Design of Heterotrimeric Collagen Triple Helices
Hartgerink, Jeffrey D.
Doctor of Philosophy
Select loci in native collagen display clusters of contiguous amino acids that recognize a diverse array of extracellular matrix (ECM) and blood serum proteins critical for homeostasis and hemostasis. The mechanism of collagen binding to these proteins has primarily been elucidated using short peptides, called collagen mimetic peptides (CMPs), that independently fold into the so called homotrimeric collagen triple helices, where all three peptide chains have identical amino acid sequence. However, the homotrimer binding mechanism cannot be extrapolated to explain protein binding in AAB and ABC-type heterotrimeric collagens that contain either two or three unique polypeptide chains without significant speculation. Given the requirement of a one amino acid offset between the peptide chains in a collagen triple helix, a mixture of two or three unique peptides can self-assemble into 8 and 27 competing triple helices, respectively. Heterotrimeric CMPs have remained synthetically inaccessible due to the challenge associated with introducing bias in this ensemble of competing states. Previously, Hartgerink lab employed axial Lys – Asp / Glu salt-bridges to successfully self-assemble an ABC heterotrimer. Here, we extend this paradigm to successfully demonstrate the design of a proof-of-principle AAB heterotrimer. Four AAB heterotrimers, each carrying unpaired Lys, Asp or Glu and a combination of Lys – Asp or Lys – Glu axial salt-bridges, were designed. Of these, only the heterotrimer containing unpaired Glu and a combination of Lys – Asp as well as Lys – Glu salt-bridges successfully self-assembled into an AAB heterotrimer. Next, a general methodology to self-assemble AAB heterotrimers containing the α2β1 and α1β1-integrin recognition sequences from collagen I and IV, respectively, and the matrix metalloproteinase-1 cleavage sequence from collagen I was developed. The protein recognition sequences were included as guests in a host peptide sequence containing a network of salt-bridges that bias the ensemble of competing triple helices to the desired AAB heterotrimer. Successful self-assembly of heterotrimers across multiple guest sequences was observed, which demonstrates the wide applicability of the host sequence design. In future, binding of these heterotrimers to the ECM and blood serum proteins has the potential to unravel the mechanism of disease evolution across multiple disease settings. We also extended the salt-bridge based design paradigm to synthesize a new class of CMP constructs. Hydroxyproline-free collagen triple helices are lucrative for expression in bacterial systems. Using a combination of Lys – Asp and Lys – Glu salt-bridges, a hydroxyproline-free ABC collagen heterotrimer was successfully designed. Remarkably, this ABC heterotrimer was stable despite one the peptides containing no proline or hydroxyproline, a requirement previously thought to be critical for stability. Additionally, an ABC heterotrimer containing a non-canonical four residue offset between the peptide chains was designed. In this heterotrimer, the non-covalent interactions at the termini are unsatisfied which renders them “sticky” to further assembly. This design lays the groundwork to create longer and therefore, stickier offsets to facilitate self-assembly of collagen-mimetic nanofibers.
Collagen mimetic peptide; AAB-type collagen heterotrimers; ABC-type collagen heterotrimers; Axial salt-bridge interaction; Lateral salt-bridge interaction; More... Collagen NMR spectroscopy; A2b1 integrin I-domain; A1b1-integrin I-domain; Von Willebrand factor; A3-domain; Matrix metalloproteinase-1 (mmp-1); Hydroxyproline-free collagen; Sticky-ended collagen; Collagen nanofibers Less...