Selective chain recognition in the C-terminal α-helical coiled-coil region of laminin

Recombinant fragments α, β, and γ were prepared comprising about 200 C-terminal residues of the corresponding polypeptide chains in the three-stranded α-helical coiled-coil domain of laminin. Circular dichroism spectra, thermal transition profiles, non-denaturing gels, analytical ultracentrifugation, and calorimetry indicated α-helicity and high thermal stabilities for the βγ heterodimer and homoassociates of β. Very little or no coiled-coil formation was found for α and γ. The thermal melting profiles and their concentration dependencies were quantitatively described by a two-state mechanism in which two unfolded chains combine to a fully α-helical dimer. For the βγ dimer the melting temperature was T(m) = 42°C at a chain concentration of 25 μM in 5 mM sodium phosphate buffer (pH 7.4). Addition of 100 mM NaCl decreased the T(m) slightly but the relative stability of βγ and ββ coiled-coils was not significantly changed, indicating that electrostatic interactions alone are not responsible for chain selection. Upon addition of 1 M urea the T(m) value dropped by about 10°C. The enthalpy changes for the formation of the coiled-coil were ΔH(o) = -304 (±30) kJ/mol for the βγ heterodimer and -198 (±20) kJ/mol for the β-homoassociates. Gibbs free energies and entropies amounted to ΔG(o) = -42.8 kJ and ΔS(o) = -876 J/mol K for the heteroassembly and -37.8 kJ/mol and -537 J/mol K for the homoassembly of β. This low preference for heteroassociation of the fragment is smaller than the chain selectivity observed for larger fragments and intact laminin. Deletion of ten residues from the C-terminal region of the γ-fragment which were recently reported as an essential assembly-site was not sufficient to abolish heteroassociation. Interaction of α-fragment with double-stranded βγ coiled-coils reflected the formation of a three-stranded coiled-coil in laminin but for the small recombinant fragments association between α and β-homoassociates was also observed. The C-terminal 100 residues in the coiled-coil domain are therefore not alone responsible for the high specificity of chain selection in laminin.