio of protein-carbohydrate and Y-axis indicates the thermal change in each injection. In the titration of ΔN10IPO with Me-Gal, no obvious thermal changes could be detected.</p

Thermodynamics values of IPO and ΔN10IPO titrated with various carbohydrates^*.

*<p>Triple repeats were analyzed and the values represented the average with standard errors in parenthesis.</p>a<p>The n value was fixed at 1.0 for fitting the curves.</p><p>ND represents not determined.</p