Subscriber access provided by Caltech Library
Article
Peptide-level interactions between proteins and smallmolecule drug candidates by HDX-MS, PLIMSTEX and modified SUPREX: the example of ApoE3 Hanliu Wang, Don L. Rempel, Daryl E. Giblin, Carl Frieden, and Michael L. Gross Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01121 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Peptide-level interactions between proteins and small-molecule drug candidates by HDX-MS, PLIMSTEX and modified SUPREX: the example of ApoE3 Hanliu Wang1,3, Don L. Rempel1, Daryl Giblin1, Carl Frieden2, Michael L. Gross1* 1
Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St.
Louis, MO 63130 2
Department of Biochemistry and Molecular Biophysics, School of Medicine, Washington
University in St. Louis, 660 S. Euclid Ave, St. Louis, MO 63110 3
Current address: Analytical Research and Development, Pfizer Inc., Chesterfield, MO
63017, United States *Corresponding author: Michael L. Gross Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130 Email:
[email protected] Abstract 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 smallmolecule 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 site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), 1 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 25
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 ∆/ %, 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. Key words protein–ligand interaction; hydrogen deuterium exchange of proteins; mass spectrometry; protein binding affinity; unfolding; Apolipoprotein E; protein-ligand interactions by mass spectrometry, titration, and HDX (PLIMSTEX); SUPREX.
2 ACS Paragon Plus Environment
Page 3 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Introduction The interaction of proteins with various ligands, their resulting stoichiometry, binding interfaces, conformational changes, thermodynamics, and kinetics are key to many biochemical processes. Although methods using fluorescence1 or Surface Plasmon Resonance (SPR),2 provide essential binding constants, they often require specific reporter labels and afford limited if any spatial or site-specific information. High resolution methods (e.g., Nuclear Magnetic Resonance (NMR)3 and X–ray crystallography4), are often difficult because they require relatively large amounts of sample and considerable expertise. Hydrogen– deuterium exchange (HDX), because it is non-perturbing and simple to execute while providing high sensitivity and spatial resolution of the protein,5-9 has become one of the most applied mass spectrometry-based footprinting approaches in studying protein conformational change and locating protein–ligand interactions. Ligand binding can directly or indirectly affect protein local hydrogen-bond networks and change solvent accessibility. Backbone amide hydrogen exchange with D2O responds to these changes. In HDX kinetics, as most commonly practiced, the protein needs only be incubated in D2O and time-dependent measurements made. Plots of deuterium uptake extent against exchange times, inform on binding regions when comparing HDX in the presence and absence of a ligand.7 To increase the utility of HDX, two methods have been advanced to determine protein– ligand binding affinities. One is PLIMSTEX (protein–ligand interactions by mass spectrometry, titration, and H/D Exchange),10 in which a protein in solution is submitted to HDX at a predetermined time with increasing ligand concentration. A plot of HDX against ligand concentration produces a “PLIMSTEX curve”, from which binding affinity can be extracted by mathematical model fitting.11 In addition, at protein concentrations much higher than the ligand dissociation constant, PLIMSTEX can be used to determine binding stoichiometry or purity of the protein.10,12 PLIMSTEX is a titration-based platform employing the same idea as conventional fluorescence-based titration methods, but without introducing a fluorophore, which often requires amino acid substitutions. An HDX method allows a protein to be investigated in solution in its native or near-native state. PLIMSTEX has been successfully applied to a variety of complicated systems in which binding affinities estimated at the global level agree well with literature values.10,11,13-15 Its 3 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 25
application at the peptide-level, however, has developed more slowly. One reason is that we chose for our development metal-binding systems that appear to involve multiple, more complicated binding processes14,15 than do the more tractable 1:1 systems. Additionally, application to tight binding requires low protein concentrations, which formerly was challenging but now readily addressed with today’s mass spectrometers. SUPREX (stability of unpurified proteins from rates of H/D exchange), is another HDXbased method for measuring protein folding energy changes and binding constants.16 Although SUPREX
was
originally
developed
for
application
with
matrix-assisted
laser
desorption/ionization (MALDI) MS, it is also suitable for electrospray ionization (ESI) MS17. In SUPREX, a protein in the presence and absence of ligand is incubated with different concentrations of denaturant and then submitted to HDX as a function of deuterium exchange times. Each SUPREX curve is constructed by plotting the protein mass shift against denaturant concentration and fit to a four-parameter sigmoidal equation by nonlinear regression to determine a transition midpoint, / . Multiple / determinations at varied exchange
times are used to calculate an m value18 (i.e., dependence of folding free energy change on denaturant) and ∆Gf, (i.e, the folding free energy in the absence of denaturant). The binding constant can then be calculated from the ∆∆Gf between bound and unbound states.19-21 Thermodynamics of domains22 or of large peptides23 can also be probed with SUPREX coupled with enzyme digestion. We recently performed the usual differential HDX kinetics experiment of bound and unbound states of apolipoprotein E (apoE) and a small molecule effector (drug candidate).24 The apoE family consists of three isoforms that differ by single amino-acid changes at two sites out of 299 residues: apoE2 (Cys112/Cys158), apoE3 (Cys112/Arg158), and apoE4 (Arg112/Arg158).25 The motivation for our interest is apoE4, a major genetic risk factor for lateonset Alzheimer’s disease (AD)26; in contrast, apoE2 and apoE3 have no such detrimental effect.27 We found binding at three regions of the C-terminal domain: 229–235 (LDEVKEQ), 234–243 (EQVAEVRAKL) and 258–265 (QARLKSWF). To explore the potential of PLIMSTEX to characterize this system for local binding, we extended it by coupling it with tandem protease digestion and obtained binding constants at peptide levels. Additionally, we
4 ACS Paragon Plus Environment
Page 5 of 25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
implemented a modified peptide-level SUPREX protocol to investigate regional bindinginduced unfolding changes, particularly of the C-terminus.
Experimental Materials and Reagents As previously reported24 and briefly described here, apoE3 was expressed as a thioredoxin fusion protein in E. coli that was grown in LB media to OD600 = 0.6. A PreScission peptide was inserted between the thioredoxin and apoE. After purification using an N-terminal 6X-Histag, thioredoxin was removed with the PreScission protease (GE Healthcare, Pittsburgh, PA). Phosphate buffer saline (PBS), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), urea, formic acid, trifluoroacetic acid, porcine pepsin, fungal XIII, HPLC grade water and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO). D2O was from Cambridge Isotope Laboratories Inc. (Andover, MA). The compound, N-5-[(3-chlorophenyl)sulfamoyl]-2-hydroxyphenyl-2-(3methyl-4-oxo-3,4-dihydrophthalazin-1-yl)acetamide (C23H19ClN4O5S, name shortened to EZ482), was from Enamine LLC (Monmouth Junction, NJ). The structure of this compound is provided in Figure 1. Sample Preparation For PLIMSTEX, 10 µM apoE3 (1X PBS containing 2 mM DTT, pH 7.4) was incubated with different concentrations of EZ-482 (dissolved in DMSO) for 1 h at 25 ⁰C prior to HDX analysis. All stock solutions contained 2% DMSO including the apoE3 sample without ligand. In the originally described SUPREX experiment, the protein was dissolved and submitted to denaturant and D2O exchange by including the denaturant in deuterated exchange buffers. In this work, the unbound and bound (10 µM apoE3 and 500 µM EZ-482; 1:50 protein:ligand) apoE3 solutions were incubated for 1 h at 25 ⁰C. Then various concentrations of urea were added to the master solutions and incubated for 1 h at 25 ⁰C again before HDX analysis. The final urea concentration was measured by a hand-held refractometer (Atago U.S.A., Bellevue, WA). HDX Protocol
5 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 25
Both PLIMSTEX and modified SUPREX were conducted with the same HDX protocol24 after sample preparation. Briefly, HDX was initiated by 10-fold dilution of 4 µL stock solution into D2O buffer (1X PBS, pD 7.4) for 2 min at 25 ⁰C, followed by mixing 2:3 (v/v) with 1 mg/mL fungal XIII, 3 M urea, and 1% formic acid on ice for additional 2 min. The quenched and fungal XIII digested peptides were then submitted to online pepsin digestion, trapped and desalted on an Agilent C8 trap column (2.1 mm × 1.5 cm, Santa Clara, CA). The sequentially digested and desalted peptides were then separated over 11 min on a C18 column (2.1 mm × 5 cm, 3 µm Hypersil Gold, Thermo Fisher, Waltham, MA). Data were collected on a Bruker maXis quadrupole time of flight (Bremen, Germany) mass spectrometer via positive-ion ESI. The entire procedure was carried out in a custom-built water-ice bath to minimize back exchange. All data points were taken in duplicate. Data Analysis Peptides from sequential digestion prior to HDX analysis were submitted to collisioninduced dissociation fragmentation (CID) on a Thermo LTQ 7 T FT-MS (San Jose, CA) in a data-dependent mode. Product-ion spectra were identified in MassMatrix (version 2.4.2),28 and manually verified. Deuterium uptake of each peptide was deconvoluted with HDExaminer (2.0, SierraAnalytics, Inc., Modesto, CA). No corrections for back exchange. For peptides whose HDX% level up at high urea concentration, some (e.g. 79−93, 175−191, and 234−243) have
