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Control of repeat-protein curvature by computational protein design.

Park K, Shen BW, Parmeggiani F, Huang PS, Stoddard BL, Baker D - Nat. Struct. Mol. Biol. (2015)

Bottom Line: First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures.Second, a set of junction modules that connect the different building blocks are designed.Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington, USA. [2] Institute for Protein Design, University of Washington, Seattle, Washington, USA.

ABSTRACT
Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature.

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Building block and junction module design. (a) Junction module design illustrated by the L24→L28 fusion design. (b) Schematic representing a two-unit junction module JNBBi→BBj and a three-unit wedge module JNBBi→BBw→BBi. (c) The idealized building block modules for L22, L24, and {L28→L29}. (d) The junction modules for L22→L24, L24→L22, L22→L28 (DLRR_F6), L24→L28 (DLRR_G2), and L24→L32→L24 (DLRR_H2). For each design, module organization, model structure, far-UV CD spectra (mean residue ellipticity, [θ] × 10−3 deg cm2 dmol−1), and thermal denaturation profile following the wavelength 218 nm are displayed from left to right. When multiple designs exist, data of the most stable one are shown. The backbone modules are indicated in different colors: magenta-L22, green-L24, yellow-L28, and orange-L29 (Fig. 1c). The designed junction modules are represented by dotted lines, and the N-terminal capping domain connected to L22 or L24 is shown in gray.
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Figure 2: Building block and junction module design. (a) Junction module design illustrated by the L24→L28 fusion design. (b) Schematic representing a two-unit junction module JNBBi→BBj and a three-unit wedge module JNBBi→BBw→BBi. (c) The idealized building block modules for L22, L24, and {L28→L29}. (d) The junction modules for L22→L24, L24→L22, L22→L28 (DLRR_F6), L24→L28 (DLRR_G2), and L24→L32→L24 (DLRR_H2). For each design, module organization, model structure, far-UV CD spectra (mean residue ellipticity, [θ] × 10−3 deg cm2 dmol−1), and thermal denaturation profile following the wavelength 218 nm are displayed from left to right. When multiple designs exist, data of the most stable one are shown. The backbone modules are indicated in different colors: magenta-L22, green-L24, yellow-L28, and orange-L29 (Fig. 1c). The designed junction modules are represented by dotted lines, and the N-terminal capping domain connected to L22 or L24 is shown in gray.

Mentions: Genes were synthesized for proteins containing 5 to 7 idealized building block modules. The N–terminal capping domain of internalin B was fused to DLRR_A and DLRR_B to enhance protein solubility and expression12,20 whereas DLRR_C was expressed without a capping motif; instead the sequences of the N and C terminal repeats were redesigned to eliminate exposed hydrophobic residues. The idealized repeat designs were expressed in E.coli and found to be soluble and to have high thermal stability (Fig. 2c).


Control of repeat-protein curvature by computational protein design.

Park K, Shen BW, Parmeggiani F, Huang PS, Stoddard BL, Baker D - Nat. Struct. Mol. Biol. (2015)

Building block and junction module design. (a) Junction module design illustrated by the L24→L28 fusion design. (b) Schematic representing a two-unit junction module JNBBi→BBj and a three-unit wedge module JNBBi→BBw→BBi. (c) The idealized building block modules for L22, L24, and {L28→L29}. (d) The junction modules for L22→L24, L24→L22, L22→L28 (DLRR_F6), L24→L28 (DLRR_G2), and L24→L32→L24 (DLRR_H2). For each design, module organization, model structure, far-UV CD spectra (mean residue ellipticity, [θ] × 10−3 deg cm2 dmol−1), and thermal denaturation profile following the wavelength 218 nm are displayed from left to right. When multiple designs exist, data of the most stable one are shown. The backbone modules are indicated in different colors: magenta-L22, green-L24, yellow-L28, and orange-L29 (Fig. 1c). The designed junction modules are represented by dotted lines, and the N-terminal capping domain connected to L22 or L24 is shown in gray.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4318719&req=5

Figure 2: Building block and junction module design. (a) Junction module design illustrated by the L24→L28 fusion design. (b) Schematic representing a two-unit junction module JNBBi→BBj and a three-unit wedge module JNBBi→BBw→BBi. (c) The idealized building block modules for L22, L24, and {L28→L29}. (d) The junction modules for L22→L24, L24→L22, L22→L28 (DLRR_F6), L24→L28 (DLRR_G2), and L24→L32→L24 (DLRR_H2). For each design, module organization, model structure, far-UV CD spectra (mean residue ellipticity, [θ] × 10−3 deg cm2 dmol−1), and thermal denaturation profile following the wavelength 218 nm are displayed from left to right. When multiple designs exist, data of the most stable one are shown. The backbone modules are indicated in different colors: magenta-L22, green-L24, yellow-L28, and orange-L29 (Fig. 1c). The designed junction modules are represented by dotted lines, and the N-terminal capping domain connected to L22 or L24 is shown in gray.
Mentions: Genes were synthesized for proteins containing 5 to 7 idealized building block modules. The N–terminal capping domain of internalin B was fused to DLRR_A and DLRR_B to enhance protein solubility and expression12,20 whereas DLRR_C was expressed without a capping motif; instead the sequences of the N and C terminal repeats were redesigned to eliminate exposed hydrophobic residues. The idealized repeat designs were expressed in E.coli and found to be soluble and to have high thermal stability (Fig. 2c).

Bottom Line: First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures.Second, a set of junction modules that connect the different building blocks are designed.Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington, USA. [2] Institute for Protein Design, University of Washington, Seattle, Washington, USA.

ABSTRACT
Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high-affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat-protein scaffolds and apply it to leucine-rich-repeat proteins. First, self-compatible building-block modules are designed that, when polymerized, generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom-designed shapes are generated by appropriately combining building-block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat-protein curvature.

Show MeSH