Interpretation of Hydrogen-Deuterium Exchange Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles

Abstract Hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS) is a widely-applied biophysical technique that probes the structure and dynamics of biomolecules without the need for site-directed modifications or bio-orthogonal labels. The mechanistic interpretation of HDX data, however, is often qualitative and subjective, owing to a lack of quantitative methods to rigorously translate observed deuteration levels into atomistic structural information. To help address this problem, we have developed a methodology to generate structural ensembles that faithfully reproduce HDX-MS measurements. In this approach, an ensemble of protein conformations is first generated, typically using molecular dynamics simulations. A maximum-entropy bias is then applied post-hoc to the resulting ensemble, such that averaged peptide-deuteration levels, as predicted by an empirical model, agree with target values within a given level of uncertainty. We evaluate this approach, referred to as HDX ensemble reweighting (HDXer), for artificial target data reflecting the two major conformational states of a binding protein. We demonstrate that the information provided by HDX-MS experiments, and by the model of exchange, are sufficient to recover correctly-weighted structural ensembles from simulations, even when the relevant conformations are observed rarely. Degrading the information content of the target data, e.g., by reducing sequence coverage, by averaging exchange levels over longer peptide segments, or by incorporating different sources of uncertainty, reduces the structural accuracy of the reweighted ensemble but still allows for useful insights into the distinctive structural features reflected by the target data. Finally, we describe a quantitative metric to rank candidate structural ensembles according to their correspondence with target data, and illustrate the use of HDXer to describe changes in the conformational ensemble of the membrane protein LeuT. In summary, HDXer is designed to facilitate objective structural interpretations of HDX-MS data, and to inform experimental approaches and further developments of theoretical exchange models.