1 Calcium-mediated allostery of the EGF fold Conan K. Wang , Hafiza

Subscriber access provided by Kaohsiung Medical University

Article

Calcium-mediated allostery of the EGF fold Conan K Wang, Hafiza Abdul Ghani, Anna Bundock, Joachim Weidmann, Peta J. Harvey, Ingrid A Edwards, Christina I Schroeder, Joakim E. Swedberg, and David J Craik ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00291 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Calcium-mediated allostery of the EGF fold

Conan K. Wang*, Hafiza Abdul Ghani, Anna Bundock, Joachim Weidmann, Peta J. Harvey, Ingrid A. Edwards, Christina I. Schroeder, Joakim E. Swedberg, David J. Craik1,*

1

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia

*Corresponding Authors: Dr Conan K. Wang or Professor David J. Craik Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia Tel: 61-7-3346 2019 Fax: 61-7-3346 2101 E-mail: [email protected] or [email protected]

Keywords: disulfide-bond, epidermal growth factor, low-density lipoprotein, folding, calcium

1 ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The epidermal growth factor (EGF)-like domain is one of the most abundant disulfide-containing domains in nature and is involved in many cellular processes critical to life. Although many EGF-like domains participate in calcium-dependent functions by responding to the local calcium concentration, little is known about how this responsiveness is programmed at the molecular level. Here, we reveal the structural and environmental determinants underpinning the folding of a synthetic analogue of the EGF-A domain (from the low-density lipoprotein receptor). We show that calcium sensitivity is enabled by an allosteric folding pathway, in which calcium binding is connected to the peptide core through local inter-residue interactions. In the absence of calcium, the fold favors disorder because the inherently weak core is insufficient to stabilize the active form, resulting in substantial loss in activity of two orders of magnitude. The EGF-A fold, which can freely transition between active and disordered states, is volatile and we found it to be intolerant of mutations, unlike other disulfide-rich peptides that have been used as stabilizing frameworks. This volatility is beneficial for modularity/plasticity and appears to have evolved for such a purpose, allowing cellular pathways to sense and respond to environmental cues.


Page 2 of 34

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION In many biological processes, including those involving signaling and metabolic pathways, calcium plays a critical role in switching endocytic receptors 'on' or 'off'.1-3 At the plasma membrane, endocytic receptors are turned on by high (mM range) extracellular calcium concentrations; whereas, in the endocytic pathway they are turned off by lower (100- to 1000fold) calcium concentrations. Usually, these receptors use calcium-binding domains, including the LA (Low-density lipoprotein type A), CUB (C1r/C1s, Uegf, Bmp1), SRCR (scavenger receptor cysteine-rich repeats), and EGF-like (epidermal growth factor-like) domains, to sense the concentration of calcium ions. Although these domains vary considerably in structure, they coordinate calcium in a similar way by using an aspartate and/or glutamate carboxyl, together with backbone carbonyl, groups.

Of these calcium-binding domains, the EGF-like domain is one of the most abundant in both animal and plant proteins. To date, 71,677 proteins have been found to contain the EGF-like domain (InterPro accession no: IPR000742); ~52% of which have been predicted to bind calcium (accession no: IPR001881), and are called calcium-binding EGF-like domains. They are integral to many extracellular proteins, which collectively have diverse functions, including cell development, blood coagulation, and cholesterol metabolism.

The first EGF domain (EGF-A) of the low-density lipoprotein receptor (LDLR) – a membrane glycoprotein involved in lipoprotein metabolism – is one example of a calcium-binding EGF-like domain. The LDLR is responsible for binding and subsequent endocytic uptake of cholesterolcontaining lipoprotein ligands, reducing cholesterol levels in blood.4 This process can be negatively regulated by degradation of the LDLR, which can be mediated by binding to 3 ACS Paragon Plus Environment


proprotein convertase substilisin/kesin type 9 (PCSK9). This interaction requires EGF-A and is calcium-dependent. It is also a validated drug target for lowering cholesterol.5-7

Here we examine the prototypical EGF-A domain of LDLR to elucidate how calcium-binding EGF-like domains sense calcium. We focus on a synthetic truncated variant (tEGF-A, Figure 1a) comprising two disulfide bonds in the native I-III and II-IV connectivity, the native calciumbinding site, and all residues involved in the interaction interface because of its relative simplicity as well as its ability to recapitulate the activity of the full-length domain.8 Its sequence is shown in Figure 1b as well as the sequences of other calcium-binding EGF-like domains. To delineate the role of specific residues, we synthesized a series of tEGF-A mutants and examined their effect on folding in solution, using NMR, and in silico, using molecular dynamics. The effect of folding was also correlated to activity by measuring binding to PCSK9. Our results suggest that an allosteric folding pathway enables the EGF-like fold to respond to calcium in order to modulate activity.


Page 4 of 34

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS

Calcium binding is important for structure and activity We examined the effect of calcium on the structure of tEGF-A using NMR, and found that in the absence of calcium the spectra exhibited poor dispersion in the amide region (Figure 2a; Supplementary Figures S1 & S2). This is characteristic of a peptide sampling many different non-structured conformations. As the calcium concentration increases, peaks in the spectra sharpen and dispersion increases, indicative of a well-ordered structure, typical of a β-sheetcontaining peptide. Increasing the pH also leads to peak sharpening, consistent with the aspartate/glutamate-mediated calcium coordination mechanism. This coordination is not entropically stable, as increasing the temperature leads to broadening of the peaks and narrowing of the amide spectral dispersion.

We used molecular dynamics simulations to explore the effect of calcium depletion (Figure 2b), and observed disorder of tEGF-A in silico that was consistent with the profile of the NMR spectra (Figure 2a). Simulations at 315 K suggested the structure is not stable without calcium, as it quickly deviated from the active conformation as the simulation progressed. However, the degree of disorder observed in the 315 K simulation is probably underestimated based on the profile of the calcium-depleted spectrum. Thus, we tested higher temperature simulations, which – although unrealistic – were used to artificially increase the sampling rate and prospectively explore potential disordered structures. (We note that accurate parameterization of metal ions is not straightforward;9 thus, due to the difficulty in accurately parameterizing the pentagonal bipyramidal coordination configuration required for calcium binding, a calcium-bound form was



not simulated). Nevertheless, these simulations combined with the results of the NMR experiments confirmed the importance of calcium for stabilizing the structure.

We confirmed that tEGF-A binds specifically to immobilized PCSK9 in a concentrationdependent manner, in the presence of 5 mM CaCl2 (Figure 2c). This interaction is calciumdependent, as evident from reduced binding at lower calcium concentrations. In the absence of calcium, a >100-fold drop in binding was observed. The importance of calcium for folding and binding supports a link between folding and activity.

The fold is volatile and easily disturbed To study the structural determinants that affect the tEGF-A fold, we synthesized a series of mutants by sequentially replacing each residue with an alanine and found many to exhibit poorly defined folds. All residues were separately and individually replaced with alanine, with the exception of glycine and cysteine as we had previously established that such a replacement is detrimental to folding.8 Eighteen analogues were produced, and the structure of each mutant was assessed by observing the dispersion and sharpness of peaks in 1D proton spectra, as illustrated in Figure 3a. Of the 18 mutants, only five (i.e. T2A, S13A, V15A, N17A, and K20A) had spectra suggestive of an ordered fold, but not all (e.g. T2A) were completely well-ordered (Figure 3b and c; Supplementary Figure S3). Based on earlier results, we expected the substitution of acidic residues that coordinated calcium with their carboxyl groups to alanine would be detrimental to folding; however, we found the substitution of residues that appeared innocuous were, in fact, unfavorable for folding; for example, those that were far away from the calcium-binding site or were highly solvent-exposed.


Page 6 of 34

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We also conducted a screen for binding to PCSK9 for all mutants and found that most resulted in substantial loss of binding, compared to wild-type, of up to 300-fold (Supplementary Table S2). This difference was comparable to that resulting from calcium depletion of the wild-type (Figure 2c). By comparison, some mutants (i.e. L19A), despite exhibiting significant disorder, had reasonable binding affinities (i.e. only ~25-fold < wild-type), suggesting the existence of a complex relationship between the adopted fold(s) in solution and activity. These results indicate that the fold is generally not robust to mutations, and is thus easily disturbed.

The disulfide bonds are not sufficient to constrain the folded state We examined whether the native disulfide connectivity is sufficient to constrain the folded state of tEGF-A. In practice, synthesis and oxidation of disulfide-rich peptides is complicated by the number of disulfide isomers that can be obtained, but this challenge can be addressed by modification of the Cys residues.10-11 For example, one approach is to use orthogonally protected cysteine pairs, and we used this strategy to confirm that the poor folding was not due to connectivity (Figure 4a). Two of the alanine mutants, which were synthesized using the nondirected approach and did not adopt a well-ordered fold (i.e. E24A and L19A), had CysI and CysIII protected with Acm groups. This allowed for a two-step oxidation procedure, in which the II-IV disulfide was formed first, followed by the I-III bond. In both cases, and for mutants of L19A, this strategy did not result in a well-folded product, as shown in Figure 4b. Similarly, the protection of CysII and CysIV with Acm groups, which changed the sequential order of disulfide bond formation, also failed to produce a well-folded peptide according to NMR.



We demonstrated that the poor folding of the mutants was not due to disulfide bond shuffling during or after peptide oxidation by using a non-reducible triazole bridge as a surrogate for the IIII disulfide bond. Two mutants based on the L19A analogue were synthesized, differing only in their triazole ring orientation. Like the L19A mutant, the final constrained peptides also had spectra characterized by poor dispersion. In these cases, the peaks appeared sharper (which could be due to changes in exchange dynamics) and of sufficient quality for more detailed structural characterization. Consequently, we elucidated the tertiary structures of these analogues (Figure 4c; Supplementary Figure S4). Unlike the native fold, the structures of the mutants appeared highly flexible and were not compact. Although the structures might resemble only some of the disordered states, we hypothesized that the lack of compactness might result from a weak hydrophobic core, resulting in preferential exposure of the core to solvent.

A weak hydrophobic core biases the disordered state The hydrophobic effect is one of the main forces driving protein folding; rather than being an attraction between hydrophobic atoms, it is more accurately defined as the result of the cohesiveness of water, and therefore the surrounding environment plays a significant role in folding. We speculated that if the hydrophobic core is weak (e.g. arises from an imbalance in hydrophobicity between the core and solvent), then the fold would be responsive to changes in solvent conditions. Thus, we examined the effect of acetonitrile (making the environment more favorable for hydrophobic exposure) on the fold using NMR (Figure 5). Previously, in the absence of acetonitrile, we had demonstrated that an increase in temperature led to increased structural flexibility and distortion (Figure 5b, left; 283–308 K titration shown in Supplementary Figure S5). However, in the presence of 30% v/v acetonitrile, the fold was more resistant to


Page 8 of 34

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


increases in temperature, having sharper peaks and more defined dispersion than in the absence of acetonitrile at the same temperatures (Figure 5b, left; Supplementary Figure S5). It is possible that acetonitrile stabilizes exposure of hydrophobic residues that are natively on the surface of tEGF-A; however, in a completely aqueous solvent, the surface residues compete for burial with hydrophobic residues that line the core, resulting in more disordered structures with reduced rigidity.

To further investigate the role of the hydrophobic core, we searched the Protein Data Bank for an EGF-like domain with a more densely packed hydrophobic core (Figure 5a, right), and found the third EGF-like domain of Jagged-1. Its overall structure is similar to that of tEGF-A (Supplementary Figure S6). Its core is defined by two tyrosine residues, one histidine, and one proline that pack tightly together. Compared to the LDLR-derived domain, the Jagged-derived domain exhibited higher stability at higher temperatures (Figure 5b and c, right; 283–308 K titration shown in Supplementary Figure S7). As expected, the peaks sharpened upon temperature increase (283–293 K). For the Jagged-derived domain, acetonitrile reduced dispersion and caused slight broadening of the peaks, consistent with it interfering with interresidue interactions at the core. We then showed, with a stronger core, the peptide to be more tolerant of substitutions. The Jagged-derived domain containing an alanine substitution (i.e. T20A) in the same position as the L19A mutant could retain a similar dispersion and sharpness of peaks as the wild-type form (Figure 5d and e; Supplementary Figure S9). Thus, these results indicate that in the LDLR-derived domain, a weak core destabilizes the ordered state

Additional local interactions couple with calcium binding and core stabilization



Although we have demonstrated that the residues at the core and those involved in calcium coordination are involved in folding, it is unclear why residues that do not participate in either role affect the integrity of the EGF-like structure. L19 and E24 are both examples of these apparently fold-neutral residues, but when either are mutated to Ala the peptide fold is destabilized and acetonitrile could not rescue the effect (Figure 5e; Supplementary Figures S10 and S11). After examining molecular dynamics simulations in more detail, we noticed that the L19A mutant exhibited greater flexibility in the β-sheet region; thus, it is possible that a reduction in the side-chain size resulting from the alanine substitution removed packing interactions with neighboring residues (i.e. N17 and E24) that were favorable for folding (Figure 5f). We hypothesized that replacement of leucine with a similarly large residue would result in those favorable interactions being retained; thus, we synthesized the L19K mutant. Although it showed increased order compared to the L19A mutant, its spectrum still largely resembled a disordered peptide (Figure 5e; Supplementary Figure S12). Further molecular modeling of the mutant indicated that although the lysine residue was able to interact with neighboring residues, it could also form additional interactions with E24 that would subsequently compete with native ionic interactions between N17 and E24. The importance of this interaction pair is supported by the disordered structure of the E24A mutant in solution. Unlike the L19A mutant, the L19K mutant exhibits improved spectral quality (Figure 5e) and adopts a native-like structure (Supplementary Figure S6) in the presence of acetonitrile, indicating that the balance of forces regulating its folding has changed. Interestingly, the propensity to adopt the ordered state (i.e. wild-type > L19K > L19A; Figure 5e) correlates well with binding affinity (wild-type > L19K > L19A; Figure 5g), providing further confirmation of the importance of folding on activity.


Page 10 of 34

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DISCUSSION In this study we used a peptide derived from the LDLR, a membrane protein that functions in a calcium-dependent manner, to understand how a signaling pathway mediated by an external calcium ion is programmed into a peptide. This peptide was designed to mimic the first calciumbinding EGF-like domain of LDLR (EGF-A), and comprises two disulfide bonds arranged in the native connectivity as well as the native calcium-binding site. We identified several factors affecting folding that synergize to allow the peptide to respond to calcium cues, and probably have broader implications for understanding protein folding.

Many protein-protein interactions are regulated by a calcium-mediated switch, allowing interconversion between active and inactive states.1 An example is the interaction between LDLR and PCSK9, which has been subjected to intense scrutiny over its role in cholesterol metabolism.5 Binding of PCSK9 to the EGF-A domain of LDLR controls LDLR degradation by an endocytic mechanism, and leads to higher levels of circulating cholesterol. Structural studies have demonstrated that a calcium ion is coordinated by EGF-A in the LDLR:PCSK9 complex, indicating that calcium has an important structural role.12 Here, the structural rigidity of our EGF-derived peptide drastically reduced upon depletion of calcium, resulting in a >100-fold reduction in binding to PCSK9. The loss of rigidity would be expected to translate into enthalpic and entropic penalties that are unfavorable for binding. In this way, the calcium-mediated switch is equivalent to a folding-mediated process. Although we recognize that other factors might contribute to the LDLR:PCSK9 interaction, it is remarkable how a small ionic signal has used protein folding to elegantly make a much larger molecule amplify its message.



We hypothesized that the sensitivity of the EGF-like fold to calcium is due to it being structurally frustrated and volatile. Thermodynamically, the energetic barrier between active and inactive states is reduced in the absence of calcium. A reason for this is that the EGF-like fold has a weak hydrophobic core relative to the physicochemical properties of its surface in the active state, with solvation forces then acting to preference disorder. Indeed, packing interactions within the hydrophobic core can have a marked effect on the stability of a protein domain,13 although there are noteworthy examples of protein stability without a hydrophobic core.14 Here, we demonstrated that the calcium-binding fold was sensitive to the 'hydrophobicity' of the solvent and that an EGF-like peptide with a richer core was more structurally stable. Interestingly, a hydrophobic residue at position 23, which forms part of the core, was demonstrated to be important for directing formation of the canonical cystine connectivity during disulfide bond oxidation of EGF-like peptides from thrombomodulin.15 Therefore, the hydrophobic core is important for folding, during both oxidation of the reduced peptide and also calcium signaling in the oxidized state.

The calcium-binding fold might have been optimized to be volatile during evolution, so finetuned for its function that it is intolerant to mutation. This is because many of the residues have specific roles in folding. We showed that the residues decorating the surface of the β-sheet (e.g. L19 and E24) are involved in packing interactions that stabilize secondary structure (i.e. residues 15 to 26), and thus the residues that form the core (e.g. Y23) of the active state. Disruption of these interactions by substitution to alanine or lysine resulted in analogues that were more disordered in solution. We honed in on residue 19 because it sits at the border between the calcium-binding site and the β-sheet packing interactions; that is, its backbone carbonyl helps


Page 12 of 34

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coordinate calcium while its side-chain packs with other exposed residues. It is thus possible that this residue is part of a latent folding pathway that allows mechanical signals to be transmitted between calcium binding and core stabilization, as illustrated in Figure 6.

Furthermore, we found that the level of structural disorder of the mutants correlated with their level of binding reduction compared to the wild-type, confirming the role of folding in mediating activity. The fact that some mutants, despite being disordered in solution, could still bind to PCSK9 suggests that binding-induced conformational change occurred, resembling the mechanism of action of intrinsically disordered proteins, many of which function in signaling and as pathway regulators.16 The importance of disorder in biological functions has definitely gained momentum over recent years.17

Although the EGF-like domain can be flexible, its structure is still constrained by disulfide bonds, thus allowing it to more readily sample the active conformation compared to a completely unconstrained peptide when the environment is 'just right'. Structural constraints, such as disulfide bonds, have attracted significant interest from peptide chemists because peptide drug leads typically need to be constrained to overcome inherent metabolic and thermodynamic limitations.18-19 Indeed, molecular dynamics simulations have shown that disulfide bonds can help stabilize structures that would not otherwise form.20 Here, we demonstrated that the disulfide bonds are not sufficient for safeguarding the EGF-like fold against structuredestabilizing mutations. Despite this, we previously showed that the EGF-derived peptide studied here has high serum stability and could inhibit PCSK9 from binding to native LDLR, confirming its potential as a drug lead.8 In general, disulfide-rich peptides have enhanced



stability compared to linear disulfide-poor peptides and are thus thought to be excellent drug leads.21-22 Additionally, they have a wide range of therapeutically interesting activities. There are now several examples of native and engineered disulfide-rich peptides that have high stability and potent activities in vitro and in vivo.18, 23-25 Although widespread in nature, there are now increasing numbers of technologies for the discovery of disulfide-rich peptides and analogues thereof;26-29 thus, it is expected that they will see more use in the future.

Based on work on numerous exceptionally stable peptides that are excellent frameworks for modification, it was initially surprising that the EGF-like peptide was so easily disturbed by small and inert chemical modifications. In the context of biology and function, it is conceivable that the calcium-binding EGF-like fold has evolved so that it is sensitive to environmental cues. By controlling folding, function can then be regulated.


Page 14 of 34

Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


METHODS

Peptide synthesis Peptides were synthesized as previously described.8 Briefly, sequences were assembled on rinkamide resin at 0.125 or 0.25 mmol-scale using Fmoc solid-phase peptide synthesis on a Symphony Multiplex Synthesizer. Deprotection of the Fmoc group was achieved with 30% v/v piperidine in dimethylformamide (DMF). Side-chain protected amino acids were activated using 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (4 eq) and N,N-diisopropylethylamine (4 eq) in DMF and coupled twice. The assembled peptides were washed with dichloromethane before drying under nitrogen. The peptides were cleaved from the resin with a 94:3:3 mixture of trifluoroacetic acid (TFA), triisopropylsilane, and water for 2 h. The peptides were then precipitated with ice-cold diethyl ether, followed by extraction with 50% v/v acetonitrile, before lyophilization. The list of peptides synthesized for this study is given in Supplementary Table S1.

Peptide purification and identification Peptides were purified by high-performance liquid chromatography (HPLC) on a Shimadzu Prominence system using a 1%/min linear gradient of 99.95% v/v acetonitrile/0.05% v/v TFA and either a Phenomenex Jupiter 5µ C18 preparative (250 x 50 mm at 50 mL/min; 250×21.2 mm at 8 mL/min) or semi-preparative (250 × 10 mm at 3 mL/min) columns. Peptide purities (>95%) were confirmed using an analytical Phenomenex Jupiter 5µ (150 × 2 mm) column at 0.3 mL/min, as described above. Peptide masses were determined by electrospray ionization mass spectrometry using a Shimadzu Prominence system.



Peptide oxidation Initial trials indicated that peptides could be oxidized using either selective or non-selective protocols. The first six alanine mutants were oxidized using orthogonal protection of the cysteine residues, whereas the remaining alanine mutants were oxidized using a non-selective method, after conditions were optimized. Specifically, trial oxidations were carried out using conditions ranging from 0–15% v/v dimethyl sulfoxide (DMSO)/80–100% v/v NH4HCO3 at pH 6.8–7.8 and including 15% v/v DMSO/0.05% v/v TFA in water. Acetamidomethyl (Acm) and triphenylmethyl (Trt) groups were used for orthogonal protection. Selective removal of Acmprotecting groups and disulfide formation was achieved by dissolving peptide (0.5–1 mg/mL) in a solution of iodine (1 mg/mL) in 80% methanol. If the reaction proceeded slowly, 1 mg/mL of iodine dissolved in 80% acetic acid was used instead. The peptide was monitored using mass spectrometry before the reaction was quenched using a solution of ascorbic acid (10 mg/mL) until discoloration of the iodine. The reaction mixture was diluted ten times with 0.05% TFA in water, and the pH adjusted to ~3 before purification with HPLC.

Copper(I)-Catalyzed Azide-Alkyne Cycloaddition Peptides were synthesized containing azido alanine and propargyl glycine. To form the intramolecular triazole bridge between these residues, copper(I)-catalyzed azide-alkyne cycloaddition (CuCAA) was employed. To that end, peptide was dissolved at 0.5 mM concentration in 1M guanidinium HCl, 100 mM potassium diphosphate at pH 8. A stock solution of THPTA was added to a final concentration of 5 mM before copper (II) sulfate was added to 1 mM. The solution was degassed by means of sonication before addition of sodium ascorbate (30


Page 16 of 34

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mM final concentration) started the reaction. An instant colour change of the solution from blue (copper(II)) to clear (copper(I)) was observed and the reaction was incubated at 37°C for 2 hrs without stirring in a closed reaction vessel. The reaction product was isolated with semi preparative HPLC. Since there is no mass change between starting material and reaction product, ultra-HPLC (UHPLC) analysis was used to judge reaction success: retention time shifts between reaction product and starting material indicated a successful intramolecular cyclization.

Cysteine residues were protected using Acm and intramolecular disulfides were formed following CuCAA and HPLC isolation. HPLC fractions (aqueous acetonitrile, 0.05% TFA) containing the triazole bridged peptide with Acm protected cysteines were treated with iodine until a yellow brown solution was obtained. The reaction was allowed to proceed under exclusion of light at room temperature for 30 min, before it was quenched by addiction of 100 mM ascorbic acid solution. The disulfide and triazole bridged peptides were then isolated on HPLC and lyophilized.

Nuclear magnetic resonance spectroscopy Peptides were dissolved in 500 µL of 90% v/v H2O/10% v/v D2O, containing 0–30 mM CaCl2. Acetonitrile-d3 was used in some cases. The pH was adjusted to 5.3–5.5 using concentrated NaOH. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 MHz or Avance-600 MHz spectrometer equipped with a cryoprobe. One-dimensional (1D) 1H spectra were recorded at temperatures ranging from 283–308 K.



Structure determinations were performed using previously described methods.30 Briefly, oneand two-dimensional NMR spectra (1H-1H TOCSY, NOESY, DQF-COSY and E-COSY, and 1

H-13C HSQC) were acquired using a Bruker Avance-600 MHz spectrometer. Restraints were

derived from intensities of NOE cross-peaks, 3J(HN-Hα) coupling constants, 3J(Hα-Hß) coupling constants, amide temperature coefficients, and deuterium exchange rates. Structure calculations were performed using CYANA 3.0.

Surface plasmon resonance All surface plasmon resonance (SPR) experiments were performed on a Biacore 3000 (GE Healthcare Lifesciences, Fairfield, CT, U.S.A.) instrument at 25°C. Biotinylated PCSK9 (Acro Biosystems) was captured on a streptavidin sensor chip using a flow rate of 5 µL/min and a 100 µg/mL protein solution in running buffer, typically achieving a 2000–3000 response unit (RU) change. Experiments on alanine mutants were performed at a flow rate of 50 µL/min, with a buffer of 25 mM Tris, 100 mM NaCl, 5 mM CaCl2, and 0.01% w/v surfactant polyoxyethylenesorbitan (GE) at pH 7.5. SPR binding curves were generated from 0.39, 1.56, 6.25, 25.00 and 100.00 µM peptide concentrations, using 30-s injection and 30-s dissociation cycles. Other SPR experiments were performed at a flow rate of 20 µL/min, with buffers composed of 20 mM Hepes and 100 mM NaCl at pH 7.5, both with and without 5 mM CaCl2. SPR binding curves were generated from 0.625, 1.25, 2.50, 5.00, 10.00, 20.00, 40.00 and 80.00 µM peptide concentrations, with 60-s injection and 120-s dissociation cycles. Peptides were dissolved in running buffer, and the concentrations determined by Nanodrop A280nm measurements using molar extinction coefficients determined using CyBase.31 Data were


Page 18 of 34

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analyzed using GE BIAevaluation software (version 4.1.1) and kinetic analysis of referencesubtracted sensorgrams.

Molecular dynamics Molecular dynamics simulations were performed as previously described.32 The structure of truncated EGF-A (PDB ID: 4NE9) was solvated in a water box measuring 50 x 50 x 50 Å. Each simulation began with equilibration using stepwise relaxation. All heavy atoms were harmonically restrained with a force constant of 2 kcal mol-1 Å-2 before a conjugate gradient minimization of 10,000 steps was applied using NAMD v2.10 and CHARMM27 force field parameters. The simulation was heated to the desired temperature and simulated for 100 ps, followed by 100 ps under constant pressure and periodic boundary conditions. A Langevin thermostat with a damping coefficient of 0.5 ps-1 was used to maintain system temperature, and a Langevin piston barostat was used to maintain system pressure at 1 atm. The particle mesh Ewald algorithm was used to compute long-range electrostatic interactions at every time step and non-bonded interactions were truncated smoothly between 10.5 and 12 Å. All covalent hydrogen bonds were constrained by the SHAKE algorithm (or the SETTLE algorithm for water), permitting an integration time step of 2 fs. Production runs of 50 ns were carried out. Coordinates were saved every 100 ps.



ACKNOWLEDGEMENTS D. Craik is an Australian Research Council (ARC) Australian Laureate (FL150100146). C. Schroeder is an ARC Future Fellow (FT160100055). J. Swedberg is a National Health and Medical Research Council (NHMRC) Early Career Fellow (APP1069819). Work in our laboratory on peptide scaffolds is supported by grants from the ARC (DP150100443) and the National Health and Medical Research Council (APP1107403). We thank A. Cooper for editorial and proof-reading assistance.

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. Table of peptide sequences and binding affinities; NMR spectra of mutants; solution structure of triazole bridge mutants; molecular dynamics simulations.


Page 20 of 34

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE CAPTIONS

Figure 1. Calcium-binding EGF-like peptides. a) Structure of a peptide derived from the EGFA domain (tEGF-A) of the low-density lipoprotein receptor (LDLR; left), and bound to PCSK9 (white; right). Selected residues are labeled. The peptide binds calcium (green), which is coordinated by the side-chain carboxyl groups of E4 and D18, backbone carbonyl atoms, and nearby water molecules. The structure shows a β-sheet spanning residues 15–26, a small α-helix from residues 4–6, and two disulfide bonds with a I-III and II-IV connectivity. These structural features are also mapped onto the peptide sequence in panel b. b) Sequence alignment of peptides based on calcium-binding EGF-like domains. These domains are found in a range of proteins, including LDLR, Fibrillin-1, Notch1, Factor VII and Factor IX.

Figure 2. Dependence of structure and binding on calcium. a) Folding was monitored using 1D proton NMR. The amide region (i.e. between 5.6 and 10.0 ppm) is shown for each condition. The orange spectra is the reference condition, which was 30 mM CaCl2, pH 5.2, 283 K. An increase in pH (7.0 and 8.5), while maintaining the calcium concentration and temperature, led to sharpening of peaks. A reduction in CaCl2 concentration (15 and 0 mM, at pH 5.2, 283 K) and an increase in temperature (288–308 K in 5 K increments at pH 5.2, 30 mM CaCl2) led to broadening of peaks and poorer dispersion. b) Molecular dynamics simulations of the calciumfree form. Calcium was removed from the calcium-bound form (PDB 4NE9) in silico and the structures at 10, 30 and 50 ns of the production runs at 315, 360 and 400 K are shown. c) Dependence of PCSK9 binding on calcium. PCSK9 was immobilized on a streptavidin sensor chip. The upper panel shows an overlay of sensorgrams for varying concentrations of tEGF-A 21 ACS Paragon Plus Environment


(ranging from 0.63–10.00 µM). The lower panel shows binding at 10 µM peptide concentration for 5.00, 0.01, and 0 mM CaCl2 concentrations from triplicate experiments.

Figure 3. Structural integrity of alanine mutants. A suite of alanine mutants was synthesized and their folds were assessed using NMR. a) Comparison of spectra for a well-ordered (wildtype tEGF-A) and a disordered fold (L19A). A well-ordered fold is characterized by sharp peaks, dispersion, and an up-field shifted signal from the Hα of a cysteine residue. b) The structure of tEGF-A (PDB ID: 4NE9), colored according to the effect of alanine substitution on folding. Residues colored red lead to a disordered fold, those in orange are tolerated, and those in grey were not mutated. c) Schematic illustrating the role of each residue in calcium coordination, PCSK9 binding, and stabilization of the active fold. Calcium is coordinated by the side-chain of E4 and D18, and carbonyl atoms of T2, L19 and G22, and nearby water molecules, forming a bipyramidal pentagonal coordination. Residues within 5 Å of PCSK9 in the bound complex (PDB ID: 4NE9) are colored blue. The same color scheme used in panel b is used to depict to role of residues in folding.

Figure 4. Directed disulfide bond formation. a) Schematic of peptides synthesized to further explore orthogonal protection of cysteine residues. The cysteine residues protected by Acm groups are indicated. Other mutations are indicated by an orange oval. In the last two mutants, the I-III disulfide was replaced with a triazole bridge, requiring the substitution of the two cysteine residues with an azidoalanine and propargylglycine pair. b) The corresponding spectrum acquired at 283 K for each mutant is shown and aligned to the right. The peptides were dissolved in 9:1 H2O:D2O, pH 5.5 with the addition of 5 mM CaCl2. c) Solution structure of L19A mutant


Page 22 of 34

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with a triazole bridge surrogate. An overlay of the five lowest-energy structures is shown to the left. The lowest energy structure is shown to the right. Cysteine residues are numbered.

Figure 5. The peptide core and additional interactions important for folding. a) Structure of tEGF-A highlighting the residues that make up the core (green). On the right is an EGF-derived peptide from Jagged-1, displaying a rich hydrophobic core (green). b) Spectra acquired at 283 and 293 K for samples dissolved in 9:1 H2O:D2O, pH 5.5 with the addition of 5 mM CaCl2. c) Spectra acquired at 283 and 293 K for samples dissolved in 7:3 H2O:CD3CN, pH 5.5 with the addition of 5 mM CaCl2. d) Alignment of EGF-derived peptides from Jagged-1 and LDLR and selected mutants. e) Spectra of the mutants acquired at 283 K for sample dissolved in either 9:1 H2O:D2O, pH 5.5 with the addition of 5 mM CaCl2 or 7:3 H2O:CD3CN, pH 5.5 with the addition of 5 mM CaCl2. f) Packing interactions of surface-exposed residues of the β-sheet (i.e. N17, L18, E24, L26). Hypothetical effect of alanine and lysine substitutions at position 19 based on molecular dynamics simulations. g) Binding affinity of L19 mutants to immobilized PCSK9. Running buffer of 10 mM Hepes pH 7.4, 100 mM NaCl, 5 mM CaCl2 was used. Binding data was acquired at 298 K.

Figure 6. A putative internal signaling pathway programmed into the calcium-binding EGF domain. Binding of calcium (a) helps stabilize residues at the binding site, which in turn stabilizes nearby packing interactions that result in secondary structure stabilization (b) and eventually the peptide core (c). The residues proposed to play a role at each stage are colored green.



References (1)

Dell'Orco, D., Koch, K. W. (2016) Fingerprints of Calcium-Binding Protein Conformational Dynamics Monitored by Surface Plasmon Resonance, ACS Chem Biol. 11, 2390-2397.

(2) Catterall, W. A., Wisedchaisri, G., Zheng, N. (2017) The chemical basis for electrical signaling, Nat Chem Biol. 13, 455-463. (3) Monteith, G. R., Prevarskaya, N., Roberts-Thomson, S. J. (2017) The calcium-cancer signalling nexus, Nat Rev Cancer. 17, 367-380. (4)

Jeon, H., Blacklow, S. C. (2005) Structure and physiologic function of the low-density lipoprotein receptor, Annu Rev Biochem. 74, 535-562.

(5)

Seidah, N. G., Abifadel, M., Prost, S., Boileau, C., Prat, A. (2017) The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9, Pharmacol Rev. 69, 33-52.

(6)

Shapiro, M. D., Fazio, S. (2017) Dyslipidaemia: The PCSK9 adventure - humanizing extreme LDL lowering, Nat Rev Cardiol. 14, 319-320.

(7)

Mullard, A. (2017) Nine paths to PCSK9 inhibition, Nat Rev Drug Discov. 16, 299-301.

(8) Schroeder, C. I., Swedberg, J. E., Withka, J. M., Rosengren, K. J., Akcan, M., Clayton, D. J., Daly, N. L., Cheneval, O., Borzilleri, K. A., Griffor, M., Stock, I., Colless, B., Walsh, P., Sunderland, P., Reyes, A., Dullea, R., Ammirati, M., Liu, S., McClure, K. F., Tu, M., Bhattacharya, S. K., Liras, S., Price, D. A., Craik, D. J. (2014) Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro, Chem Biol. 21, 284-294. (9) Li, W., Wang, J., Zhang, J., Wang, W. (2015) Molecular simulations of metal-coupled protein folding, Curr Opin Struct Biol. 30, 25-31. (10)

Zheng, Y., Zhai, L., Zhao, Y., Wu, C. (2015) Orthogonal Cysteine-Penicillamine Disulfide Pairing for Directing the Oxidative Folding of Peptides, J Am Chem Soc. 137, 15094-15097.

(11) Empting, M., Avrutina, O., Meusinger, R., Fabritz, S., Reinwarth, M., Biesalski, M., Voigt, S., Buntkowsky, G., Kolmar, H. (2011) "Triazole bridge": disulfide-bond replacement by rutheniumcatalyzed formation of 1,5-disubstituted 1,2,3-triazoles, Angew Chem Int Ed Engl. 50, 5207-5211.


Page 24 of 34

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12)

Kwon, H. J., Lagace, T. A., McNutt, M. C., Horton, J. D., Deisenhofer, J. (2008) Molecular basis for LDL receptor recognition by PCSK9, Proc Natl Acad Sci U S A. 105, 1820-1825.

(13) Ventura, S., Vega, M. C., Lacroix, E., Angrand, I., Spagnolo, L., Serrano, L. (2002) Conformational strain in the hydrophobic core and its implications for protein folding and design, Nat Struct Biol. 9, 485-493. (14)

Gates, Z. P., Baxa, M. C., Yu, W., Riback, J. A., Li, H., Roux, B., Kent, S. B., Sosnick, T. R. (2017) Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core, Proc Natl Acad Sci U S A. 114, 2241-2246.

(15)

Ng, A. S., Kini, R. M. (2013) Structural determinants in protein folding: a single conserved hydrophobic residue determines folding of EGF domains, ACS Chem Biol. 8, 161-169.

(16)

Csizmok, V., Follis, A. V., Kriwacki, R. W., Forman-Kay, J. D. (2016) Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling, Chem Rev. 116, 6424-6462.

(17)

DeForte, S., Uversky, V. N. (2016) Order, Disorder, and Everything in Between, Molecules. 21,

1090.

(18)

Ji, Y., Majumder, S., Millard, M., Borra, R., Bi, T., Elnagar, A. Y., Neamati, N., Shekhtman, A., Camarero, J. A. (2013) In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide, J Am Chem Soc. 135, 11623-11633.

(19) Craik, D. J., Fairlie, D. P., Liras, S., Price, D. (2013) The future of peptide-based drugs, Chem Biol Drug Des. 81, 136-147. (20)

Zhang, Y., Schulten, K., Gruebele, M., Bansal, P. S., Wilson, D., Daly, N. L. (2016) Disulfide Bridges: Bringing Together Frustrated Structure in a Bioactive Peptide, Biophys J. 110, 1744-1752.

(21)

Gongora-Benitez, M., Tulla-Puche, J., Albericio, F. (2014) Multifaceted roles of disulfide bonds. Peptides as therapeutics, Chem Rev. 114, 901-926.

(22)

Kancherla, A. K., Meesala, S., Jorwal, P., Palanisamy, R., Sikdar, S. K., Sarma, S. P. (2015) A Disulfide Stabilized beta-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin, ACS Chem Biol. 10, 1847-1860.

(23) Sable, R., Durek, T., Taneja, V., Craik, D. J., Pallerla, S., Gauthier, T., Jois, S. (2016) Constrained Cyclic Peptides as Immunomodulatory Inhibitors of the CD2:CD58 Protein-Protein Interaction, ACS Chem Biol. 11, 2366-2374. 25 ACS Paragon Plus Environment


(24) Wang, C. K., Gruber, C. W., Cemazar, M., Siatskas, C., Tagore, P., Payne, N., Sun, G., Wang, S., Bernard, C. C., Craik, D. J. (2014) Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis, ACS Chem Biol. 9, 156-163. (25)

Qiu, Y., Taichi, M., Wei, N., Yang, H., Luo, K. Q., Tam, J. P. (2017) An Orally Active Bradykinin B1 Receptor Antagonist Engineered as a Bifunctional Chimera of Sunflower Trypsin Inhibitor, J Med Chem. 60, 504-510.

(26) Chen, S., Gopalakrishnan, R., Schaer, T., Marger, F., Hovius, R., Bertrand, D., Pojer, F., Heinis, C. (2014) Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides, Nat Chem. 6, 1009-1016. (27)

Getz, J. A., Cheneval, O., Craik, D. J., Daugherty, P. S. (2013) Design of a cyclotide antagonist of neuropilin-1 and -2 that potently inhibits endothelial cell migration, ACS Chem Biol. 8, 1147-1154.

(28) Zhang, Y., Eigenbrot, C., Zhou, L., Shia, S., Li, W., Quan, C., Tom, J., Moran, P., Di Lello, P., Skelton, N. J., Kong-Beltran, M., Peterson, A., Kirchhofer, D. (2014) Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor, J Biol Chem. 289, 942-955. (29) Getz, J. A., Rice, J. J., Daugherty, P. S. (2011) Protease-resistant peptide ligands from a knottin scaffold library, ACS Chem Biol. 6, 837-844. (30)

Wang, C. K., Northfield, S. E., Huang, Y. H., Ramos, M. C., Craik, D. J. (2016) Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold, Eur J Med Chem. 109, 342-349.

(31)

Wang, C. K., Kaas, Q., Chiche, L., Craik, D. J. (2008) CyBase: a database of cyclic protein sequences and structures, with applications in protein discovery and engineering, Nucleic Acids Res. 36, D206-210.

(32) Wang, C. K., Swedberg, J. E., Northfield, S. E., Craik, D. J. (2015) Effects of Cyclization on Peptide Backbone Dynamics, J Phys Chem B. 119, 15821-15830.


Page 26 of 34

Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1



Figure 2


Page 28 of 34

Page 29 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3



Figure 4


Page 30 of 34

Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5 31 ACS Paragon Plus Environment


Figure 6


Page 32 of 34

Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Figure



80x40mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 34 of 34

1 Calcium-mediated allostery of the EGF fold Conan K. Wang , Hafiza

Recommend Documents