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Analysis of MEMO1 Binding Specificity for ErbB2 using Fluorescence Polarization and Molecular Dynamics Simulations Madeline L. Newkirk, Kristen J. Rubenstein, Jessica Y. Kim, Courtney L. Labrecque, Justin Airas, Cooper Ashley Taylor, Hunter D. Evans, Quincy McKoy, Carol A. Parish, and Julie A Pollock Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00582 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Biochemistry

Analysis of MEMO1 Binding Specificity for ErbB2 using Fluorescence Polarization and Molecular Dynamics Simulations Madeline L. Newkirk†, Kristen J. Rubenstein†, Jessica Y. Kim, Courtney L. Labrecque, Justin Airas, Cooper A. Taylor, Hunter D. Evans, Quincy McKoy, Carol A. Parish, and Julie A. Pollock* Department of Chemistry, University of Richmond, Richmond, VA 23173, United States †

These authors contributed equally to the work.

Corresponding author * Email: [email protected]. Telephone: (804) 484-1578. Fax: (804) 287-1897.

ErbB2 signaling pathways are linked to breast cancer formation, growth, and aggression; therefore, understanding the behavior of proteins associated with these pathways as well as regulatory factors that influence ErbB2 function is essential. MEMO1 is a redox active protein that is shown to associate with phosphorylated ErbB2 and mediate cell motility. We have developed a fluorescence polarization assay to probe the interaction between MEMO1 and an ErbB2-derived peptide containing a phosphorylated tyrosine residue. This interaction is shown to be pH dependent and stronger with longer peptides as would be expected for protein-protein interactions. We have quantitatively mapped the binding interface of MEMO1 to the peptide using the fluorescence polarization assay and molecular dynamics simulations. We have confirmed that phosphorylation of the peptide is essential for binding and through mutagenesis have identified residues that contribute to favorable interactions. Our results highlight the importance of the protein-protein interactions of MEMO1 that complement the oxidase activity. In the future, these studies will provide a method for screening for selective modulators of MEMO1, which will allow for additional biological investigations.

Keywords: MEMO1, ErbB2, phosphorylation cascades, breast cancer, fluorescence polarization, molecular dynamics 1 ACS Paragon Plus Environment

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INTRODUCTION MEMO1 (mediator of ErbB2-driven cell motility 1), a 297-amino-acid protein, coprecipitates with ErbB2, a receptor tyrosine kinase that regulates cell growth, differentiation, and migration.1-3 Aberrant ErbB signaling is a feature of many human cancers and is associated with more aggressive, metastatic tumors and a poorer prognosis for patients.4 Specifically, ErbB2 plays an integral role in human breast carcinoma development.5 In patients whose tumors overexpress ErbB2 receptor kinase or demonstrate ErbB2 gene amplification, a poor outcome is expected in 25-30% of cases.6 Recent research demonstrates that MEMO1 is also necessary for breast tumor growth and may modulate signaling cascades involving ErbB2 and other important growth regulatory protein kinases such as mitogen-activated protein kinase (MAPK) and protein kinase B/AKT.2 Ligand binding to the C-terminal tail in the extracellular regions of ErbB2 initiates signaling and the activation of the cytoplasmic kinase resulting in autophosphorylation at several sites in the tail region, termed YA, YB, YC, YD, and YE, corresponding to tyrosine residues 1023, 1139, 1196, 1222, and 1248, respectively.7, 8 Phosphorylation at these sites promotes association with various effector proteins, including MAPK and AKT, which initiate a series of downstream signaling events that ultimately promote cell growth and migration.2 Studies of the functional role of these phosphorylation sites led to the identification of MEMO1, which coprecipitates with ErbB2 when one of the autophosphorylation sites, tyrosine 1222 (YD) is phosphorylated (pYD).9 MEMO1 controls ErbB2-regulated microtubule dynamics and may be required for the activation of MAPK and AKT that underlies the constitutive expression of estrogen receptor α (ERα) gene targets implicated in many breast tumors.9, 10 In addition, MEMO1 is found in complexes containing proteins known to promote tumor growth such as

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Biochemistry

ERα, insulin-like growth factor 1 receptor (IGF1R), epidermal growth factor receptor (EGFR), and insulin receptor substrate 1 (IRS1).2, 11, 12 With this study, we set out to elucidate the mechanism of ErbB2 recognition by MEMO1. Fluorescence polarization and thermal stability assays were developed to investigate the binding affinity and stability of complex formation between MEMO1 and a truncated ErbB2-derived peptide. Additionally, mutagenesis was used to identify residues essential to the ErbB2/MEMO1 interaction. The MEMO1 binding site was further mapped using molecular docking and by performing molecular dynamics studies.

MATERIALS AND METHODS Site-Directed, Ligase Independent Mutagenesis. Twelve point mutations of MEMO1 (UniProt Accession ID: Q9Y316, species: human) were generated using site-directed, ligase independent mutagenesis (SLIM) protocols as previously described.13, 14 Primers used for mutagenesis can be found in Supplemental Table S1. All mutants were confirmed by sequencing at Eurofins Genomics.

Protein Expression. The plasmid vector pET-15b encoding for WT (Genscript) or mutant MEMO1 was transformed into chemically competent BL21(DE3) Star cells (Thermo Fisher). Single colonies were incubated overnight at 37 °C with shaking in Lennox Broth containing ampicillin (100 µg/mL). Fresh media (500 mL) was inoculated with 5 mL of the overnight culture and grown at 37 °C to an OD600 ≈ 0.6. Protein expression was induced with IPTG (0.5 mM) for an additional 3 h. Cells were collected by centrifugation (4000 rpm, 10 min), and pellets were stored at -20 °C.


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Protein Purification. Cell pellets were resuspended in 10 mL lysis buffer (50 mM sodium phosphate, pH 7.6, 200 mM NaCl, 5 mM imidazole, 5% glycerol) supplemented with 0.5 mM PMSF. Cells were lysed by sonication, then centrifuged at 12,000 rpm for 30 min at 4 °C to remove cellular debris. The clarified lysate was purified using Ni-NTA affinity chromatography. The column was charged with NiCl2 (50 mM) and equilibrated with lysis buffer. The cell lysate was added to the column and washed with wash buffer (50 mM sodium phosphate, pH 7.6, 200 mM NaCl, 50 mM imidazole, 5% glycerol). The protein was eluted with elution buffer (50 mM sodium phosphate, pH 7.6, 200 mM NaCl, 250 mM imidazole, 5% glycerol). The purified MEMO1 was desalted using a 10DG column (Biorad) into storage buffer (50 mM sodium phosphate, pH 7.6, 200 mM NaCl, 5% glycerol). Protein purity was verified by SDS-PAGE (Supplemental Fig. S1), and concentration was calculated from the absorbance at 280 nm using calculated molar extinction coefficients for wild type MEMO1 or the mutant proteins (Supplemental Table S2).15

Peptides. All peptides corresponding to the ErbB2 tail were custom synthesized by GenScript. Three N-terminal fluorescein-labeled peptides (FL-pYD10, 10-mer: fluorescein-1217Phe-AspAsn-Leu-Tyr-pTyr-Trp-Asp-Gln-Asp1226-NH2; FL-pYD5, 5-mer: fluorescein-1220Leu-TyrpTyr-Trp-Asp1224-NH2, FL-YD10, 10-mer: fluorescein-1217Phe-Asp-Asn-Leu-Tyr-Tyr-TrpAsp-Gln-Asp1226-NH2) and two non-labeled peptides (pYD10, 10-mer: H-1217Phe-Asp-AsnLeu-Tyr-pTyr-Trp-Asp-Gln-Asp1226-NH2; pYD5, 5-mer: H-1220Leu-Tyr-pTyr-Trp-Asp1224NH2) were used in the experiments as indicated.


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Fluorescence Polarization. Saturation curves were created by titrating fluorescent peptide with MEMO1.16-18 Buffers (listed in full in Figure 2 and Table 1, where NaP is sodium phosphate buffer, CAPS is N-cyclohexyl-3-aminopropanesulfonic acid buffer, and NaOAc-AcOH is sodium acetate-acetic acid buffer) were combined with MEMO1 (native or mutant, 4.5 µM), and two-fold dilutions were performed. “Background” data points were also collected in the absence of protein to measure the fluorescence of completely unbound fluorescent ligand. Fluoresceinlabeled peptide (FL-pYD10, FL-pYD5, or FL-YD10) was added to each dilution, as well as to the background condition, to a final concentration of 0.1 µM. Each dilution was pipetted in triplicate into a 96 well half-area opaque plate (90 µL per well) including a group blank (buffer only). The plate was incubated for 10 minutes at room temperature. Fluorescence measurements were made using a SpectraMax i3 plate reader (Molecular Devices) at 4.11 mm above the plate with fixed excitation (485 nm) and emission (525 nm). Millipolarization (mP) was calculated using the Softmax Pro software from the parallel and perpendicular intensities (Qpara and Qperp, respectively): = 1000 ∗

∗

(1)

∗

mP were subsequently converted to units of anisotropy (A): =

∗

(2)

The G factor was determined experimentally to be 1.4 by measuring the parallel and perpendicular intensities of a 1 nM solution of fluorescein using the following equation:

=

∗

(3)

where 27 mP is the reported polarization value of 1 nM fluorescein.19


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These data were then manipulated to correct for variations in total intensity (Q) that may have resulted from interaction with MEMO1.20, 21 Total intensity was calculated from the intensity of the parallel and perpendicular light accounting for the effects of polarized emission: ! = !"#$# + 2!"'$"

(4)

The intensity of the fully bound fluorescent ligand (Qb) was calculated by determining the yintercept of the plot of Q vs.

(

[*+*,]

, where Qf is the intensity of the free (unbound) ligand, Q is

the total intensity, and [MEMO1] is the corresponding protein concentration. The anisotropy of the fully bound fluorescent ligand (Ab) was calculated by determining the y-intercept of the plot of A vs.

( (//( )

1 [*+*,]

, where Af represents the anisotropy of the unbound fluorescent ligand. From

these values, the adjusted anisotropy (M) was calculated for all measured data points: 2 =

454(

3

41 54

6(

/1 7 /(

61 454(

3

41 54

6(

61

(5)

7

Adjusted anisotropy was then used to calculate the total amount of bound ligand, from which saturation curves were produced. The fraction of bound ligand (fB) was calculated: 89 =

//(

(6)

/1 /(

Total ligand bound (: = [;?@]89 ) vs. [MEMO1] curves were fit with the following nonlinear regression in GraphPad Prism 7 :=

(ABCDC E FG )H(ABCDC E FG ) I(A)(BCDC E )

(7)

where X and Ltotal are MEMO1 and total ligand concentration in µM, respectively.

Competitive Displacement Fluorescence Polarization Assay. Serial two-fold dilutions of the unlabeled phosphorylated peptides, pYD10 (0 – 150 µM) or pYD5 (0 – 600 µM) were made in 6 ACS Paragon Plus Environment

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Biochemistry

sodium phosphate buffer (50 mM, pH 6.4). A negative control was included without unlabeled peptide to measure the anisotropy of the total binding of the displacer (labeled peptide). Labeled phosphorylated peptide (FL-pYD10) and MEMO1 were combined separately, incubated for 10 minutes at room temperature, and added to each competitor dilution to a final concentration 0.1 µM and 1 µM, respectively. Each dilution was pipetted in triplicate into a 96 well half-area opaque plate (90 µL in each well) including a plate blank (buffer only). The plate was incubated for 10 minutes at room temperature before measurement using the SpectraMax i3 plate reader (Molecular Devices). Millipolarization and anisotropy was calculated as described above. Curves of anisotropy (A) vs. log[competitor] (X) were fit with the following nonlinear regression in GraphPad Prism 7 ;J=KLMN = ;J= 10OPQFR ∗ 1 + = :JWWJ +

[SB"TUN] FV

XP"9PYYPZ

(8) (9)

N([5\D]^_` )

where [FLpYD10] = 100 nM, KD = 532 nM.

Copper Loading. Purified MEMO1 protein (5 μM) was incubated with 50 μM CuSO4 in 50 mM sodium phosphate buffer, pH 6.4, for 30 min at 4 °C. Excess copper was removed by dialysis against 50 mM sodium phosphate buffer, pH 6.4, for 3 hours at 4 °C. Pre-loaded protein was used directly in the FP assay as described above.

Protein Thermal Stability by Differential Scanning Fluorimetry. Buffers (listed in full in Table 1) were combined with purified protein (native or mutant, 0.3 mg/mL) and SYPRO Orange protein stain (1:500 relative dilution) to a final volume of 100 µL. To explore its effects on


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MEMO1 stability, unlabeled pYD10 was added in some conditions at a concentration of 4 µM. Each protein sample was loaded in triplicate into a 96-well, clear PCR plate (25 µL per well), and the plate was sealed. The samples were then heated from 15 to 95 ºC at a rate of 0.5 ºC/min in a Bio-Rad RT-PCR machine. SYPRO Orange fluorescence was monitored, and the midpoint of the plot of the first derivative of fluorescence versus temperature was used to determine the melting temperature (Tm).

Structural Retrieval and Preparation for Subsequent Molecular Dynamics. The structure of apo MEMO1 was obtained from the Protein Databank.22 To the best of our knowledge, this is the only experimental MEMO1 structure.9 The original file (PDB 3BCZ) consisted of 293 amino acids. The Schrodinger Protein Preparation workflow was used to add missing hydrogen atoms to the experimental structure as well as to ensure physiologically correct protonation states, such as ensuring the neutral Νδ1-protonated π tautomer was utilized.23 No other changes were made to the structure of the protein.

Analysis of Binding Sites in MEMO1. The SiteMap package from Schrodinger was used to identify and score putative binding sites in apo MEMO1 based upon shape complementarity and solvent accessibility.24, 25 SiteMap identifies binding sites based upon an analysis of hydrophobic surface area, surface concavity, as well as the location of hydrogen-bond donor and acceptor regions.

Preparation of Binary Complexes. The GLIDE docking algorithm was used to flexibly dock various 5- and 10-mer peptides containing a phosphorylated tyrosine at peptide residue 3 (5-mer)


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Biochemistry

and 6 (10-mer) to apo-MEMO1 (Fig. 1).24, 26, 27 The Glide receptor grid was centered on MEMO1 and a 40x40x40 Å boundary box that included the entire complex was used. The Standard and Extra Precision docking modes were utilized to screen and narrow docked poses, respectively.

Figure 1. A. 5-mer (pYD5) and B. 10-mer (pYD10) ErbB2 peptide tails/ligands used in generating binary complexes via docking.

Molecular Dynamics (MD). All simulations were performed under identical constraints. The Amber ff14SB force field was applied to all simulations.28, 29 Phosphorylated residues were modeled using the phosaa10 force field.30 AmberTools’ tleap was used to neutralize each system with Na+/Cl- ions and solvate a truncated octahedron periodic box with TIP3P water molecules.31-33 At the initiation of each simulation, there was at least 12.0 Å of solvent between the solute and edge of the unit cell. The GPU-accelerated pmemd code of Amber 16 was used to perform all steps of MD for each simulation.31, 34 The initial structures, obtained by Glide docking (vide supra), were minimized, heated, equilibrated, and then subjected to unrestrained MD. The minimization process consisted of seven stages, each comprising a maximum of 5,000 steps. The first 1,000 steps were of steepest descent minimization, and the remaining 4,000 steps 9 ACS Paragon Plus Environment

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were of conjugate gradient minimization. The first of the seven stages was given a restraining force of 10.0 kcal/mol/Å2 on the heavy atoms of the solute and this was methodically lowered to 0.0 kcal/mol/Å2 by stage seven. After minimization, each structure was heated from 10 K to 300 K with a restraining force of 10.0 kcal/mol/Å2 on the solute. Equilibration consisted of lowering the restraining force every 500 ps from 10.0 kcal/mol/Å2 to 0.0 kcal/mol/Å2 over seven stages. This is a standard protocol for minimizing and equilibrating a structure for subsequent MD and a necessary step to avoid computational instabilities caused by steric clashes with added solvent, addition of hydrogen atoms, etc. After the kinetic energy equilibrated between the solvent and structure with no restraining force, unrestrained MD at constant pressure (1 atm) and temperature (300 K) was commenced. The SHAKE algorithm was used to restrain all covalent bonds to hydrogen atoms.35 This improved the computational efficiency of the simulation and allowed the use of a 2-fs time-step in the MD simulation. Unrestrained MD was used to explore the conformational flexibility of the apo MEMO1 (2 µs total; 10 seeds of 200 ns each). To explore 5-mer binding to MEMO1, we performed 500 ns of simulation initiated using 3 seeds applied to 6 different initial Glide poses (9.0 µs of simulation). A seed represents the set of initial velocities assigned to each atom within the solvated system at the beginning of the simulation. To explore 10-mer binding, we ran preliminary 200 ns simulations initiated from three seeds using three different Glide poses. From these initial results, we chose one Glide pose for which we ran 200 ns simulations starting with 3 different seeds, and a second Glide pose with which we ran 200 ns simulations starting with 2 different seeds (for a total of 1.0 µs of MD). We chose Glide poses for subsequent MD based on the Glide score (Supplemental Fig. S9-10). The Glide score is an empirical scoring function that assess electrostatic and van der Waals terms as well as goodness of fit and ligand-receptor interactions.


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Biochemistry

In all cases, Glide docking oriented the phosphorylated tyrosine towards solvent. We also used the GRAMM- docking algorithm, which converted the pY to TYR and oriented it away from the binding pocket and towards solvent.36, 37 In order to compare with previous reports,1 we also manually oriented the 5- and 10-mer peptide into the binding pocket with the phosphorylated tyrosine pointing in toward the active site. We used these manually docked poses to perform an additional 4 seeds of 250 ns simulation (5-mer) and 5 seeds of 500 ns simulation (10-mer). Analysis of all simulations, including the calculation of root-mean-square deviations (RMSD), center of mass (COM) distances, RMS residue fluctuations (RMSFs), average structures, and clustering was performed using the cpptraj module included in AmberTools 16.31, 38, 39

Clustering was also performed with the Multiscale Modeling for Structural Biology

(MMTSB) tool set.40 Binding free energies and pairwise per residue free energy decomposition values (∆Gbind) were calculated using the MMPBSA python package in AmberTools 16.41 Binding free energies and per residue free energy decomposition values calculated for all 5- and 10-mer containing simulations (5-mer = 22 trajectories; 10-mer = 10 trajectories). We computed these energies every 0.3 ns for a total of 666 and 1666 regularly spaced structural snapshots from each of the 200 and 500 ns trajectories, respectively.42 Structures and trajectories were visualized using VMD and PyMOL.43, 44 We used a generalized Born (GB) model implemented by Onufriev et al.45, 46 to calculate the electrostatic portion of the solvation free energy. The MMGBSA analysis does not provide absolute binding free energies but rather approximate relative values useful for comparisons between similar structures such as studied here. The Onufriev and Case model has previously shown promise at accurately ranking binding affinities derived from ensembles generated by MD simulations.47 This method is widely used for protein-ligand complexes.48 Normal mode analysis was used to calculate entropy values over 10 regularly


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spaced snapshot from all twenty-two 5-mer and ten 10-mer containing trajectories (Table 2). The entire protein-ligand complex was included in the entropy analysis. To do this we sampled the structures every 20, 25, or 50 ns for the 200, 250, or 500 ns trajectories, respectively. When entropy is included in the overall binding free energy values, the relative binding affinities are suggested to be scaled to more experimentally realistic values.49 Normal mode entropy calculations have also been found to improve the correlation and ranking of binding affinities.49 More rigorous techniques for calculating binding free energies using MD simulation are available (such as free energy perturbation50 and thermodynamic integration51); however, these methods are computationally demanding and impractical for large biomolecules. Thus, for molecules of the size considered in the present study, the MM-GBSA method is considered reasonably accurate and computationally accessible.

RESULTS AND DISCUSSION Fluorescence polarization of wild-type MEMO1 Previous pull-down experiments suggested that amino acids 1217-1226 within the cytoplasmic tail of ErbB2 including phosphorylated Tyr-1222 (pYD) were essential for MEMO1/ErbB2 complex formation.1 Feracci, et.al. observed similar binding of MEMO1 to the 10-mer and 16-mer ErbB2-derived peptides.1 Therefore, we designed a peptide (FL-pYD10) with the same corresponding sequence appended with an N-terminal fluorescein tag for our studies; we chose the smaller 10-mer for maximal change in anisotropy upon protein binding. The saturation curve produced from the titration of FL-pYD10 with wild type MEMO1 (Fig. 2A, red circles) representing the ratio of free to bound ligand at any given protein concentration was used to determine the KD (0.532 ± 0.087 µM) of the protein-peptide interaction after 10 minutes of incubation.52 There was not considerable change in the binding constant after incubation of 12 ACS Paragon Plus Environment

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Biochemistry

the plate for 1 hour (KD = 0.668 ± 0.130 µM) indicating equilibrium was reached within 10 minutes. For consistency, we performed the rest of our experiments using a 10 minute incubation time as described in the materials and methods. As expected, the interaction between MEMO1 and the non-phosphorylated peptide (FLYD10) did not produce a binding curve (Fig. 2A, gray squares) confirming the necessity of the modified residue and consistent with previous isothermal titration calorimetry (ITC) studies performed on a 16-mer peptide.9 We did not observe any background binding of free 5carboxyfluorescein (Supplemental Fig. S2).

Figure 2. Results of FP Assay with wild type MEMO1. A. Binding curve of FL- pYD10 (0.1 µM, red) and FL-YD10 (0.1 µM, gray) titrated with wild type MEMO1 (0 – 4.5 µM) in sodium phosphate buffer (pH 6.4). Anisotropy was adjusted as described in the methods section to account for changes in fluorescence intensity (see Supplemental Fig. S3 for raw data). Average data points from triplicate experiments (± S.D.) for FL-pYD10 are fit with a non-linear regression giving KD = 0.532 µM. B. Binding curves of FL-pYD10 (0.1 µM) titrated with wild type MEMO1 (0-4.5 µM) in sodium phosphate buffers, pH 5.7 – 7.4. Average data points from triplicate experiments (± S.D.) are fit with non-linear regressions with KDs listed (right). C. KD values (± S.D.) calculated from FP assays with FL-pYD10 (0.1 µM) and wild type MEMO1 (0 – 4.5 µM) in various buffers as indicated. D. Displacement of FL-pYD10 by unlabeled peptide (10-mer pYD10, green or 5-mer pYD5, red) in sodium phosphate buffer (pH 6.4). Average data points from triplicate experiments (± S.D.) are fit with non-linear regression. 13 ACS Paragon Plus Environment

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In addition to the role of MEMO1 in interacting with ErbB2 and other phosphorylated proteins, it was determined to be a copper-dependent redox protein.53 These two functions may be synergistic or antagonist and therefore, we wanted to examine the effects of copper on the binding association between MEMO1 and the fluorescein-labeled peptide. When wild-type MEMO1 was preloaded with Cu2+, the binding affinity decreased approximately 2-fold (KD = 0.996 ± 0.119 µM, Supplemental Fig. S4) but was not abolished completely. Since copper binding does not increase peptide binding, we focused the majority of our studies on the apoprotein. A comparison of wild type MEMO1/pYD10 binding under a range of sodium phosphate buffer pH conditions (pH 5.7 – 7.4) suggests that binding affinity increases with buffer acidity (Fig. 2B). Given that pKa of the histidine side chain is approximately 6.0, this pH dependence suggests the potential necessity of protonated histidines in the active site of MEMO1 for the stabilization of pYD,9 a conclusion further supported by the effective elimination of complex formation in CAPS-NaOH buffer, pH 10.0 (KD > 5 µM). Our results align well with previous pull-down studies with a 16-mer peptide that show better binding at lower pH.9 Furthermore, binding is affected by the presence of sodium acetate and sodium chloride (Fig. 2C). These data imply a destabilizing effect of highly ionic aqueous environments and suggests the interaction may be facilitated by electrostatic interactions. Titration of MEMO1 with a shorter 5-residue fluorescein labeled peptide (FL-pYD5) did not produce a binding curve (Supplemental Fig. S5). In addition, analysis of competitive FP data revealed that MEMO1 binds approximately 14-times more tightly to the ErbB2 10-mer pYD10 than the ErbB2 5-mer, pYD5 (Fig. 2D, KI = 7.3 μM and 107.8 μM, respectively). This


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Biochemistry

points to the cooperative nature of the residues surrounding ErbB2’s phosphorylated Tyr-1222 and supports previous findings that shorter truncations do not bind productively to MEMO1.1 In addition, interaction of MEMO1 with protein partners within a cellular environment may provide more stability for the interaction.

Thermal stability of wild-type MEMO1 Due to its differential binding to folded versus denatured proteins, the relative fluorescence of SYPRO Orange protein stain was used to determine MEMO1 melting temperature using a Differential Scanning Fluorimetry thermal melt assay.54 Table 1 illustrates the stabilizing effect of an acidic environment, with pH and melting temperature being inversely correlated. Under these experimental conditions, the observed increase in melting temperature of MEMO1 correlates with the pH dependent decrease in KD reported above. Alternatively, the addition of imidazole significantly decreases melting temperature in a concentration-dependent manner (Supplemental Fig. S6). Notably, there does not appear to be a significant effect of ErbB2 peptide presence on MEMO1 stability as measured in this assay (Table 1). This suggests that there is likely not a significant structural change in MEMO1 when binding to this peptide, which is in agreement with our apo- and peptide-bound protein dynamics, presented below. Table 1. MEMO1 melting temperatures determined by DSF thermal stability assay. Tm in various buffers/additive conditions as indicated, with or without pYD10 (4 µM). Tm (°C) 59.5 58.0 59.0 58.5 58.5 56.3 55.0 54.5 53.5

Buffer (pH) 50 mM NaP (5.7) 50 mM NaP (5.8) 50 mM NaP (6.0) 50 mM NaP (6.2) 50 mM NaP (6.4) 50 mM NaP (6.6) 50 mM NaP (6.8) 50 mM NaP (7.0) 50 mM NaP (7.2)


Tm (+ 4 µM pYD10) (°C) 59.5 58.3 59.0 58.5 58.5 56.5 55.5 55.0 54.0

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50 mM NaP (7.4) 100 mM NaOAc-AcOH (4.7) 100 mM NaOAc-AcOH (6.0) 100 mM Imidazole-HCl (7.0) 100 mM CAPS-NaOH (10.0) 50 mM NaP (6.4) + 50 mM NaCl 50 mM NaP (6.4) +100 mM NaCl 50 mM NaP (6.4) +200 mM NaCl

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52.2 57.5 53.0 49.2 N/A 58.5 58.0 58.0

52.8 58.3 59.0 49.3 N/A 58.3 58.2 58.0

MEMO1 apo dynamics Very little is known about the molecular dynamics of MEMO1 because the structure has only recently been reported.9 We began our investigation of the atomistic behavior of MEMO1 by focusing on the apo protein. We initiated unrestrained MD on apo MEMO1 via 10 different 200 ns simulations with different randomly selected seeds. These shorter simulations with different random seeds were used to speed surface coverage. The resulting ensemble of structures suggests that apo MEMO1 is a relatively non-flexible protein that remains in a conformation similar to the experimental structure (PDB: 3BCZ) throughout the 2 microseconds of simulation. All RMSD values for the entire apo-MEMO1 ensemble fall within 1.6 Å relative to the solvated and equilibrated experimental structure. Representative root mean square deviation (RMSD) data are shown in Figure 3A and all RMSD trajectories can be found in the Supplemental Table S3. RMSD values computed relative to the experimental structure were indistinguishable from the RMSD values computed relative to the solvated and equilibrated initial structure used to initiate the simulation. (Supplemental Table S3). To further quantify the dynamics of the apo protein, we clustered the ensemble using cpptraj39and MMTSB.40 MMTSB allows for hierarchical clustering whereas with cpptraj we used a density-based clustering algorithm. Cpptraj clustering resulted in a single large cluster containing 19853 members that included the starting structure. MMTSB clustering produced 29 families of which the most populated six clusters encompassed 60% of the ensemble (Supplemental Table S4). However, 16 ACS Paragon Plus Environment

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Biochemistry

the representative structures from each of these clusters were geometrically similar and showed very little structural differences. A superimposition of these representative structures is shown in Figure 3B and Supplemental Figure S7. Taken together, clustering and RMSD trajectory analysis reveal an apo-MEMO1 that adopts a single stable secondary conformation that is wellrepresented by the experimental structure, with some flexibility in disordered loop regions as shown circled in Figure 3B. Root mean square fluctuation (RMSF) values from the apo simulations indicate that there are clusters of residues that are the most mobile: Val5-Val6; His81-His82-Val83-Pro84-Leu85; Leu124-Gln125-Asp127-Glu128-Asp129; Arg196-Glu204Gly207 and Trp282-Gln283 (Supplemental Figure S8).


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Figure 3. A. Representative RMSD of the apo-MEMO1 ensemble relative to the solvated and equilibrated experimental structure. Data for the additional 9 trajectories can be found in Supplemental Table S3. B. Superimposition of the 6 most populated clusters obtained by MMTSB clustering. Red circles highlight regions of disorder as suggested by clustering and RMSF analysis. C. Sitemap analysis of the MEMO1 structure.

Analysis of MEMO1 binding sites The SiteMap package from Schrodinger was used to identify and score putative binding sites in apo MEMO1 based upon shape complementarity and solvent accessibility.24, 25 Three sites were identified (Fig. 3C). The highest scoring site is the same as the site identified previously by Qiu et al. as the vestigial binding site.9 This site contains conserved histidines 18 ACS Paragon Plus Environment

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Biochemistry

(His-49, His-81, and His-192) and a cysteine (Cys-244). Interestingly, SiteMap results indicate that this largest, most probable site encompasses not only the conserved residues but also extends along the backbone of the protein, suggesting the ability to bind both small molecules, peptides, and proteins. Site 2 and 3 are lower scoring (SiteMap score of ~0.495 for Site 2 and 3; relative to 0.998 for Site 1), contiguous sites, occurring on the other side of the protein from Site 1. There is limited hydrophobic character in these lower scoring sites.

Generating docked poses of phosphorylated 5- and 10-mer ligands The GLIDE docking program identified six and three high-ranking poses of the 5-mer and 10-mer, respectively, bound to MEMO1. All of the 5-mer poses oriented the peptide in Site 1; for the 10-mer, 2 of the 3 poses were oriented in Site 1 while the highest ranked 10-mer pose oriented the peptide along a gap between an α-helix (residue 209-226) and a β-sheet (residue 267-275) on the back of the protein. All poses and Glide Scores are shown in the Supplementary Figures S9-S10. Only poses that oriented the ligand into Site 1 were used to initiate MD simulations. Exploration of other MEMO1 sites is beyond the scope of the current work but will be explored in a subsequent study. We began simulations from all Site 1 poses in order to enhance surface sampling and provide energetic and dynamic verification of the docked poses. Interestingly, GLIDE did not generate a 5- or 10-mer pose with the pYD oriented towards the histidines and cysteine, as reported in a previous docking report.1 This is perhaps not surprising – the phosphate moiety will have a strong interaction with bulk solvent and the empirical GLIDE scoring function contains terms that reward solvent exposure of charged groups. To test for alternative orientations of pYD, we also initiated MD from a manually docked 5- and 10-mer peptide with the pYD residue pointing inwards and towards the His/Cys pocket. 19 ACS Paragon Plus Environment

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Peptide:MEMO1 complex dynamics Our MD results suggest that ligand binding does not induce significant conformational change in MEMO1. This is in agreement with our melting studies described above. RMSD values relative to the initial structure show slightly larger deviations in comparison to apoMEMO1 RMSD values; however, if the RMSD is broken down into contributions from the protein and the ligand, we can see that the higher values are ascribed to ligand motion. Overall, the RMSD plots for both the 5- and 10-mer binary complexes showed stable simulations that are sampling the same overall protein conformation (Fig. 4A and Supplemental Tables S5-S6).

Figure 4. A. Representative RMSD values for the 10-mer:MEMO1 system, protein and peptide, relative to the equilibrated structure used to initiate the simulation. All trajectories are shown in Supplemental Tables S5-S6. B. Representative MEMO1:10-mer structure highlighting residues 20 ACS Paragon Plus Environment

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Biochemistry

that contribute the most to binding as determined by the MMGBSA approximation of interaction energies. MEMO1 shown in teal ribbon, pYD10 peptide shown in red and computationally significant residues are labeled. Per residue interaction energies are shown below in Table 4. (Representative structure taken from 10-mer simulation initiated from Glide Pose 1, Seed 1, frame 142.)

We used the MM-GBSA approximation to quantify differences in binding free energy between the 5- and 10-mer (Table 2). MM-GBSA analysis was performed on all twenty-two 5mer containing trajectories, i.e. eighteen 500 ns 5-mer:MEMO1 trajectories initiated from six unique Glide poses and four 250 ns trajectories initiated from manually oriented 5-mer:MEMO1 poses with the pYD pointing into the His/Cys pocket. As well, we performed MM-GBSA analysis on ten 10-mer containing trajectories, i.e. five 200 ns 10-mer:MEMO1 trajectories initiated from 2 unique Glide poses and five 500 ns initiated from 2 different manually oriented 10-mer:MEMO1 poses with the pYD oriented into the His/Cys pocket. Binding energy trajectories and per residue free energy decomposition data for each simulation can be found in Supplemental Tables S7-S10. From these simulations, we can see that binding to Site 1 is favorable regardless of the orientation of the phosphotyrosine moiety. In fact, simulations initiated from manually oriented ligands reoriented the pYD back towards solvent, in some cases as early as the minimization and equilibration phase. MM-GBSA results suggest that the 10-mer binds more tightly to MEMO1 than the 5-mer, in agreement with the experimental FP results described above. The MM-GBSA binding free energy averaged over all 10-mer trajectories was 10 kcal/mol stronger than for the 5-mer. Due to the differences in size between the 5- and 10mer, we also estimated the normal mode entropic correction to the MM-GBSA energy (Table 2). We do find that the 10-mer has a larger average conformational entropy penalty (-33 kcal/mol) relative to the 5-mer (-27 kcal/mol) and this leads to entropy corrected average free energy estimates of 9.4 and 7.4 kcal/mol for the 5-mer and the 10-mer, respectively. This suggests a 21 ACS Paragon Plus Environment

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more favorable 10-mer complex, in agreement with experiment. It is important to note that the entropy-corrected values are not fully representative of absolute binding energies, but give a more accurate description of relative binding between molecular systems. Upon inclusion of entropic effects, we do not see favorable (negative) MMGBSA free energies, but this may be a sampling issue, i.e. if we were to run more simulations and/or include additional snapshots in our entropy analysis the average may approach the experimental observable of favorable binding. We note that the ∆∆G between the 5-mer and the 10-mer, as computed using MMGBSA plus normal mode entropy, suggests that the 10-mer is 1.5 kcal/mol more stable than the 5-mer (Table 2). This is in good agreement with our experimental result of 2.7 kcal/mol in favor of the 10-mer (Fig. 2D, KI = 7.3 μM (10-mer) and 107.8 μM (5-mer)).55


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Table 2. The average binding affinity, ∆Gbind (kcal/mol), of the 5- and 10-mer peptides (Fig. 1) bound to MEMO1, as estimated by MM-GBSA computations. Simulations initiated from the “reoriented” pose had the pYD residue pointing into the active site whereas Glide docked poses oriented the pYD towards solvent. Normal mode entropy analysis was use to approximate T∆S. Simulation Initiated from Structure:

Average ∆G (kcal/mol)

5-mer (500 ns) Pose 1 Seed 1 -4.98 Pose 1 Seed 2 -23.13 Pose 1 Seed 3 -15.73 Pose 2 Seed 1 -24.02 Pose 2 Seed 2 -15.08 Pose 2 Seed 3 -27.24 Pose 3 Seed 1 -29.12 Pose 3 Seed 2 -25.52 Pose 3 Seed 3 -33.47 Pose 4 Seed 1 -32.37 Pose 4 Seed 2 -27.66 Pose 4 Seed 3 -16.66 Pose 5 Seed 1 -15.23 Pose 5 Seed 2 -14.41 Pose 5 Seed 3 -13.57 Pose 6 Seed 1 -11.60 Pose 6 Seed 2 -20.58 Pose 6 Seed 3 -12.59 5-mer reoriented pose (250ns) Seed 1 -11.16 Seed 2 3.20 Seed 3 -15.15 Seed 4 -19.03 Average -18.41 10-mer (200 ns) Pose 1 Seed 1 -37.85 Pose 1 Seed 2 -34.49 Pose 1 Seed 3 -23.57 Pose 2 Seed 1 -25.05 Pose 2 Seed 2 -31.48 10-mer reoriented (500ns) Pose 1 Seed 1 -32.58 Pose 1 Seed 2 -25.09 Pose 1 Seed 3 -15.58 Pose 1 Seed 4 -21.86 Pose 2 Seed 1 -16.83 Average -26.44

Standard Error of the Mean (kcal/mol)

Sampled T∆S (kcal/mol)

Standard Error of the Mean T∆S (kcal/mol)

Adjusted ∆G (kcal/mol)

0.31 0.19 0.19 0.37 0.19 0.24 0.32 0.26 0.24 0.24 0.21 0.14 0.13 0.14 0.08 0.16 0.12 0.11

-27.53 -31.39 -27.54 -29.42 -26.01 -26.32 -29.17 -29.91 -32.10 -29.17 -28.30 -29.40 -23.70 -21.34 -23.24 -21.73 -26.86 -22.51

0.95 1.14 1.82 1.52 1.70 1.77 1.41 0.73 0.96 2.04 1.57 2.57 1.54 3.81 2.15 3.62 1.44 2.00

22.55 8.26 11.80 5.40 10.93 -0.92 0.05 4.39 -1.37 -3.20 0.64 12.74 8.47 6.93 9.67 10.13 6.28 9.92

0.24 0.32 0.64 0.34 0.24

-29.51 -27.39 -28.08 -29.24 -27.27

1.42 1.79 2.26 1.27 1.80

18.35 30.59 12.93 10.21 8.85

0.26 0.30 0.30 0.35 0.33

-39.36 -39.02 -31.92 -31.98 -36.75

0.72 3.21 2.09 3.01 1.31

1.51 4.53 8.35 6.93 5.27

0.27 0.27 0.35 0.23 0.30 0.30

-35.25 -28.45 -35.69 -30.71 -28.99 -33.81

1.04 1.36 2.06 1.53 1.74 1.81

2.67 3.36 20.11 8.85 12.16 7.37


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MEMO1 Mutational Analysis Identification of MEMO1 as a structural homologue of a class of non-heme iron dioxygenases led to an investigation into the importance of specific residues in its vestigial active site including His-49, His-81, His-192 and Cys-244.9 We set out to investigate the respective binding contributions of these residues, as well as five others situated nearby and/or identified through our MD studies above (Asp-189, Trp-16, Arg-196, Arg-198, and Tyr-54), in the MEMO1/ErbB2-peptide complex formation (Supplemental Fig. S11). Each residue was mutated to Ala using site-directed ligase-independent mutagenesis. Asp-189, Cys-244, and Tyr54 were also conservatively mutated to Asn, Ser, and Phe, respectively. Unfortunately, two mutants (H49A and C244A) did not fold properly after expression and efforts to refold were unsuccessful; therefore, these were excluded from further experimentation. We analyzed each point mutant by FP and thermal stability. Figure 5A-B and Supplemental Table S11 compare the binding curves, KD values, and melting temperatures of the mutants and wild type MEMO1 in 50 mM sodium phosphate buffer (pH 6.4). Similar to the wild type protein, incubation with the ErbB2-derived 10-mer peptide does not influence the melting temperature (Supplemental Table S11). While there is some minimal variation in the overall stability of the mutant MEMO1 proteins, melting temperature does not change linearly with binding affinity. W16A and C244S unfold at the lowest temperatures. While the mutation of Asp to Ala in position 189 (D189A) completely disrupts binding, mutation to asparagine in the same position slightly increases the ErbB2/MEMO1 interaction. This slight enhancement could be attributed to the removal of a negative charge, which would be unfavorable with the


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Biochemistry

negatively charged peptide. The loss of binding with D189A exposes the potential importance of hydrogen bonding in this position. Indeed, when we examine our MD results, Asp-189 spends approximately 10% of the simulation within hydrogen bonding distance from the glutamine residue in the peptide. Similarly, substitution of Tyr with Ala (Y54A) results in the elimination of pYD binding, whereas a Phe in the same position (Y54F) confers only a minor reduction in activity. Due to the relatively similar structures of Tyr and Phe, it is likely that aromatic stacking in this position is a significant factor in the strength of the interaction between MEMO1 and the ErbB2 peptide. When we examine the distance between the center of mass (COM) of the peptide with Tyr-54 in our two lowest energy 10-mer trajectories, we find that Tyr-54 spends the majority of time distant from the center of mass of the peptide (Supplemental Fig. S12, Supplemental Table S12). However, when we examine the COM distances between Tyr-54 and aromatic residues of the peptide throughout all simulations, one of our trajectories shows interaction distances that could demonstrate aromatic stacking (periods of ~100 ns at distances between 4 and 10 Å) (Supplemental Table S13). Mutation of either of the positively charged Arg residues reduces the binding affinity; experimentally Arg196 seems to play a more important role although computationally both residues contributed similarly to the estimated MM-GBSA binding affinity (Supplementary Tables S11 and S14 and Fig. 5B). By analyzing the distance between the COM of specific residues and the COM of the 10-mer over the full trajectories, we see the peptide and Arg196/Arg-198 spend a considerable amount of time within 5 to 10 Å (Supplemental Fig. S13, Supplemental Tables S14-S15). These dominant electrostatic interactions between the Arg residues and the pYD10 peptide aligns well with our observation that increased ionic strength decreases binding affinity (Fig. 2C). Mutation of Trp to Ala (W16A) completely abolished


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binding to the peptide and an examination of our two lowest energy 10-mer MD trajectories shows that Trp-16 spends considerable amount of time near Tyr-5 or Trp-7 on the 10-mer peptide (Supplemental Table S16).

Figure 5. A. Binding curves of FL-pYD10 (0.1 µM) titrated with wild type or mutant MEMO1 (0-4.5 µM) in sodium phosphate buffer (pH 6.4). B. Binding curves of FL-pYD10 (0.1 µM) titrated with mutant MEMO1 (0-4.5 µM) in sodium phosphate buffer (pH 6.4). Average data points from triplicate experiments (± S.D.) are fit with non-linear regression. KD values are listed in Supplemental Table S11.

Per residue free energy decomposition analysis (Table 3) of the 10-mer:MEMO1 complex shows very favorable interactions for peptide residues Tyr-5 and Trp-7 and protein residues Trp-16, His-81, and Phe-197, as well as for charged residues Arg-196 and Arg-198. A similar atomistic picture unfolds for the 5-mer with significant favorable interactions occurring for peptide residues Tyr-2 and Trp-4 and protein residues Trp-16, His-81, Arg-196, Phe-197 and Arg-198 (Supplemental Table S14). A visualization of the resultant trajectories shows significant hydrogen bonding between pYD and/or Asp-2 on the peptides and the Arg residues on MEMO1 (Fig. 6A and 6B; Supplemental Table S15). In addition, Tyr-54 shows some interaction with Asp-10 in the 10-mer peptide in some of the trajectories (Fig. 6C; Supplemental Table S15). Curiously, almost all ligand protein interactions involving the phosphorylated tyrosine residues are unfavorable with per residue free energy contributions 26 ACS Paragon Plus Environment

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Biochemistry

ranging from 0.27 to 8.34 kcal/mol. In only three of the thirty-two 5- and 10-mer simulations does the pYD show a favorable contribution and in these cases the values are small, ranging from -0.23 to -0.67 kcal/mol. A visualization of the trajectories along with a comparison of the MM-GBSA estimations suggests that the phosphorylated tyrosine residue is very flexible and can bind either to MEMO1 or to bulk solvent. It is also possible that being bracketed on either side of the pYD by bulky aromatic groups (Tyr and Trp) leads to steric interactions that favor a solvent exposed pYD. A visualization of the MD trajectories shows favorable T-shaped pi–pi orientations between pYD and Trp7 (Fig. 6D and Supplemental Table S15).

Table 3. Per residue free energy decomposition values (kcal/mol) of all peptide residues as well as residues in MEMO1 that produced significantly favorable (negative) energy interactions upon binding of MEMO1 to the 10-mer peptide. P=pose; S=seed; RP = reoriented pose. The most favorable 10-mer and MEMO1 residue contributors for a given trajectory are shown in bold. Simulation P1S1 P1S2 P1S3 P2S1 P2S2 RPS1 RPS2 RPS3 RPS4 RP#2 initiated from: Residue # peptide 1.45 1.43 -0.20 1.13 PHE1 0.32 1.42 5.91 0.70 1.33 1.18 -1.12 0.32 -1.10 0.09 ASP2 -0.42 0.19 2.07 0.17 2.48 4.25 -0.05 -0.08 0.43 -0.24 ASN3 0.09 -0.13 -0.55 0.08 0.97 -1.96 -0.68 -1.34 -0.10 -1.61 LEU4 0.04 -0.92 -3.81 -0.24 -3.55 -1.95 -1.24 -2.13 -0.74 -1.66 -1.73 -1.08 TYR5 -1.01 -4.05 -6.23 -4.85 3.02 2.85 3.01 PTR6 1.57 0.94 0.51 3.09 1.47 -0.33 7.19 -3.94 -4.36 -1.70 -2.67 -3.23 -1.91 TRP7 -2.41 -0.11 -3.51 -6.23 -0.74 -0.52 -0.72 0.14 ASP8 0.14 -0.20 0.54 -0.40 2.22 0.37 -2.82 -1.00 -0.25 -1.94 -0.92 -4.19 GLN9 -1.21 -0.95 -0.61 -0.65 1.27 1.27 -0.14 -0.13 1.47 0.88 -0.67 3.28 1.35 -0.50 ASP10 MEMO -1.11 -1.67 0.00 0.01 -0.25 -0.48 TRP16 -5.80 -0.08 -0.83 -4.62 -1.78 -0.24 0.01 0.01 -0.31 -0.09 TYR54 -0.20 0.02 -1.37 -0.03 -0.25 -0.45 -0.07 -0.41 -0.25 -0.49 HIS81 -1.28 -0.27 -0.49 -0.34 -0.45 -1.67 -0.28 -0.40 -0.11 -0.44 HIS82 -1.23 -0.32 -0.42 -0.82 0.56 0.47 0.12 0.16 ASP189 0.12 0.12 -1.05 -0.03 0.41 -1.03 -6.24 -3.58 -0.66 -4.78 -1.79 -19.48 ARG196 -8.90 -10.30 -8.35 -11.60 -2.05 -3.23 -1.26 -0.70 PHE197 -0.07 -0.81 -1.88 -0.44 -2.06 -1.72 -4.48 -11.10 -9.74 -6.33 -6.59 -7.20 ARG198 -5.22 -8.25 -4.16 -3.57


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B

A

Asp 2 1.90 Å

1.85 Å Arg 198 Arg 196 2.50 Å

1.76 Å

pYD

C

D

Trp7 Asp 10 1.88 Å

2.66 Å

3.66 Å

Tyr 54

pYD

Figure 6. Residues important for peptide MEMO1 binding. A. Asp2 interactions with Arg198 via a double hydrogen bond interaction. Bond lengths 1.90 and 1.85 Å. B. pYD hydrogen bonding with NH2 group on Arg 196. Bond lengths 1.76 and 2.50 Å. C. MEMO1 Tyr 54 forming a bifurcated hydrogen bond with peptide Asp 10. Bond lengths 1.88 and 2.66 Å. D. T-shaped pi stacking interaction between pYD and peptide Trp7. Bond lengths shown are measured in representative structures. Representative structure in A and C taken from lowest energy structure in the 10-mer trajectory initiated from Glide Pose 1, Seed 1. Representative structure in B and D taken from lowest energy structure in the 10-mer trajectory initiated from reoriented Pose 1, Seed 1. Maximum, minimum and averaged distances from complete trajectories can be found in Supplemental Table S15.

Surprisingly, contradictory to pull-down studies reported in the literature, mutation of the His/Cys pocket (H81A, H192A, C244S) resulted in minimal changes to the experimental binding 28 ACS Paragon Plus Environment

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Biochemistry

affinity.1 When we examine the distance between the COM distances between the 10-mer peptide and these specific residues in our lowest energy trajectories, we find that they remain distant from the peptide and most likely do not have a significant role in peptide binding (Supplemental Fig. S14-15, Supplemental Table S12). Previous work showed that the mutations H49A, H81A, and H192A abolished redox activity.53 From our binding studies, both experimentally and computationally, the most important residues in the interaction of MEMO1 with phosphorylated ErbB2 appear to be Trp-16, Arg-196, Asp-189, Arg-198, and Tyr-54. These two conclusions lead us to believe that there may be a cooperative relationship between the protein scaffolding and redox activity of MEMO1; a hypothesis could be made that one section of the protein is essential for forming protein-protein interactions that places MEMO1 in a complex where it may oxidize other partners. Future studies will be necessary to elucidate when MEMO1 is essential as a binding partner and when it performs oxidase activity.

CONCLUSIONS Increased MEMO1 protein levels are found in greater than 40% of human breast tumors correlating with poor prognostic factors and metastasis.53 Therefore, the characterization of the molecular interactions between MEMO1 and binding partners is important for understanding its role in breast cancer. In this study, we have utilized complementary methods to quantitatively characterize the molecular interaction between MEMO1 and an ErbB2-derived peptide. We have identified critical residues on the binding interface of MEMO1 (Arg-196, Arg-198, Trp-16, Asp189, and Tyr-54) and characterized the stability of the protein both computationally and experimentally.


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FP is a powerful, homogenous tool for studying molecular binding interactions. It requires less protein than isothermal titration calorimetry (ITC), utilizes less expensive equipment than ITC and surface plasmon resonance (SPR), and is easily adaptable for highthroughput screening assays.52 The FP assay we have developed could easily be adapted to examine additional protein partners of MEMO1. Although the ErbB2-binding site is the best established, MEMO1 is found in additional protein complexes including FGFR1, S1PR1, IRS1, ER, and IGR1R, where the molecular interactions are not well understood.11, 12, 56-58 Simple modification to the peptides would allow for probing of these interactions. In addition, FP is commonly utilized to identify small molecule inhibitors of protein-protein interactions including the 14-3-3 family of phosphobinding proteins.59, 60 The identification of a small molecule modulator of MEMO1 would create further opportunities to probe the function of this overexpressed protein in aggressive breast cancers.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding author * Email: [email protected]. Telephone: (804) 484-1578. Fax: (804) 289-8233. ORCID Julie A. Pollock: 0000-0003-0153-9991 Carol A. Parish: 0000-0003-2878-3070 Cooper A. Taylor: 0000-0003-0617-2009 Author Contributions


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J.A.P conceived the project. J.A.P, C.A.P, and K.J.R. designed experiments and analyzed data. C.A.P., M.L.N., K.J.R., J.Y.K., C.L.L., J.A., C.A.T., H.D.E., and Q.M. performed experiments. J.A.P., C.A.P., M.L.N, and K.J.R. drafted the paper. All authors edited the manuscript. Funding This research was supported by funding from the Virginia Academy of Sciences Mary Louise Andrews Award for Cancer Research, the Jeffress Memorial Trust, and the University of Richmond (J.A.P.). M.L.N. was the recipient of a summer fellowship from the Endocrine Society and Puryear-Topham-Pierce fellowship from the chemistry department at University of Richmond. C.A.P. acknowledges support from the NSF RUI program (Grant CHE-1213271) and the Donors of the American Chemical Society Petroleum Research Fund. Computational resources were provided, in part, by the MERCURY supercomputer consortium under NSF grant CHE-1626238. J.Y.K., C.L.L., J.A., C.A.T., and H.D.E. acknowledge support from the University of Richmond Arts and Sciences Undergraduate Research Committee. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors thank those at the University of Richmond who are vital to the success of the undergraduate research program including Phil Joseph and Robert Plymale.

ABBREVIATIONS MEMO1, mediator of ErbB2-driven cell motility 1; SLIM, site-directed, ligase independent mutagenesis; pY, phosphorylated tyrosine; Tm, melting temperature; MD, molecular dynamics; FP, fluorescence polarization; DSF, differential scanning fluorimetry


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Analysis of MEMO1 Binding Specificity for ErbB2 using Fluorescence Polarization and Molecular Dynamics Simulations Madeline L. Newkirk†, Kristen J. Rubenstein†, Jessica Y. Kim, Courtney L. Labrecque, Justin Airas, Cooper A. Taylor, Hunter D. Evans, Quincy McKoy, Carol A. Parish, and Julie A. Pollock*


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Figure 1. A. 5-mer (pYD5) and B. 10-mer (pYD10) ErbB2 peptide tails/ligands used in generating binary complexes via docking. 175x77mm (300 x 300 DPI)

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Figure 2. Results of FP Assay with wild type MEMO1. A. Binding curve of FL- pYD10 (0.1 µM, red) and FLYD10 (0.1 µM, gray) titrated with wild type MEMO1 (0 – 4.5 µM) in sodium phosphate buffer (pH 6.4). Anisotropy was adjusted as described in the methods section to account for changes in fluorescence intensity (see Supplemental Fig. S3 for raw data). Average data points from triplicate experiments (± S.D.) for FL-pYD10 are fit with a non-linear regression giving KD = 0.532 µM. B. Binding curves of FL-pYD10 (0.1 µM) titrated with wild type MEMO1 (0-4.5 µM) in sodium phosphate buffers, pH 5.7 – 7.4. Average data points from triplicate experiments (± S.D.) are fit with non-linear regressions with KDs listed (right). C. KD values (± S.D.) calculated from FP assays with FL-pYD10 (0.1 µM) and wild type MEMO1 (0 – 4.5 µM) in various buffers as indicated. D. Displacement of FL-pYD10 by unlabeled peptide (10-mer pYD10, green or 5mer pYD5, red) in sodium phosphate buffer (pH 6.4). Average data points from triplicate experiments (± S.D.) are fit with non-linear regression. 190x100mm (96 x 96 DPI)


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Figure 3. A. Representative RMSD of the apo-MEMO1 ensemble relative to the solvated and equilibrated experimental structure. Data for the additional 9 trajectories can be found in Supplemental Table S3. B. Superimposition of the 6 most populated clusters obtained by MMTSB clustering. Red circles highlight regions of disorder as suggested by clustering and RMSF analysis. C. Sitemap analysis of the MEMO1 structure. 128x144mm (96 x 96 DPI)


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Figure 4. A. Representative RMSD values for the 10-mer:MEMO1 system, protein and peptide, relative to the equilibrated structure used to initiate the simulation. All trajectories are shown in Supplemental Tables S5S6. B. Representative MEMO1:10-mer structure highlighting residues that contribute the most to binding as determined by the MMGBSA approximation of interaction energies. MEMO1 shown in teal ribbon, pYD10 peptide shown in red and computationally significant residues are labeled. Per residue interaction energies are shown below in Table 4. (Representative structure taken from 10-mer simulation initiated from Glide Pose 1, Seed 1, frame 142.) 146x102mm (300 x 300 DPI)


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Figure 5. A. Binding curves of FL-pYD10 (0.1 µM) titrated with wild type or mutant MEMO1 (0-4.5 µM) in sodium phosphate buffer (pH 6.4). B. Binding curves of FL-pYD10 (0.1 µM) titrated with mutant MEMO1 (04.5 µM) in sodium phosphate buffer (pH 6.4). Average data points from triplicate experiments (± S.D.) are fit with non-linear regression. KD values are listed in Supplementary Table S11. 182x53mm (96 x 96 DPI)


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Figure 6. Residues important for peptide MEMO1 binding. A. Asp2 interactions with Arg198 via a double hydrogen bond interaction. Bond lengths 1.90 and 1.85 Å. B. pYD hydrogen bonding with NH2 group on Arg 196. Bond lengths 1.76 and 2.50 Å. C. MEMO1 Tyr 54 forming a bifurcated hydrogen bond with peptide Asp 10. Bond lengths 1.88 and 2.66 Å. D. T-shaped pi stacking interaction between pYD and peptide Trp7. Bond lengths shown are measured in representative structures. Representative structure in A and C taken from lowest energy structure in the 10-mer trajectory initiated from Glide Pose 1, Seed 1. Representative structure in B and D taken from lowest energy structure in the 10-mer trajectory initiated from reoriented Pose 1, Seed 1. Maximum, minimum and averaged distances from complete trajectories can be found in Supplemental Table S15. 240x207mm (300 x 300 DPI)


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