Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

We describe a platform utilizing two methods based on hydrogen–deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein–ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides <i>site-specific</i> binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, Δ<i>C</i>_1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein–ligand interactions

Continuous and Pulsed Hydrogen–Deuterium Exchange and Mass Spectrometry Characterize CsgE Oligomerization

We report the use of hydrogen–deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE

The Binding of Apolipoprotein E to Oligomers and Fibrils of Amyloid‑β Alters the Kinetics of Amyloid Aggregation

Deposition of amyloid-β (Aβ) in Alzheimer’s disease (AD) is strongly correlated with the <i>APOE</i> genotype. However, the role of apolipoprotein E (apoE) in Aβ aggregation has remained unclear. Here we have used different apoE preparations, such as recombinant protein or protein isolated from cultured astrocytes, to examine the effect of apoE on the aggregation of both Aβ_1–40 and Aβ_1–42. The kinetics of aggregation, measured by the loss of fluorescence of tetramethylrhodamine-labeled Aβ, is shown to be dramatically slowed by the presence of substoichiometric concentrations of apoE. Using these concentrations, we conclude that apoE binds primarily to and affects the growth of oligomers that lead to the nuclei required for fibril growth. At higher apoE concentrations, the protein also binds to Aβ fibrils, resulting in fibril stabilization and a slower rate of fibril growth. The aggregation of Aβ_1–40 is dependent on the apoE isoform, being the most dramatic for apoE4 and less so for apoE3 and apoE2. Our results indicate that the detrimental role of apoE4 in AD could be related to apoE-induced stabilization of the soluble but cytotoxic oligomeric forms and intermediates of Aβ, as well as fibril stabilization

ApoE: In Vitro Studies of a Small Molecule Effector

Apolipoprotein E4 (apoE4), one of three isoforms of apoE, is the major risk factor for developing late onset Alzheimer’s disease. The only differences among these isoforms (apoE2, apoE3, and apoE4) are single amino acid changes. Yet these proteins are functionally very different. One approach to ameliorating the effect of apoE4 with respect to Alzheimer’s disease would be to find small molecular weight compounds that affect the behavior of apoE4. Few studies of this approach have been carried out in part because there was no complete structure of any full-length apoE isoform until 2011. Here, we focus on one small molecular weight compound, EZ-482, and explore the effects of its binding to apoE. Using hydrogen–deuterium exchange, we determined that EZ-482 binds to the C-terminal domains of both apoE3 and apoE4. The binding to apoE4, however, is accompanied by a unique N-terminal allosteric effect. Using fluorescence methods, we determined an apparent dissociation constant of approximately 8 μM. Although EZ-482 binds to the C-terminal domain, it blocks heparin binding to the N-terminal domain. The residues of apoE that bind heparin are the same as those involved in apoE binding to LDL and LRP-1 receptors. The methods and the data presented here may serve as a template for future studies using small molecular weight compounds to modulate the behavior of apoE