Modelling proteins’ hidden conformations to predict antibiotic resistance

Abstract

TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM’s specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models’ prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime in vitro and in vivo. Therefore, we expect this framework to have numerous applications in drug and protein design.

Orthogonal Mass Spectrometry-Based Footprinting for Epitope Mapping and Structural Characterization: The IL-6 Receptor upon Binding of Protein Therapeutics

“…FPOP is capable of reporting on protein transient dynamics, including fast folding 42 and alteration in side-chain flexibility 43 . We also showed that FPOP reveals fast fluctuations occurring remotely upon ligand binding, which is undetectable by slower footprinting methods 24 .…”

“…The more rapid approaches offer an opportunity to understand protein dynamics and minor structural fluctuations. 43 …”

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly

“…We ran 28 simulations, each 1 μs in length, following a previously published protocol (Hart et al, 2016). Each of these simulations was initiated from chain A of PDB ID 4YPI.…”

Conformational dynamics and enzyme evolution

“…a correlation between the increased cefotaxime-degrading ability of the variants and the increased rigidity of the V-loop of TEM in the designed variants [98].…”

“…In particular, as global structural features appear to be conserved over large spans of evolutionary time [84], it is non-trivial to use simply structural comparisons to rationalize the origins of the large observed changes in activity. Bowman and coworkers have used Markov state models (MSMs) [96,97] in order to construct maps of the conformational ensembles adopted by variants of the modern TEM b-lactamase [98], in order to explore the extent to which the conformational diversity of this enzyme can play a role in modulating activity. These MSMs allowed for the identification of 'hidden' conformational states that appear to be functionally relevant in determining specificity but are not visible from static crystal structures.…”