On the folding of a structurally complex protein to its metastable active state.

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a ß-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a ß-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.

Capturing the Membrane-Triggered Conformational Transition of an α-Helical Pore-Forming Toxin.

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 ß-tongue inserts first while the insertion of helix αA1 is membrane assisted. We conclude that the ß-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.

Using the folding landscapes of proteins to understand protein function.

Proteins fold on a biologically-relevant timescale because of a funnel-shaped energy landscape. This landscape is sculpted through evolution by selecting amino-acid sequences that stabilize native interactions while suppressing stable non-native interactions that occur during folding. However, there is strong evolutionary selection for functional residues and these cannot be chosen to optimize folding. Their presence impacts the folding energy landscape in a variety of ways. Here, we survey the effects of functional residues on folding by providing several examples. We then review how such effects can be detected computationally and be used as assays for protein function. Overall, an understanding of how functional residues modulate folding should provide insights into the design of natural proteins and their homeostasis.

Structural Perturbations Present in the Folding Cores of Interleukin-33 and Interleukin-1ß Correlate to Differences in Their Function.

The interleukin-1 cytokines belong to the ß-trefoil fold family and play a key role in immune responses to infections and injury. We simulate the structure-based models of two interleukin-1 cytokines, IL-33 and IL-1ß, and find that IL-33 has a lower barrier to folding than IL-1ß. We then design the folding motif (FM) of the ß-trefoil fold and identify structural deviations of IL-33 and IL-1ß from this FM. In previous work, we found that structural deviations from the FM that are large enough to lower folding free energy barriers were part of ligand binding sites. In contrast, we find that structural perturbations in IL-33 and IL-1ß which reduce the folding free energy barrier are located in the folding core and do not bind ligands. In both proteins, such core residues are interleaved with surface residues which are proximal to receptor binding sites. However, IL-33 has a lower folding barrier because its core perturbations are larger than those in IL-1ß. In order to understand the role of these core perturbations, we perform atomistic simulations of both proteins and find that the larger core perturbations may allow IL-33 to communicate signals differently across the protein. Integrating previous data, we also hypothesize that the larger IL-33 core perturbations may help accommodate its more charged binding site and may also aid in its inactivation by caspase-mediated cleavage. Together, our results suggest that protein folding landscapes are modulated not only by larger functional features such as binding sites but also by the details of protein function and fate. Furthermore, a comparative study of such landscapes may be a facile way to identify subtle differences in allosteric connectivity between two similar proteins.

In the multi-domain protein adenylate kinase, domain insertion facilitates cooperative folding while accommodating function at domain interfaces.

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.