Analytical and functional aspects of protein-ligand interactions: Beyond induced fit and conformational selection.

Ligand-dependent changes in protein conformation are foundational to biology. Historical mechanistic models for substrate-specific proteins are induced fit (IF) and conformational selection (CS), which invoke a change in protein conformation after ligand binds or before ligand binds, respectively. These mechanisms have important, but rarely discussed, functional relevance because IF vs. CS can differentially affect a protein's substrate specificity or promiscuity, and its regulatory properties. The modern view of proteins as conformational ensembles in both ligand free and bound states, together with the realization that most proteins exhibit some substrate promiscuity, demands a deeper interpretation of the historical models and provides an opportunity to improve mechanistic analyses. Here we describe alternative analytical strategies for distinguishing the historical models, including the more complex expanded versions of IF and CS. Functional implications of the different models are described. We provide an alternative perspective based on protein ensembles interacting with ligand ensembles that clarifies how a single protein can 'apparently' exploit different mechanisms for different ligands. Mechanistic information about protein ensembles can be optimized when they are probed with multiple ligands.

Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs.

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the ß1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Hydrogen-deuterium exchange mass spectrometry of membrane proteins in lipid nanodiscs.

Hydrogen deuterium exchange mass spectrometry (H/DX MS) provides a quantitative comparison of the relative rates of exchange of amide protons for solvent deuterons. In turn, the rate of amide exchange depends on a complex combination of the stability of local secondary structure, solvent accessibility, and dynamics. H/DX MS has, therefore, been widely used to probe structure and function of soluble proteins, but its application to membrane proteins was limited previously to detergent solubilized samples. The large excess of lipids from model membranes, or from membrane fractions derived from in vivo samples, presents challenges with mass spectrometry. The lipid nanodisc platform, consisting of apolipoprotein A-derived membrane scaffold proteins, provides a native like membrane environment in which to capture analyte membrane proteins with a well defined, and low, ratio of lipid to protein. Membrane proteins in lipid nanodiscs are amenable to H/DX MS, and this is expected to lead to a rapid increase in the number of membrane proteins subjected to this analysis. Here we review the few literature examples of the application of H/DX MS to membrane proteins in nanodiscs. The incremental improvements in the experimental workflow of the H/DX MS are described and potential applications of this approach to study membrane proteins are described.

Human kidney on a chip assessment of polymyxin antibiotic nephrotoxicity.

Drug-induced kidney injury, largely caused by proximal tubular intoxicants, limits development and clinical use of new and approved drugs. Assessing preclinical nephrotoxicity relies on animal models that are frequently insensitive; thus, potentially novel techniques - including human microphysiological systems, or "organs on chips" - are proposed to accelerate drug development and predict safety. Polymyxins are potent antibiotics against multidrug-resistant microorganisms; however, clinical use remains restricted because of high risk of nephrotoxicity and limited understanding of toxicological mechanisms. To mitigate risks, structural analogs of polymyxins (NAB739 and NAB741) are currently in clinical development. Using a microphysiological system to model human kidney proximal tubule, we exposed cells to polymyxin B (PMB) and observed significant increases of injury signals, including kidney injury molecule-1 KIM-1and a panel of injury-associated miRNAs (each P < 0.001). Surprisingly, transcriptional profiling identified cholesterol biosynthesis as the primary cellular pathway induced by PMB (P = 1.22 ×10-16), and effluent cholesterol concentrations were significantly increased after exposure (P < 0.01). Additionally, we observed no upregulation of the nuclear factor (erythroid derived-2)-like 2 pathway, despite this being a common pathway upregulated in response to proximal tubule toxicants. In contrast with PMB exposure, minimal changes in gene expression, injury biomarkers, and cholesterol concentrations were observed in response to NAB739 and NAB741. Our findings demonstrate the preclinical safety of NAB739 and NAB741 and reveal cholesterol biosynthesis as a potentially novel pathway for PMB-induced injury. To our knowledge, this is the first demonstration of a human-on-chip platform used for simultaneous safety testing of new chemical entities and defining unique toxicological pathway responses of an FDA-approved molecule.

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs.

Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3-12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F'- and G'-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4-membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.