Abstract
Protein interactions mediated by the intrinsically disordered proteins (IDPs) are generally associated with lower affinities compared to those between globular proteins. Here, we characterize the association between the intrinsically disordered HigA2 antitoxin and its globular target HigB2 toxin from Vibrio cholerae using competition ITC experiments. We demonstrate that this interaction reaches one of the highest affinities reported for IDP-target systems (K D = 3 pM) and can be entirely attributed to a short, 20-residue-long interaction motif that folds into α-helix upon binding. We perform an experimentally based decomposition of the IDP-target association parameters into folding and binding contributions, which allows a direct comparison of the binding contribution with those from globular ultra-high affinity binders. We find that the HigA2-HigB2 interface is energy optimized to a similar extent as the interfaces of globular ultra-high affinity complexes, such as barnase-barstar. Evaluation of other ultra-high affinity IDP-target systems shows that a strategy based on entropy optimization can also achieve comparably high, picomolar affinities. Taken together, these examples show how IDP-target interactions achieve picomolar affinities either through enthalpy optimization (HigA2-HigB2), resembling the ultra-high affinity binding of globular proteins, or via bound-state fuzziness and entropy optimization (CcdA-CcdB, histone H1-prothymosin α).
Highlights
In order to perform their biological function proteins interact with their partners and establish a complex network of protein-protein interactions
We found that the nanobodies Nb6 (binding to the C-terminal helix of HigB2 (Hadži et al, 2017a)) and Nb10 compete with the HigA2 binding and performed the competition ITC experiments by titrating HigA2 into the preformed nanobody-HigB2 complex
Global fitting of the direct and the competition ITC isotherms leads to the accurate estimation of the HigA2-HigB2 affinity, which is KD 16 pM at 25°C (ΔGassoc −14.7 kcal mol−1)
Summary
In order to perform their biological function proteins interact with their partners and establish a complex network of protein-protein interactions. Interactions between such well-folded proteins can be viewed through the classical paradigm of the lock-and-key mechanism, where the interaction surfaces are highly complementary and no major conformational changes are observed upon binding (Chothia and Janin, 1975). Some conformational changes can be observed upon binding of globular proteins, which have important implications for their function. The most extreme examples of conformational changes associated with protein binding can be found among the intrinsically disordered proteins (IDPs), which do not have a compact three-dimensional structure in their native state. Upon binding to target proteins IDPs become structured to varying degrees ranging from completely ordered complexes, usually involving formation of α-helices, to those which retain a high degree of disorder
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