Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which hydrophobic effect contributes to the unique and compact native state of proteins is however confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant however binds cognate DNA with only a marginally higher affinity than the wild-type, though non-trivial differences are observed in the binding to non-cognate DNA. Our work provides direct experimental evidence for the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications in protein design and functional manipulation.

The dimensions of intrinsically disordered proteins (IDPs) are sensitive to small energetic-entropic differences between intramolecular and protein–solvent interactions. This is commonly observed on modulating solvent composition and temperature. However, the inherently heterogeneous conformational landscape of IDPs is also expected to be influenced by mutations that can (de)stabilize pockets of local and even global structure, native and non-native, and hence the average dimensions. Here, we show experimental evidence for the remarkably tunable landscape of IDPs by employing the DNA-binding domain of CytR, a high-sequence-complexity IDP, as a model system. CytR exhibits a range of structure and compactness upon introducing specific mutations that modulate microscopic terms, including main-chain entropy, hydrophobicity, and electrostatics. The degree of secondary structure, as monitored by far-UV circular dichroism (CD), is strongly correlated to average ensemble dimensions for 14 different mutants of CytR and is consistent with the Uversky–Fink relation. Our experiments highlight how average ensemble dimensions can be controlled via mutations even in the disordered regime, the prevalence of non-native interactions and provide testable controls for molecular simulations.

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

Understanding how molecular interactions within the plasma membrane govern assembly, clustering, and conformational changes in single-pass transmembrane (TM) receptors has long presented substantial experimental challenges. Our previous work on activating immune receptors has combined direct biochemical and biophysical characterizations with both independent and experimentally restrained computational methods to provide novel insights into the key TM interactions underpinning assembly and stability of complex, multisubunit receptor systems. The recently published cryo-EM structure of the intact T cell receptor (TCR)-CD3 complex provides a unique opportunity to test the models and predictions arising from these studies, and we find that they are accurate, which we attribute to robust simulation environments and careful consideration of limitations related to studying TM interactions in isolation from additional receptor domains. With this in mind, we revisit results in other immune receptors and look forward to how similar methods may be applied to understand receptors for which little or no structural information is currently available.