Extant fold-switching proteins are widespread Porter, Lauren L.; Looger, Loren L.
Proceedings of the National Academy of Sciences - PNAS,
06/2018, Letnik:
115, Številka:
23
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A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch ...folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ∼100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ∼100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5–4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health.
AlphaFold2 has revolutionized protein structure prediction by leveraging sequence information to rapidly model protein folds with atomic‐level accuracy. Nevertheless, previous work has shown that ...these predictions tend to be inaccurate for structurally heterogeneous proteins. To systematically assess factors that contribute to this inaccuracy, we tested AlphaFold2's performance on 98‐fold‐switching proteins, which assume at least two distinct‐yet‐stable secondary and tertiary structures. Topological similarities were quantified between five predicted and two experimentally determined structures of each fold‐switching protein. Overall, 94% of AlphaFold2 predictions captured one experimentally determined conformation but not the other. Despite these biased results, AlphaFold2's estimated confidences were moderate‐to‐high for 74% of fold‐switching residues, a result that contrasts with overall low confidences for intrinsically disordered proteins, which are also structurally heterogeneous. To investigate factors contributing to this disparity, we quantified sequence variation within the multiple sequence alignments used to generate AlphaFold2's predictions of fold‐switching and intrinsically disordered proteins. Unlike intrinsically disordered regions, whose sequence alignments show low conservation, fold‐switching regions had conservation rates statistically similar to canonical single‐fold proteins. Furthermore, intrinsically disordered regions had systematically lower prediction confidences than either fold‐switching or single‐fold proteins, regardless of sequence conservation. AlphaFold2's high prediction confidences for fold switchers indicate that it uses sophisticated pattern recognition to search for one most probable conformer rather than protein biophysics to model a protein's structural ensemble. Thus, it is not surprising that its predictions often fail for proteins whose properties are not fully apparent from solved protein structures. Our results emphasize the need to look at protein structure as an ensemble and suggest that systematic examination of fold‐switching sequences may reveal propensities for multiple stable secondary and tertiary structures.
Fold‐switching proteins, which remodel their secondary and tertiary structures in response to cellular stimuli, suggest a new view of protein fold space. For decades, experimental evidence has ...indicated that protein fold space is discrete: dissimilar folds are encoded by dissimilar amino acid sequences. Challenging this assumption, fold‐switching proteins interconnect discrete groups of dissimilar protein folds, making protein fold space fluid. Three recent observations support the concept of fluid fold space: (1) some amino acid sequences interconvert between folds with distinct secondary structures, (2) some naturally occurring sequences have switched folds by stepwise mutation, and (3) fold switching is evolutionarily selected and likely confers advantage. These observations indicate that minor amino acid sequence modifications can transform protein structure and function. Consequently, proteomic structural and functional diversity may be expanded by alternative splicing, small nucleotide polymorphisms, post‐translational modifications, and modified translation rates.
Fold switching suggests that protein fold space is more fluid than previously realized because homologous amino acid sequences can assume multiple folds with different secondary structure arrangements. In this figure, fold switching allows groups of homologous sequences (colored contours) to span different regions of protein fold space.
Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are ...associated with a number of diseases, including tuberculosis, cancer, Alzheimer's, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.
Fold-switching proteins respond to cellular stimuli by remodeling their secondary structures and changing their functions. Whereas several previous reviews have focused on various structural, ...physical-chemical, and evolutionary aspects of this newly emerging class of proteins, this minireview focuses on how fold switching modulates protein function and regulates biological processes. It first compares and contrasts fold switchers with other known types of proteins. Second, it presents examples of how various proteins can change their functions through fold switching. Third, it demonstrates that fold switchers can regulate biological processes by discussing two proteins, RfaH and KaiB, whose dramatic secondary structure remodeling events directly affect gene expression and a circadian clock, respectively. Finally, this minireview discusses how the field of protein fold switching might advance.
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•Fold-switching proteins remodel their secondary structures in response to stimuli•Six observed varieties of remodeling foster diverse functional changes•Fold-switching proteins regulate gene expression and a circadian clock's periodicity•Several approaches hold promise for the discovery of more fold switchers
Fold-switching proteins remodel their secondary structures and change their functions in response to cellular stimuli. Kim and Porter discuss how the dramatic conformational changes in these proteins help to regulate biological processes, such as bacterial gene expression and the periodicity of the cyanobacterial circadian clock.
Although homologous protein sequences are expected to adopt similar structures, some amino acid substitutions can interconvert α-helices and β-sheets. Such fold switching may have occurred over ...evolutionary history, but supporting evidence has been limited by the: (1) abundance and diversity of sequenced genes, (2) quantity of experimentally determined protein structures, and (3) assumptions underlying the statistical methods used to infer homology. Here, we overcome these barriers by applying multiple statistical methods to a family of ~600,000 bacterial response regulator proteins. We find that their homologous DNA-binding subunits assume divergent structures: helix-turn-helix versus α-helix + β-sheet (winged helix). Phylogenetic analyses, ancestral sequence reconstruction, and AlphaFold2 models indicate that amino acid substitutions facilitated a switch from helix-turn-helix into winged helix. This structural transformation likely expanded DNA-binding specificity. Our approach uncovers an evolutionary pathway between two protein folds and provides a methodology to identify secondary structure switching in other protein families.
Distinguishing features of fold‐switching proteins Chakravarty, Devlina; Schafer, Joseph W.; Porter, Lauren L.
Protein science,
March 2023, 2023-03-00, 20230301, Letnik:
32, Številka:
3
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Though many folded proteins assume one stable structure that performs one function, a small‐but‐increasing number remodel their secondary and tertiary structures and change their functions in ...response to cellular stimuli. These fold‐switching proteins regulate biological processes and are associated with autoimmune dysfunction, severe acute respiratory syndrome coronavirus‐2 infection, and more. Despite their biological importance, it is difficult to computationally predict fold switching. With the aim of advancing computational prediction and experimental characterization of fold switchers, this review discusses several features that distinguish fold‐switching proteins from their single‐fold and intrinsically disordered counterparts. First, the isolated structures of fold switchers are less stable and more heterogeneous than single folders but more stable and less heterogeneous than intrinsically disordered proteins (IDPs). Second, the sequences of single fold, fold switching, and intrinsically disordered proteins can evolve at distinct rates. Third, proteins from these three classes are best predicted using different computational techniques. Finally, late‐breaking results suggest that single folders, fold switchers, and IDPs have distinct patterns of residue–residue coevolution. The review closes by discussing high‐throughput and medium‐throughput experimental approaches that might be used to identify new fold‐switching proteins.
Metamorphic proteins and how to find them Porter, Lauren L.; Artsimovitch, Irina; Ramírez-Sarmiento, César A.
Current opinion in structural biology,
June 2024, 2024-06-00, 20240601, Letnik:
86
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In the last two decades, our existing notion that most foldable proteins have a unique native state has been challenged by the discovery of metamorphic proteins, which reversibly interconvert between ...multiple, sometimes highly dissimilar, native states. As the number of known metamorphic proteins increases, several computational and experimental strategies have emerged for gaining insights about their refolding processes and identifying unknown metamorphic proteins amongst the known proteome. In this review, we describe the current advances in biophysically and functionally ascertaining the structural interconversions of metamorphic proteins and how coevolution can be harnessed to identify novel metamorphic proteins from sequence information. We also discuss the challenges and ongoing efforts in using artificial intelligence-based protein structure prediction methods to discover metamorphic proteins and predict their corresponding three-dimensional structures.
The program SSDraw generates publication‐quality protein secondary structure diagrams from three‐dimensional protein structures. To depict relationships between secondary structure and other protein ...features, diagrams can be colored by conservation score, B‐factor, or custom scoring. Diagrams of homologous proteins can be registered according to an input multiple sequence alignment. Linear visualization allows the user to stack registered diagrams, facilitating comparison of secondary structure and other properties among homologous proteins. SSDraw can be used to compare secondary structures of homologous proteins with both conserved and divergent folds. It can also generate one secondary structure diagram from an input protein structure of interest. The source code can be downloaded (https://github.com/ncbi/SSDraw) and run locally for rapid structure generation, while a Google Colab notebook allows easy use.
Although most proteins conform to the classical one‐structure/one‐function paradigm, an increasing number of proteins with dual structures and functions have been discovered. In response to cellular ...stimuli, such proteins undergo structural changes sufficiently dramatic to remodel even their secondary structures and domain organization. This “fold‐switching” capability fosters protein multi‐functionality, enabling cells to establish tight control over various biochemical processes. Accurate predictions of fold‐switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Recently, we developed a prediction method for fold‐switching proteins using structure‐based thermodynamic calculations and discrepancies between predicted and experimentally determined protein secondary structure (Porter and Looger, Proc Natl Acad Sci U S A 2018; 115:5968–5973). Here we seek to leverage the negative information found in these secondary structure prediction discrepancies. To do this, we quantified secondary structure prediction accuracies of 192 known fold‐switching regions (FSRs) within solved protein structures found in the Protein Data Bank (PDB). We find that the secondary structure prediction accuracies for these FSRs vary widely. Inaccurate secondary structure predictions are strongly associated with fold‐switching proteins compared to equally long segments of non‐fold‐switching proteins selected at random. These inaccurate predictions are enriched in helix‐to‐strand and strand‐to‐coil discrepancies. Finally, we find that most proteins with inaccurate secondary structure predictions are underrepresented in the PDB compared with their alternatively folded cognates, suggesting that unequal representation of fold‐switching conformers within the PDB could be an important cause of inaccurate secondary structure predictions. These results demonstrate that inconsistent secondary structure predictions can serve as a useful preliminary marker of fold switching.
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