Gagnon, I. A. | bioRxiv Science

bioRxiv2025

NMR Spectroscopy for the Validation of AlphaFold2 Structures

The introduction of AlphaFold has fundamentally changed our ability to predict the structure of proteins from their primary sequence of amino acids. As machine learning (ML) and artificial intelligence (AI) based protein prediction continues to advance, we examine the potential of hybrid techniques that combine experiment and computation that may yield more accurate structures than AI alone with significantly reduced experimental burden. We have developed heuristics comparing N-edited NOESY spectra and AlphaFold predicted structures that seek to determine whether the predicted structure reasonably describes the structure of the protein which generated the NOESY. These heuristics are fundamentally different than those used before in that they concentrate on interresidue contacts and not specific HH pairs. We present a large collection of data connecting entries across the BMRB, PDB and AlphaFold Database that includes experimentally derived structures and corresponding spectra, establishing it as a means to develop and test hybrid methods utilizing AlphaFold and NMR spectra to perform structure determination. These data test the new heuristics ability to identify inaccurate AlphaFold structures. A support vector machine was developed to test the consistency of NMR data with predicted structure and we show its application to the structure of an unsolved engineered protein, LoTOP.

biophysics↗

bioRxiv2022

HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding

To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process. Significance StatementTonB dependent transporters such as BtuB are found in the outer membranes of Gram-negative bacteria. They import scarce nutrients essential for growth, such as B12, the substrate of BtuB. Many transport steps remain enigmatic. Recent studies have emphasized force-mediated unfolding or the breakage of the "Ionic Lock", a moiety far from the B12 binding site. A strong dependence on the membrane environment has been noted. Accordingly, we measured hydrogen exchange on BtuB still embedded in native outer membranes and found that B12 binding is sufficient to break the Ionic Lock. The amino terminus then extends into the periplasm to bind TonB. But we find no evidence of pore formation, which likely requires energy transduction from the inner membrane by TonB.

biochemistry↗