Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment

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Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide #-Hairpin Alexandre Iourievich Ilitchev, Maxwell J. Giammona, Carina Olivas, Sarah Louise Claud, Kristi Lazar Cantrell, Chun Wu, Steven K. Buratto, and Michael T. Bowers J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b05925 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of the American Chemical Society

Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide βHairpin Alexandre I. Ilitchev†, Maxwell J. Giammona†, Carina Olivas‡, Sarah L. Claud⊥, Kristi L. Lazar Cantrell⊥, Chun Wu‡, Steven K. Buratto†, Michael T. Bowers†* † Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, United States ‡ Department of Chemistry and Biochemistry, Rowan University, Glassboro, New Jersey 08028, United States ⊥ Department of Chemistry, Westmont College, Santa Barbara, CA 93108, United States

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ABSTRACT Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Co-aggregation of heterogeneous peptide species can occur however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP106-126) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of non-homologous protein assembly as they co-aggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only formed pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic β-hairpin conformation. Further, when PrP106-126 was combined with nonamyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing β-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic β-hairpin structure, it did so in the presence of PrP106-126 and underwent a conformational transition to β-sheet in solution. We also find that PrP106-126 forms hetero-oligomers with the IAPP8-20 fragment but not with the “aggregation hot spot” IAPP20-29 fragment. PrP106-126 apparently induces IAPP into a -hairpin structure within the PrP:IAPP hetero-dimer complex and then, through ligand exchange, catalytically creates the amyloidogenic -hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model which provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.


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INTRODUCTION The prion hypothesis posits that disease can be initiated and proliferated by misfolded protein species.1-2 While the fundamental debate surrounding the validity of this hypothesis appears to have been settled,3-5 the extent to which misfolded structures can have this infectious effect on healthy functioning protein is still not fully understood.6-8 Much of the current work in the field of amyloid aggregation focuses on self-association and homo-oligomerization of a single protein species and how one misfolded protein may induce structural changes in healthy functioning copies. There is mounting evidence, however, that aggregation is not limited to homo-oligomers, and that distal amyloid protein can act similarly to prions and induce and proliferate seemingly unrelated illnesses.8-10 The interplay and co-aggregation of Tau and Aβ protein in Alzheimer’s disease is a known instance of two proteins, or protein fragments, with limited sequence homology creating hetero-oligomeric plaques composed of both species.11-12 Numerous studies have also linked aggregation of Aβ with the peptide directly linked to type-II diabetes, islet amyloid polypeptide (IAPP),13-19 and distinct hetero-fibril aggregates have been observed in vitro.20-23 In vivo aggregation prone IAPP has been found in the brains of Alzheimer’s patients, inextricably linking the two illnesses.24-25 Finally, both non-toxic and toxic forms of the prion protein (PrPC and PrPSc) have been found to associate with both Aβ and IAPP, indicating that prion-like interplay can occur between numerous amyloidogenic protein species.26-30 IAPP and the critical amyloidogenic and neurotoxic fragment of PrP (PrP106-126)31-34 have been of particular interest as a model of amyloid interaction as they exhibit little to no sequence homology and yet mixtures may still produce heterogeneous fibrils and aggregate more aggressively than each individual peptide by itself.30,35 Understanding the nature of this process would provide key insight into the broader driving forces of amyloid species acting as prions. 3 ACS Paragon Plus Environment


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The primary sequences of IAPP and PrP106-126 are given in Scheme 1. Molecular modeling

Scheme 1. Sequences of the peptides studied in this work; human IAPP (hIAPP) is shown in black, rat IAPP (rIAPP) is shown in blue, and the 106-126 prion protein fragment (PrP106-126) is shown in green. The presence of a disulfide bond is indicated by a bracket. Both human and rat IAPP contain an amidated C-terminus. Sequence differences in rIAPP from hIAPP are shown in red. studies have suggested that hetero-oligomers of hIAPP and PrP106-126 are primarily driven by hydrophobic interactions, and that the amyloidogenic region of human IAPP (hIAPP), 20-29, acts as a significant association site with PrP106-126.35 In order to probe oligomer formation in vitro and to elucidate more about the structural changes that occur to both species in their early oligomeric states, we consider not only the interaction of hIAPP with PrP106-126 but also the interaction of non-amyloidogenic rat IAPP with PrP106-126. As 5 of the 7 sequence differences between hIAPP and rIAPP occur in the 20-29 region (including 3 β-sheet breaking proline substitutions in rIAPP) one would expect a substantial difference in the interaction between these two peptides with PrP106-126 if the 20-29 region of IAPP is involved. To test this hypothesis we have employed a mixture of ion mobility spectrometry - mass spectrometry (IMS-MS) and replica exchange molecular dynamics (REMD). IMS-MS allows for the observation of native structures and oligomers through the use of a soft nano-electrospray source and very soft 4 ACS Paragon Plus Environment

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instrumental treatment. These conditions allow for sampling of solution-phase structures which are distinguished from gas-phase conformations based on collision cross-section.36 To gain molecular insight on early dimer formation, REMD simulations of PrP106-126 with hIAPP/rIAPP were performed. Additionally, Far-UV circular dichroism (CD) experiments were utilized to observe conformational transitions in solution with AFM complimenting these measurements by allowing for the concurrent observation of higher order oligomer structures not easily observed by ion mobility or CD. Finally, we interrogate the interaction of PrP106-126 with the 1-8, 8-20, 2029 and 29-37 fragments of hIAPP in order to shed light on which region of IAPP is favored during hetero-oligomerization. Together these complimentary techniques and reaction mixtures give deep insight into hetero-oligomer assembly in vitro and the possible mechanism for prionlike cross talk between amyloid systems.

MATERIALS AND METHODS Sample Preparation. Rat and human IAPP, as well as 1-8, 8-20, 29-37 hIAPP fragments were purchased from Genscript (Piscataway, NJ). 20-29 hIAPP and 106-126 PrP fragments were purchased from Anaspec (Fremont, CA). Rat IAPP, human IAPP, and 29-37 hIAPP have amidated C-termini. Peptide samples (>95% purity via HPLC) were used without further purification. Dry samples were dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-isopropanol (Fisher Scientific, Los Angeles, CA) to a concentration of 1 mM and 10 μL aliquots were taken and dried under vacuum overnight. Aliquots were re-suspended in 1:1 water:methanol (pH = ~7.4 unless otherwise noted) to the desired concentration immediately before use. Ion Mobility Mass Spectrometry. All samples were analyzed on a home-built nanoelectrospray ion-mobility mass spectrometer (IMS-MS), described in detail previously.37 Sample 5 ACS Paragon Plus Environment


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spray tips are created by pulling borosilicate glass capillaries to a fine point using a capillary puller (Sutter Instrument Co., Novato, CA, USA) and are coated with gold on an Emitech K550X sputter coater (Quorum Technologies, East Sussex, UK). Sample is then pulled from the spray tips by a nanoelectrospray ionization source into an ion funnel via a stainless steel inlet (~70 μm orifice). The funnel collects and stores the ions, which can either be channeled directly to a quadrupole mass analyzer to generate a mass spectrum or pulsed at regular intervals through a 4.5 cm drift cell in order to generate an arrival time distribution (ATD). The uniform electric field applied across the drift cell is tuned in order to determine the experimental collision cross section as a function of reduced mobility. In some experiments the energy with which the ions are pulsed into the drift cell is changed. This allows interrogation of the various mass spectral peaks and ATDs. An increase in injection energy transiently heats the injected species allowing either dissociation of oligomers or conformer rearrangement. Details of this process can be found elsewhere.38 All IMS-MS experiments were performed a minimum of 4 times with a coefficient of variance less than 5% for all reported cross sections. Additional IMS-MS details are provided in the Supporting Information. Atomic Force Microscopy. After 1 hour of incubation, peptide samples prepared for the IMS-MS experiments were diluted 10x to a total sample concentration of 5 μM and deposited on freshly cleaved V1-grade muscovite mica (TedPella, Redding, CA). Deposited samples were dried under vacuum overnight and analyzed using tapping-mode AFM on a MFP-3D atomic force microscope (Asylum Research, Goleta, CA) using a silicon probe with a nominal spring constant of 7 N/m and resonant frequency of 155 kHz (MikroMasch USA, Lady’s Island, SC). All images were collected, in air, in the repulsive force regime. The samples were not washed following deposition on the mica, allowing accurate determination of macroscopic structures in 6 ACS Paragon Plus Environment

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solution.39 Far-UV Circular Dichroism. CD data were collected on a Jasco J-810 CD spectrometer equipped with a temperature-controlled holder. The CD measurements were collected at 1 nm intervals from 260 to 190 nm with a 4 s response time and a 1 nm bandwidth. All spectra were collected in a 1 mm quartz cuvette at 20ºC. The CD spectra of freshly dissolved (t = 0) PrP 106-126, hIAPP, and rIAPP were measured at a concentration of 50 μM in 1:1 water:methanol. The samples were stored at room temperature and run again after one day (t = 24 hours). Ten scans were collected at each time point, and the data were averaged. A buffer baseline was subtracted from the averaged data. Mixtures of freshly dissolved 1:1 (50:50 μM) hIAPP:PrP106-126 and 1:1 (50:50 μM) rIAPP:PrP106-126 were measured in 1:1 water methanol as well. The samples were monitored over time to detect the formation of β-sheet. Replica Exchange Molecular Dynamics. The AMBER 8 simulation suite was used in replica exchange molecular dynamics (REMD) simulations of PrP106-126 with hIAPP/rIAPP.40-41 The starting monomer conformation of PrP106-126 with +2 charge was taken from our previous study.33 The starting monomer conformations for +3 hIAPP and +4 rIAPP were taken from our previous REMD simulations in implicit solvent.42 The two monomers (a PrP106-126 monomer plus a either an hIAPP or rIAPP monomer) were started 20 Å apart in order to model their association (Figure S1). These peptides were modeled using the AMBER all-atom point-charge protein force field, ff96.43 Solvation effects of water solvent were represented by the implicit solvent model (IGB = 5) plus the surface term (gbsa = 1, 0.005 kcal/Å2/mol) with an effective salt concentration of 0.2 M.44 Studies in the Dill group have examined a number of force field/ implicit solvent combinations and have concluded that ff96/IGB5 offers a good balance between helical and sheet propensities.45-46 This combination, in conjunction with REMD simulation 7 ACS Paragon Plus Environment


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yielded impressive results in the folding of both small α, β and α/β proteins with a well-defined native fold and natively unfolded peptides including hIAPP and rIAPP,33,42,47-49 and in predicting correct inter-domain orientation of a large multi-domain protein (CheA).50 The simulation protocol closely followed the one described in the methods section of reference 42 and the salient points are highlighted in the supporting information.


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RESULTS AND DISCUSSION Interaction of PrP106-126 with human and rat IAPP reveals similar hetero-oligomeric structures. Previous molecular dynamics simulations of PrP106-126 and hIAPP heterooligomerization indicated that hydrophobic interaction is one of the primary driving forces of coaggregation, with particular interaction occurring between PrP106-126 and the 20-29 aggregationprone region of hIAPP.35 In order to experimentally probe this result we have looked at the interaction of PrP106-126 with both hIAPP and rIAPP, since rat IAPP contains several proline substitutions within the region in question (Scheme 1). It has been suggested that these prolines are likely the primary reason that rat IAPP is non-amyloidogenic.51-52

Figure 1. Positive mode mass spectra of (A) human IAPP, PrP106-126, and a 1:1 mixture of each as well as (B) rat IAPP, PrP106-126, and a 1:1 mixture of each. Total peptide concentration in each sample was 50μM. Peaks are annotated with oligomer size to charge ratios (n/z), where contributions of both peptides to the total oligomer number are noted in the bottom panels.



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If the critical inter-peptide interaction does occur within the 20-29 region of hIAPP, we would expect hIAPP to co-associate with PrP106-126 while rIAPP would not. What we observe, however, is that both human and rat IAPP form similar quantities of co-aggregates, as seen in their respective mass spectra (Figure 1). Additionally, the hetero-oligomer peaks exhibit the same distribution of features in their ATDs with similar cross-sections for all conformations (Figure 2). These results imply that the interaction between the two IAPP species and PrP106-126 is similar and most likely does not involve the 20-29 region, since it is disparate for hIAPP and rIAPP.

Figure 2. Arrival time distributions of hetero-oligomer peaks from 25:25 μM solution of PrP106-126 with either human (A,C) or rat (B,D) IAPP. Each peak is fit using the procedure described in the Supporting Information. Peak shapes for a single conformer at the cross section noted are given by the dotted lines. The peaks are labeled with oligomer size to charge ratios (n/z) and the cross section of each feature.


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Figure 3. Arrival time distributions of +4 monomer peaks for (A) 50 μM human IAPP (~976 m/z), (B) human IAPP in a 25:25 μM hIAPP:PrP106-126 solution mixture, (C) 50 μM rat IAPP (~980 m/z), and (D) rat IAPP in a 25:25 μM rIAPP:PrP106-126 solution mixture. The presence of PrP106-126 not only amplifies the extended, β-sheet conformation in hIAPP but also induces a similar conformation in rIAPP. Each peak is fit using the procedure described in the Supporting Information, as indicated by the dotted lines. The cross section of each feature is noted.

PrP106-126 induces β-hairpin conformation in both human and rat IAPP. The interaction of PrP106-126 with both IAPP species not only forms new hetero-oligomers but also induces changes in the conformational distribution of the IAPP monomer peaks. Our group has previously reported that the key structural difference between human and rat IAPP is that hIAPP 11 ACS Paragon Plus Environment


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Figure 4. Mass spectra of a 1:1 mixture of hIAPP:PrP106-126 at increasing drift cell injection energies (A). All non-hetero-oligomeric peak identities are assigned in Figure 1. As injection energy is increased the relative amplitudes of the hetero-oligomer peaks decrease, indicating that they are being dissociated into hIAPP and PrP106-126 monomers. This is corroborated in the ATDs (B-D) as higher order hetero-oligomers disappear at high injection energies while more of the extended hIAPP monomer appears. Each ATD peak is fit using the procedure described in the Supporting Information, as indicated by the dotted lines. ATDs are annotated by oligomer size to charge (n/z) and cross section. 12 ACS Paragon Plus Environment

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adopts two conformations, one extended and one compact, while rIAPP only adopts a compact structure.42 Replica exchange molecular dynamics results indicate the extended structure is an amyloidogenic β-hairpin conformation of hIAPP.53 In the presence of PrP106-126 more of this extended structure is detected for hIAPP than is typically present in pure hIAPP samples (Figure 3.A,B). Additionally, the ATD of +4 rIAPP shows the appearance of a new peak with a similar cross-section to the hIAPP β-hairpin, implying PrP106-126 catalytically transforms helix-coil rIAPP to the β-hairpin structure. In order to determine whether PrP106-126 is acting as a catalyst on the structural distribution of IAPP monomers observed in the IMS-MS results, we modulated the injection energy into the drift cell and observed the effect on dissociation of the hetero-oligomers and on the distribution of hIAPP monomer structures. As we increase injection energy, we see a decrease in the relative amount of hetero-dimer peaks in the mass spectrum (Figure 4.A), demonstrating that we are dissociating these oligomeric structures into individual PrP106-126 and hIAPP monomers. We also observe the appearance of a previously unseen +1 charge state of PrP106-126 at high injection energies. This charge state is likely the product of dissociation of the (1+1) / +5 hetero-dimer, with the partner product being a +4 hIAPP species. Additionally, we see an increase in the extended conformation of the +4 monomer of hIAPP in the ATDs and simultaneously see a loss of higher order aggregates of hIAPP:PrP106-126 (Figure 4.B-C). Injection energy studies on pure hIAPP (Reference 42, Figure 4) indicate increased injection energy decreases the relative abundance of the extended conformer of hIAPP relative to the compact form. Hence the data in Figure 4 B,C suggest that the IAPP moiety in the hetero-oligomer PrP/hIAPP dimer exists as an extended conformer. They also suggest further reaction of these hetero-dimers may lead to conformational conversion of the compact helix-coil form of hIAPP to the amyloidogenic 13 ACS Paragon Plus Environment


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extended β-hairpin form.

Figure 5. CD spectra of (A) 50 μM PrP106-126, (B) 50 μM hIAPP, and (C) 50 μM rIAPP in 1:1 water:methanol at t = 0 and t = 24 hours. CD spectra of mixtures of (D) 1:1 (50:50 μM) hIAPP:PrP106-126 and (E) 1:1 (50:50 μM) rIAPP:PrP106-126 in 1:1 water:methanol at variable times up to t = 24 hours.

Far-UV Circular Dichroism spectra of mixtures of PrP106-126 with both human and rat IAPP show prion induced conformational conversion of IAPP from α-helix to -sheet. The CD spectra of PrP106-126, hIAPP, rIAPP and mixtures of PrP106-126 with hIAPP and rIAPP are given in Figure 5. The spectra for PrP106-126 are given in panel 5A and are similar to a previous result obtained in 10mM HEPES buffer, pH 7.4.54 The strong negative band centered at 203 nm at t = 0 is characteristic of an unfolded protein.55 After 24 hours the spectrum changes to one characteristic of -sheet with a negative band near 217 nm and a positive band at 193 nm, indicating a random coil to -sheet transition has occurred in solution.56 The spectra for human 14 ACS Paragon Plus Environment

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IAPP and rat IAPP are given in panels 5B and 5C respectively. These peptides (especially rat) show minima at 208 and 222 nm characteristic of -helical secondary structure.56 After 24 hours hIAPP has transitioned to primarily -sheet while rIAPP is unchanged, consistent with the facts that hIAPP readily forms fibrils and rIAPP does not.57-58 The spectra for mixtures of PrP106-126 and both human and rat IAPP are given in panels 5D and 5E. The results for the hIAPP mixture were expected; after 24 hours the peptides in solution are primarily -sheet. The results for the rIAPP mixture are both more revealing and interesting. They indicate a relatively rapid transition from dominant -helical structure to -sheet within 1.5 hours. At the same time point the hIAPP mixture was still primarily -helical. These rat IAPP observations are wholly consistent with the IMS-MS findings that indicate PrP106-126 induces the -hairpin extended conformation in rIAPP. The results for both IAPP peptides indicate that PrP106-126 transforms them from primarily -helix to primarily -sheet/-hairpin, strongly implicating that this occurs in the PrP/IAPP dimer complex which either then dissociates to yield IAPP -hairpin or ligand exchanges to form IAPP -hairpin dimer, in both cases regenerating PrP106-126. Atomic force microscopy shows disparate macroscopic hetero-oligomeric aggregates for human and rat IAPP. In order to ascertain what effect the shift in oligomer and conformation distribution had regarding pre-fibrillar and other macroscopic structures formed in solution, atomic force microscopy was conducted in parallel to the IMS-MS experiments. As seen in Figure 6, human IAPP forms extensive proto-fibrils and PrP106-126 generates short, pre-fibril structures in solution whereas rat IAPP creates exclusively disordered globular structures. When hIAPP is combined with PrP106-126 the overall density of fibrils detected by AFM decreases, potentially signifying that the homo-oligomeric aggregates are dissociating in order to form 15 ACS Paragon Plus Environment


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hetero-oligomeric structures and these hetero-oligomeric structures do not proceed to form fibrils. Mixing of rat IAPP with PrP106-126 qualitatively appears to have little to no effect on either rIAPP or PrP106-126 structures, instead showing a dilute distribution of both, again with no heterooligomeric structures apparent. Since the CD results indicate a relatively rapid transformation from -helix to -sheet in these mixtures it appears that PrP106-126 is very effective in converting rIAPP to the extended -hairpin conformer but these conformers do not subsequently go on to form proto-fibrils, unlike the hIAPP -hairpins.

Figure 6. Comparative AFM images of (A.i) 5 μM PrP106-126, (A.ii) 5 μM hIAPP, (A.iii) 1:1 (2.5:2.5 μM) hIAPP:PrP106-126, and (B.i) 5 μM PrP106-126, (B.ii) 5 μM rIAPP, (B.iii) 1:1 (2.5:2.5μM) rIAPP:PrP106-126. All samples were incubated at IMS experiment concentrations for 1 hour and subsequently diluted 10x before deposition onto the mica surface. Qualitative observations of the heterogeneous peptide mixtures do not appear to show any novel macroscopic assemblies. 16 ACS Paragon Plus Environment

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Particle height analysis (Figure S2) shows a combination of PrP106-126 and rIAPP particles in the mixture with few, if any, newly formed aggregate particles, consistent with the above analysis. Overall, these results illustrate that the 1:1 interaction between PrP106-126 and either hIAPP or rIAPP does not lead to large hetero-oligomeric proto-fibrils in solution. Hence, while PrP106-126 induces conformational changes in IAPP monomers, these monomers continue to preferentially self-associate. These results are consistent with a mechanism where heterooligomerization acts catalytically and generates structures with exposed coordination sites for homo-oligomeric assembly, especially in hIAPP. Fragment interaction analysis implicates hIAPP8-20 as the primary interaction site of PrP106-126 and hIAPP. In order to localize the primary interacting region of IAPP with PrP106-126 a piece-wise analysis of hIAPP fragments was performed. The 1-8, 8-20, 20-29, and 29-37 regions of hIAPP have all been shown to be capable of self-assembly.59-62 We have therefore individually combined each of these fragments with PrP106-126 to ascertain their aggregation propensity with the prion fragment (Figure S3). Of these, only 8-20 hIAPP formed new peaks corresponding to a 1:1 adduction of hIAPP8-20 with PrP106-126 (Figure 7.A), forming aggregates up to (2+2) PrP106-126:hIAPP8-20 (Figure 7.B). The observation of this interaction is interesting for several reasons. Both PrP(His111) and hIAPP(His18) aggregation appear to be modulated significantly by the protonated state of their histidine residues: PrP adopts extended, aggregation prone conformations in acidic environments due to histidine protonation and hIAPP has been shown to lose its aggregation propensity upon histidine protonation due to charged side-chain repulsion.63-66 When the pH of the PrP106-126:hIAPP8-20 mixture is lowered, a marked loss of highorder hetero-oligomer peaks is seen in the ATDs, indicating that the unprotonated state of 17 ACS Paragon Plus Environment


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histidine is essential to the hetero-aggregation process (Figure S4).

Figure 7. Combined mass-spectra of hIAPP8-20, PrP106-126, and a mixture of PrP106-126:hIAPP8-20 (A). Newly formed peaks in the mixture are shown at 15x magnification. The ATD of the newly formed (1+1) / +3 peak is given in inset (B). The two fragments form a range of oligomeric structures up to a (2+2) / +6 tetramer. Each ATD peak is fit using the procedure described in the Supporting Information, as indicated by the dotted line.

A possible scenario explaining these observations requires the -hairpin of PrP106-126 to associate with the 8-20 region of IAPP. In full length IAPP this region is helical and the association of the PrP hairpin could then extend it, allowing a localized -sheet type interaction between the two peptides in the dimer complex. Once the 8-20 region of IAPP opens up it becomes thermodynamically reasonable for the C-terminal region of the peptide to also open, creating a -hairpin IAPP moiety interacting with the PrP106-126 -hairpin in the dimer complex, 18 ACS Paragon Plus Environment

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forming a PrP(hairpin):IAPP(hairpin) motif. Dissociation or ligand exchange of this complex then regenerates PrP106-126 -hairpin and increased IAPP -hairpin monomer or dimer.

Figure 8. Arrival time distributions of the hetero-oligomers of hIAPP and rIAPP with PrP106-126 (A) as well as the +4 monomer of human (B) and rat IAPP (C) by themselves and in 1:1 mixtures with PrP106-126. The mixtures are shown at both pH = 7.5 and pH = 4.5. Low pH appears to inhibit the conformational effects of PrP106-126 on both IAPP species, although the effect is more pronounced for human than rat IAPP. Each peak is fit using the procedure described in the Supporting Information. pH studies corroborate interaction of PrP106-126 with hIAPP at 8-20 region as acidity modulates hetero-oligomer formation. Previous studies have proposed that the protonated state of His18 is a fundamental contributor to the aggregation propensity of hIAPP,65 particularly because aggregation has been shown to be inhibited in acidic environments.67-68 Insulin granules 19 ACS Paragon Plus Environment


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do not show pronounced intra-vesicular amyloid aggregation, despite containing a high concentration of hIAPP, and it is believed to be in part due to the acidic (pH = ~5) environment within the vesicle.69 The interaction of PrP106-126 with the 8-20 region could therefore be sensitive to the protonated state of both hIAPP at His18 as well as His111 of PrP106-126. When we probed the full length hIAPP:PrP106-126 interaction at solution pH = 4.5 we observed two results of note. First, the oligomer distribution shifts for hIAPP. While hetero-oligomers are still formed between the PrP fragment and hIAPP, they contained far fewer higher order oligomers than at pH = 7.5 (Figure 8.A). For rIAPP, with an arginine at position 18, little change is observed since Arg is protonated at both pH values. Additionally, the +4 monomer distribution of rIAPP and hIAPP at pH = 4.5 now mirrors that of pure rat or human IAPP at pH = 7.5 (Figure 8.B,C). Therefore, it appears that the protonated state of histidine plays a crucial role in the hetero-oligomerization of IAPP species with PrP106-126, indicating that there is a loss of aggregate stability and aggregation propensity as histidine protonation occurs.

The REMD simulations between IAPP monomers and PrP106-126 monomers show hIAPP forms a four stranded β-sheet and a compact helix-4-stranded-sheet with PrP106-126, and rIAPP also forms the compact helix-4-stranded-sheet with PrP106-126. Our earlier studies indicate that the +2 PrP106-126 monomer at neutral pH mainly adopts a β-hairpin conformation and a compact coil conformation.33 The +3 and +4 hIAPP peptide at neutral pH have three major conformations: helix-hairpin, compact β-sheet rich and extended β-hairpin. The +4 rIAPP at neutral pH has two major conformations: helix-coil and coil-rich (Figure 5 of Ref. 42). Given that +3 hIAPP and +4 hIAPP adopt similar conformations in solution, only +3 hIAPP was used in our REMD simulation. Four heterodimer systems (Figure 9) were constructed for 600 ns 20 ACS Paragon Plus Environment

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Figure 9. Heterodimer formation observed in the REMD simulations. (A) hIAPP hairpin + PrP106-126, hairpin, (B) hIAPP hairpin + PrP106-126 Coil, (C) hIAPP helix-hairpin + PrP106-126 hairpin, and (D) rIAPP helix-coil + PrP106-126 hairpin. The left column (i) shows the initial setup of the four systems while the right column (ii) shows the most abundant conformation from the REMD simulations. Conformations of the initial +3 hIAPP, +4 rIAPP, and +2 PrP106-126 moieties are taken from our earlier studies.33,42 The N-terminal is indicated by a red ball and the Cterminal is indicated by a blue ball.

REMD simulations: hIAPP hairpin + PrP106-126, hairpin (Figure 9.A), hIAPP hairpin + PrP106-126 coil (Figure 9.B), hIAPP helix-hairpin + PrP106-126 hairpin (Figure 9.C), and rIAPP helix-coil + PrP106-126 hairpin (Figure 9.D). Detailed analyses of these simulations, including secondary and tertiary structure, are included in the supporting material (Figure S5-S12). The most abundant heterodimers from the clustering analysis are shown in Figure 9. When hIAPP hairpin interacted with PrP106-126 hairpin, a direct association between the β-strands was observed to form a four 21 ACS Paragon Plus Environment


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stranded complex (Figure 9.A). Interestingly, when hIAPP hairpin interacted with the PrP106-126 coil, PrP106-126 underwent the coil-to-sheet transition, leading to the formation a similar four stranded complex (Figure 9.B).

The coil-to-β-sheet conversion was clearly shown in the

secondary structure development of PrP106-126 (Figure S7.B). When hIAPP helix-hairpin interacted with PrP106-126 hairpin, the hairpin-hairpin association was observed to form a helix-4stranded-sheet compact heterodimer (Figure 9.C). For the interaction of rIAPP helix-coil with PrP106-126 hairpin, it is interesting to note that the coil region (20-37) of rIAPP underwent coil-toβ-sheet conversion, leading to the formation of a compact helix-4-stranded-sheet (Figure 9.D). The coil to β-sheet conversion was clearly shown in the secondary structure development of rIAPP (Figure S6.B). Consistently, the calculated collision cross sections (CCS) for the four stranded β-sheet and the compact helix-4-stranded-sheet of hIAPP+PrP106-126 dimers are ~1025 Å2 and ~948 Å2, which are close to the values of the two major peaks (1017 Å2 and 931 Å2) in the ATD of

hIAPP+PrP106-126 (Figure 2.C). The calculated CCS of the compact helix-4-

stranded-sheet of rIAPP+PrP106-126 dimer is ~932 Å2, which is again close to the value of the small peak in the ATD of hIAPP+PrP106-126 (Figure 2.D). Therefore, the four stranded β-sheet and the compact helix-4-stranded-sheet are thus excellent candidates for the species observed in the experiment. The direct observation of coil to β-sheet conversion provides a good explanation for the hetero-oligomeric structures seen in the IMS-MS which appear to have β-sheet content and for the CD results where -helix evolves to -sheet in all systems.


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CONCLUSIONS Comparative studies of human and rat IAPP in association with an important prion protein fragment show that the hetero-oligomerization propensity and aggregation pathways of disparate amyloid peptides are largely contingent on hydrophobicity rather than sequence homology. We have proposed a new model of hIAPP and PrP106-126 interaction based on previous research in the field as well as our current findings (Figure 10).33,42,53 PrP106-126 and hIAPP appear to associate either into a compact or extended structure which we attribute to either a helix-coil/β-hairpin or β-hairpin/hairpin conformation respectively, with the β-hairpin/hairpin being the most stable (Figure 4.C). Given that we do not see hetero-oligomeric proto-fibril formation by AFM, we propose that the extended hetero-oligomer exists in equilibrium with the hIAPP homo-oligomer, acting catalytically to induce β-hairpin hIAPP association. This is a new mechanistic and potentially general model for amyloid interaction in vivo. The key point being that the “prion” effect can take place very early in the assembly process and the observation of large heterocomplexes is not required for one amyloid system to affect, or even drive, the assembly of another amyloid. Our results indicate that the protonation state of histidine on both hIAPP and PrP106-126 contributes in important ways to the types of hetero-oligomeric aggregates we observe. This is significant as many amyloid species are sensitive to pH shifts due to histidine inclusions. We see an increase in the amyloidogenic β-hairpin conformation of hIAPP when it is in the presence of PrP106-126 at pH=7.5. Rat IAPP, which natively does not adopt a β-hairpin, also exhibits a hairpin conformation in the presence of the prion fragment based on the IMS-MS results. The formation of this extended confirmation is also consistent with the observed -helix to -sheet conversion observed by CD and is in full agreement with our MD simulations. Previous 23 ACS Paragon Plus Environment


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molecular dynamics simulations on hIAPP and PrP106-126 mixtures in the literature have suggested PrP106-126 interacts primarily with the 20-29 region of hIAPP.35 However, this region contains proline at positions 25, 28, and 29 for rIAPP, which should inhibit interaction with PrP106-126 and our results suggest it is only marginally involved in the PrP/IAPP interaction, if at all. We observe that these structural transformations are diminished especially for human but also rat IAPP at pH 4.5. Previous work in our group has shown that homo-oligomerization of the 8-20 region of hIAPP occurs and this assembly is dependent on the presence of unprotonated histidine at residue 18.70 Through fragment-fragment interaction we have found that the primary site of association between hIAPP and PrP106-126 occurs within this same 8-20 region, and that this interaction can be modulated by changes in the acidity of the solution. These findings are consistent with the 8-20 region, and His18 in particular, being a critical contributor to the amyloidogenesis of islet amyloid species and to the hetero-oligomerization of IAPP with PrP. It is interesting that low pH has previously been shown to destabilize aggregation-prone β-hairpin oligomers in PrP106-126, demonstrating that the unprotonated state of His111 plays a key role in the structure of the prion protein, an observation consistent with the results presented here.71-72 Overall, our findings indicate that hydrophobic interactions between regions of different peptides with no sequence overlap are sufficient to drive helix to sheet conversion and subsequent transition into amyloidogenic conformations. This is a critical new mechanistic result for “prion-like” inter-amyloid interaction dynamics and highlights the potential ubiquitous behavior of amyloids as prion-like species.


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Figure 10. Proposed reaction schematic of hetero-oligomer formation between hIAPP and PrP106-126. Illustrations of structures adapted from Ref. 33 and 42 and the MD results presented here. Hetero-oligomer formation is shown in (A) where we propose that the two 1:1 associated ATD peaks are an extended β-hairpin/β-hairpin and a mixed coil/hairpin. Homo-oligomerization of IAPP occurs either through standard hIAPP monomer interaction33,42 (B) or is catalytically induced through the extended hetero-oligomer (C). We propose this process is reversible, as the AFM data shows fewer fibril structures in 1:1 PrP106-126:hIAPP than in solutions containing only hIAPP (Figure 6.A), implying that homo-oligomeric hIAPP fibrils are dissociating to form hetero-oligomers.



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ASSOCIATED CONTENT Supporting Information. Ion mobility experimental details, atomic force microscopy analysis details, replica exchange molecular dynamics simulation details, initial monomer structures of REMD simulations, AFM particle height analysis, comparative mass spectra of PrP106-126 with hIAPP fragments, REMD simulation results, solvent comparison for hIAPP:PrP106-126 mixtures, injection energy studies of 1/+4 hIAPP in hIAPP:PrP106-126 mixtures, and comparative arrival time distributions of 1:1 PrP106-126 : hIAPP8-20 hetero-oligomers at pH = 7.5 and pH = 4.5. AUTHOR INFORMATION Corresponding Author *Michael T. Bowers, Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, USA. E-mail: [email protected]. Phone: 805-893-2893. Fax: 805-893-8703. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT Support from the National Science Foundation under grant CHE-1565941 (M.T.B.), and MRI1429467 and XSEDE MCB 160164/160173/170088 (C.W.) and the Stauffer Summer Chemistry Endowment (S.L.C.) are gratefully acknowledged. 26 ACS Paragon Plus Environment

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Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment

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