Predictions and experimental validation of fold-switching proteins

National Library Of Medicine

Investigators

Linked publications & trials

Abstract

No introductory-level biochemistry textbook is complete without a chapter about how a proteins primary sequence of amino acids determines its fold. So far, nearly all computational work has focused on predicting a single protein structure from the proteins amino acid sequence. Our research challenges the one-sequence-one-structure paradigm. In 2018, we found nearly 100 examples of proteins that can adopt more than one stable fold. The structural heterogeneity of these fold-switching proteins allows them either to perform more than one function or to be highly regulated in cells. These functional changes appear to have significant relevance to human health as several fold-switching proteins are associated with human diseases such as cancer and bacterial infections. Now we are taking this research to the next level by (1) developing computational approaches to predict which amino acid sequences can switch folds and (2) testing our predictions experimentally. This year, we have furthered objective (1) by finding that it is possible to use JPRED4, a homology-based secondary structure predictor, to predict structural variation in proteins with highly similar or even identical sequences but different folds. This method is highly robust. When tested against a set of single-fold proteins, we obtain Matthews correlations coefficients between 0.7 and 0.82. We have published two papers on this topic. One was published in Biopolymers in January of 2021; the other was accepted for publication in Biopolymers 2 weeks ago. Furthermore, we have used our predictive method to accurately predict fold switching in a family of transcriptional regulators found in bacteria: RfaH/NusG. RfaH regulates the expression of virulent proteins in E. coli while NusG is a non-specific transcriptional regulator. RfaH achieves its specificity by changing the fold of its C-terminal domain (CTD). In its autoinhibited state, this CTD folds into an alpha-helical bundle, but upon binding RNA polymerase and a specific DNA sequence called ops, the CTD switches folds to a beta-barrel that binds to a ribosomal subunit, fostering efficient translation. We have clustered approximately 15,000 nonredundant RfaH/NusG sequences, and our predictive methods correspond well with their genomic annotations. Specifically, our methods predict that 97% of annotated RfaHs switch folds, and 99.5% of annotated NusGs do not. We have collected experimental evidence supporting our predictions. We have used this data to solve two NMR structures of RfaH/NusG constructs and collect additional NMR spectra. Furthermore, we have obtained circular dichroism (CD) spectra of 15 constructs and have found that all spectra were consistent with our predictions. We will submit this paper for publication in late August or early September of 2021. A draft is posted on bioRxiv. Additionally, we have identified potential fold evolution in a large family of bacterial response regulators. We are completing this analysis and will submit it for publication by the end of 2021. Furthermore, we have demonstrated that our predictive method successfully identifies fold switching in Orf9b, a SARS-CoV-2 protein shown to switch folds by another group. Our work also suggests that Orf9b homologs likely switch folds and foster disease (higher number of viral copies) using a similar mechanism. This work was published in Protein Science in May of 2021. Finally, we have written a Review of the functional roles of fold-switching proteins. It was accepted for publication in Structure and published in January of 2021.