A chemical approach to the construction of multimeric protein assemblies

Chemical and enzymatic ligation approaches provide a general route to the synthesis of proteins whose primary structure is not restricted to the genetic code. Thus, one can replace key residues in a protein with unnatural amino acids whose chemical or structural properties can be tailored to probe specific aspects of function. In addition, isotopically labeled amino acids can be incorporated at one or more positions within the primary structure of the protein, thereby allowing highly focused structural studies of the molecule using techniques such as multidimensional NMR spectroscopy. This new-found freedom will certainly afford expanded opportunities in established areas of protein chemistry such as structure–function related studies. However, simple amino acid substitutions of this type represent only the most elementary application of these enabling technologies. Chemical ligation, in particular, has the potential to make significant contributions in areas of biology that are currently completely inaccessible to researchers. Indeed, it is in the creation of pragmatic new chemistry-driven strategies for the study of complex biological systems that chemical ligation may be of most value.Chemical ligation is already finding a growing application in the construction of covalently tethered protein complexes designed to mimic or artificially recreate a naturally occurring multiprotein complex. Chemically stabilized protein molecules of this type are of genuine importance, as non-covalent protein complexes are frequently difficult to isolate from natural sources and are often problematic to study in vitro. The artificial protein assemblies illustrated in Figure 2Figure 2 utilize both non-native topology and non-amide bonding in their design, and were intended to mimic a naturally occurring system. In the first example, the functionally critical cytoplasmic domain of the integrin αIIb β3 is modeled using a specifically designed synthetic protein hybrid [12xDesign and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin αIIb β3 (platelet GPIIb-IIIa). Muir, T.W., Williams, M.J., Ginsberg, M.H., and Kent, S.B.H. Biochemistry. 1994; 33: 7701–7708Crossref | PubMedSee all References][12]. Integrins are members of a heterodimeric transmembrane receptor superfamily, which also includes the cytokine and immunoglobulin-like receptor families. Generation of soluble truncated versions of these receptors for structural studies is complicated by the non-covalent nature of the subunit association; in the case of αIIb β3 the individual truncated α and β cytoplasmic tails will not associate to form a functional complex. The synthetic model protein was therefore designed to be highly soluble, allowing structural analysis, and to contain structural elements which would promote the association of the two cytoplasmic tails. This was achieved by covalently linking both cytoplasmic tails via a parallel amphiphilic helical coiled-coil, incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint, fixing the tails in a parallel and proximal orientation (Figure 2aFigure 2a). The 126 amino acid soluble model protein contains two C termini and was readily synthesized from two peptide building blocks using a thioether-based ligation strategy. Structural studies suggest that the cytoplasmic tails within the model protein interact through a defined tertiary structure [12xDesign and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin αIIb β3 (platelet GPIIb-IIIa). Muir, T.W., Williams, M.J., Ginsberg, M.H., and Kent, S.B.H. Biochemistry. 1994; 33: 7701–7708Crossref | PubMedSee all References][12] and that this may represent a biologically relevant conformation (unpublished data).Figure 2Synthetic proteins of non-linear architecture produced by chemical ligation. Both molecules are color coded to indicate the peptide fragments from which they were constructed. (a) Soluble model of the cytoplasmic domain of the integrin αIIb β3. The molecule contains two C termini and was constructed from the unprotected peptides, Cys-helix-αIIb (50 residues) and BrAc-helix-β3 (76 residues; BrAc=bromoacetyl), via a thioether-forming ligation strategy [12xDesign and chemical synthesis of a neoprotein structural model for the cytoplasmic domain of a multisubunit cell-surface receptor: integrin αIIb β3 (platelet GPIIb-IIIa). Muir, T.W., Williams, M.J., Ginsberg, M.H., and Kent, S.B.H. Biochemistry. 1994; 33: 7701–7708Crossref | PubMedSee all References][12]. (b) Covalently linked c-Myc–Max heterodimer (b, basic region; H, helix) constructed from four peptide fragments (30–50 residues long) by convergent chemical ligation [18xTotal chemical synthesis of a unique transcription factor-related protein: c-Myc–Max. Canne, L.E., Ferre-D'Amare, A.R., Burley, S.K., and Kent, S.B.H. J. Am. Chem. Soc. 1995; 117: 2998–3007CrossrefSee all References][18]. The individual c-Myc and Max subunits were first constructed using the thioester approach, and subsequently linked together using a second oxime-forming ligation chemistry (see Table 1Table 1 for details on each chemistry). The two ligation chemistries are mutually exclusive, allowing fully unprotected peptides to be used throughout the assembly.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn the second example, a unique transcription factor protein, c-Myc–Max, was chemically synthesized from four peptide building blocks using a modular chemical ligation strategy [18xTotal chemical synthesis of a unique transcription factor-related protein: c-Myc–Max. Canne, L.E., Ferre-D'Amare, A.R., Burley, S.K., and Kent, S.B.H. J. Am. Chem. Soc. 1995; 117: 2998–3007CrossrefSee all References][18]. Non-covalent heterodimers of the c-Myc and Max DNA-binding domains, which consist of a basic region followed by a helix-loop-helix and then a leucine zipper, are believed to be functionally important transcription factors in vivo, but it has not been possible to make the heterodimer by means of standard recombinant DNA technology. Thioester-forming ligation reactions were initially used to assemble the individual c-Myc and Max domains, which were then stitched together in a regioselective manner using an oxime-based ligation approach. The resulting covalently linked 20 kDa c-Myc–Max heterodimer contained two N termini (Figure 2bFigure 2b) and was found to bind promoter DNA in a sequence-specific manner.The two examples outlined above demonstrate that chemical ligation provides the means to generate artificial versions of protein complexes, which allow the biochemical and structural properties of the system to be evaluated, often for the first time. This unique access to systems of unnatural topology in principle allows large multisubunit protein complexes to be dissected into functionally discrete modules, permitting the biology and structure of each to be studied. Thus, the continuing integration of chemistry and biology through techniques such as chemical ligation is likely to have a huge impact on both established and embryonic areas of study in both parent fields.