pubmed.ncbi.nlm.nih.gov

Computational design and experimental verification of a symmetric protein homodimer - PubMed

  • ️Thu Jan 01 2015

Computational design and experimental verification of a symmetric protein homodimer

Yun Mou et al. Proc Natl Acad Sci U S A. 2015.

Abstract

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.

Keywords: computational protein design; docking; homodimer; nuclear magnetic resonance.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Steps used to design the C2-symmetric homodimer. The initial scaffold, ENH, used for docking is shown in gray, and the homodimer model used for all interface designs is shown in bronze. The number of models created in each step is given in parentheses.

Fig. 2.
Fig. 2.

CD analysis of ENH-c2b. (A) CD spectrum of ENH-c2b at room temperature: solid line, before thermal denaturation; dashed line, after thermal denaturation. (B) Thermal denaturation curve measured at 222 nm: open circles, experimental data; line, fitted curve obtained using a two-state transition model.

Fig. 3.
Fig. 3.

Characterization of ENH-c2b oligomeric. (A) Sedimentation velocity experiment at 5 μM with curve fit using the c(M) (continuous distribution of molecular mass) model. (B) Tryptophan homo-FRET assay: circles, experimental data; dashed line, curve fit obtained using a monomer-dimer equilibrium model.

Fig. 4.
Fig. 4.

Solution NMR structure ensemble showing the 10 lowest energy models for the core 51 amino acids (PDB ID code 2MG4). C, C terminus.

Fig. 5.
Fig. 5.

Comparison of ENH-c2b averaged minimized solution NMR structure (green) and design model (gray). (A) Superposition of a single chain from the NMR structure and design model. (B) Alternative view of A: ∼180° of rotation about the vertical axis. (C) Superposition of the entire dimer NMR structure and design model. (D) Alternative view of C: ∼90° of rotation about the horizontal axis. (E) Superposition of the left chain of the NMR structure with the left chain of the design model showing the entire dimer structure. (F) Alternative view of E: ∼90° of rotation about the horizontal axis. N, N terminus.

Fig. 6.
Fig. 6.

NMR spectrum showing intermolecular NOE restraints obtained by a 3D 13C/15N-filtered NOESY-1H-13C-HSQC experiment. Contour plots of [ω1(1H), ω3(1H)]-strips of Ala20Cβ, Ala16Cβ, Leu39Cδ, and Leu19δ are shown. Chemical shifts indicated on the top and bottom correspond to ω2(13C) and ω3(1H) dimensions, respectively. For clarity, only the aliphatic region in the ω1(1H) dimension is shown. Unambiguous restraints identified for Ala16, Ala20, Leu19, and Leu39 residues are labeled.

Similar articles

Cited by

References

    1. Hwang H, Vreven T, Janin J, Weng Z. Protein-protein docking benchmark version 4.0. Proteins. 2010;78(15):3111–3114. - PMC - PubMed
    1. Xu D, Tsai CJ, Nussinov R. Hydrogen bonds and salt bridges across protein-protein interfaces. Protein Eng. 1997;10(9):999–1012. - PubMed
    1. Bogan AA, Thorn KS. Anatomy of hot spots in protein interfaces. J Mol Biol. 1998;280(1):1–9. - PubMed
    1. Ritchie DW. Recent progress and future directions in protein-protein docking. Curr Protein Pept Sci. 2008;9(1):1–15. - PubMed
    1. Whitehead TA, Baker D, Fleishman SJ. Computational design of novel protein binders and experimental affinity maturation. Methods Enzymol. 2013;523:1–19. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources