title: One-Pot N2C/C2C/N2N Ligation To Trap Weak Protein-Protein Interactions.
abstract: Weak transient protein-protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity-induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N-terminus to N-terminus) and C2C (C-terminus to C-terminus) ligation approaches. By using N2C (N-terminus to C-terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t1/2 of 4.8 min and near quantitative conversion. The Ypt1-GDI conjugate revealed that binding of Ypt1 G-domain causes opening of the lipid-binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.
authors.affiliation: Chemical Genomics Center of the Max Planck Society, Otto-Hahn-Strasse 15, 44227, Dortmund, Germany.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 6, 44227, Dortmund, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Ehrt
authors.affiliation: TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Strasse 6, 44227, Dortmund, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Koch
authors.affiliation: Chemical Genomics Center of the Max Planck Society, Otto-Hahn-Strasse 15, 44227, Dortmund, Germany. yaowen.wu@mpi-dortmund.mpg.de.
authors.forename: Yao-Wen
authors.initials: YW
authors.lastname: Wu
mesh_terms: D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020730:Guanine Nucleotide Dissociation Inhibitors; D029701:Saccharomyces cerevisiae Proteins; C060755:YPT1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins
keywords: C-terminal 1,2-aminothiol; N-terminal thioester; Ras homodimer; native chemical ligation; protein-protein interactions