The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale.

How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.

Predicting multiple conformations via sequence clustering and AlphaFold2.

AlphaFold2 (ref. 1) has revolutionized structural biology by accurately predicting single structures of proteins. However, a protein's biological function often depends on multiple conformational substates2, and disease-causing point mutations often cause population changes within these substates3,4. We demonstrate that clustering a multiple-sequence alignment by sequence similarity enables AlphaFold2 to sample alternative states of known metamorphic proteins with high confidence. Using this method, named AF-Cluster, we investigated the evolutionary distribution of predicted structures for the metamorphic protein KaiB5 and found that predictions of both conformations were distributed in clusters across the KaiB family. We used nuclear magnetic resonance spectroscopy to confirm an AF-Cluster prediction: a cyanobacteria KaiB variant is stabilized in the opposite state compared with the more widely studied variant. To test AF-Cluster's sensitivity to point mutations, we designed and experimentally verified a set of three mutations predicted to flip KaiB from Rhodobacter sphaeroides from the ground to the fold-switched state. Finally, screening for alternative states in protein families without known fold switching identified a putative alternative state for the oxidoreductase Mpt53 in Mycobacterium tuberculosis. Further development of such bioinformatic methods in tandem with experiments will probably have a considerable impact on predicting protein energy landscapes, essential for illuminating biological function.

Protein-protein and protein-lipid interactions of pore-forming BCL-2 family proteins in apoptosis initiation.

Apoptosis is a common cell death program that is important in human health and disease. Signaling in apoptosis is largely driven through protein-protein interactions. The BCL-2 family proteins function in protein-protein interactions as key regulators of mitochondrial poration, the process that initiates apoptosis through the release of cytochrome c, which activates the apoptotic caspase cascade leading to cellular demolition. The BCL-2 pore-forming proteins BAK and BAX are the key executors of mitochondrial poration. We review the state of knowledge of protein-protein and protein-lipid interactions governing the apoptotic function of BAK and BAX, as determined through X-ray crystallography and NMR spectroscopy studies. BAK and BAX are dormant, globular α-helical proteins that participate in protein-protein interactions with other pro-death BCL-2 family proteins, transforming them into active, partially unfolded proteins that dimerize and associate with and permeabilize mitochondrial membranes. We compare the protein-protein interactions observed in high-resolution structures with those derived in silico by AlphaFold, making predictions based on combining experimental and in silico approaches to delineate the structural basis for novel protein-protein interaction complexes of BCL-2 family proteins.

A killer metamorphosis: catching BAK in action at the membrane.

Permeabilization of the outer mitochondrial membrane initiates apoptotic cell death. B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) and BCL-2-associated X (BAX) mediate mitochondrial poration, but how this process unfolds remains poorly defined. Two studies in this issue investigate the transition of dormant, inactive BAK monomer to a highly dynamic membrane-associated, pore-forming oligomer.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-ß component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.