Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.

Escherichia coli cytolysin A (ClyA) is an α-helical pore-forming toxin (PFT) which lyses target cells by forming membrane permeabilizing pores. The rate-determining step of this process is the conversion of the soluble ClyA monomer into a membrane inserted protomer. We elucidate the mechanism of this conformational transition using molecular dynamics simulations of coarse-grained models of ClyA and a membrane. We find that a membrane is necessary for the conformational conversion because membrane-protein interactions counteract the loss of the many intraprotein hydrophobic interactions that stabilize the membrane-inserting segments in the ClyA monomer. Of the two membrane-inserting segments, the flexible and highly hydrophobic β-tongue inserts first while the insertion of helix αA1 is membrane assisted. We conclude that the β-tongue is designed to behave as a quick-response membrane sensor, while helix αA1 improves target selectivity for cholesterol-containing cell membranes by acting as a fidelity check.

Natural proteins have evolved amino acid sequences that provide a native‐structural bias to folding. Structure‐based models (SBMs) of proteins ignore all non‐native interactions and encode this bias by including only interactions present in the native state. SBMs have been remarkably successful at describing the folding mechanisms and folding rates of natural proteins. Non‐native interaction‐enhanced SBMs (eSBMs) of proteins have been developed to understand non‐native traps observed experimentally in several contexts (due to binding sites in the protein, in the crowded environment of the cell, etc.). The mathematical description of the non‐native interactions is problem specific and different in each eSBM. We review the commonly used eSBMs and make a detailed comparison of their non‐native potential energy terms. Designed proteins have not had the benefit of evolution and are more likely to have non‐native intermediates. Experiments have detected the presence of non‐native interactions in the folding kinetics of the designed protein Top7. We compare the intermediate ensembles obtained from folding simulations of Top7 using three different eSBMs and comment on the robustness of these intermediates to changes in the form of non‐native interactions. Although the population of some intermediates is dependent upon the nature of non‐native interactions, the consensus structural information that arises from the different eSBMs is consistent with experiments. Furthermore, the intermediate ensembles indicate the biochemical basis for the experimentally observed non‐native interactions. In summary, eSBMs, as a group, are likely to be a powerful tool for understanding the structural and biochemical basis of non‐native interactions in protein folding.