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.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125;
Yun
Y
Mou
Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, CA 91125;
Po-Ssu
PS
Huang
Technology Commons, College of Life Science and Center for Systems Biology, National Taiwan University, Taipei 10617, Taiwan;
Fang-Ciao
FC
Hsu
Instrumentation Center, College of Science, National Taiwan University, Taipei 10617, Taiwan; shingjonghuang@ntu.edu.tw steve@mayo.caltech.edu.
Shing-Jong
SJ
Huang
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125 shingjonghuang@ntu.edu.tw steve@mayo.caltech.edu.
Stephen L
SL
Mayo
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry; D006358:Hot Temperature; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D025941:Protein Interaction Mapping; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012996:Solutions; D014157:Transcription Factors / Q000737:chemistry
D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D012996:Solutions; D014157:Transcription Factors
computational protein design; docking; homodimer; nuclear magnetic resonance