insulin mimetic peptide disrupts the primary binding site of the

Insulin mimetic peptide S519

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Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Callum F. Lawrence‡, Mai B. Margetts‡, John G. Menting‡, Nicholas A. Smith§, Brian J. Smith§, Colin W. Ward‡, and Michael C. Lawrence‡#

From the ‡Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, AUSTRALIA; §La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, AUSTRALIA, and #Department of Medical Biology, University of Melbourne, Parkville, Victoria 3010, AUSTRALIA Running title: Insulin mimetic peptide S519

To whom correspondence should be addressed: Michael C. Lawrence, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, AUSTRALIA. Tel.: +61-3-9345-2693, email: [email protected]. Keywords: Insulin receptor, insulin, peptides, X-ray crystallography, protein structure, molecular modelling, mimetic peptide, Fv antibody fragment

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2 and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2 / Site 1 fusion peptide S519 which binds the insulin receptor with sub-nanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely, the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small-molecule agents directed towards the

insulin receptor and/or the Type 1 insulin-like growth factor receptor.

INTRODUCTION

In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extra-cellular region and which had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, or 31. Some Site 1 peptides were able to activate the receptor tyrosine kinase and act as agonists in an insulin-dependent fat cell assay, whereas Site 2 and Site 3 peptides were found to act as antagonists both in phosphorylation and fat cell assays. The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short- or long disulfide loop (1). Optimized versions of the Site 1 and Site 2 peptides were subsequently described (2), in particular, those that were linked in tandem in various ways. Several of these linked peptides were found to function as either potent antagonists (e.g., peptide S661, a Site 1-Site 2 combination) or as potent agonists (e.g., peptide S597, a Site 2-Site 1 combination), depending on the order in which the individual Site 1 and Site 2 peptides were linked (2,3).

http://www.jbc.org/cgi/doi/10.1074/jbc.M116.732180The latest version is at JBC Papers in Press. Published on June 8, 2016 as Manuscript M116.732180

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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In earlier studies (4,5), we provided isothermal titration calorimetry (ITC2) data that suggested how Site 1 peptides might bind to the insulin receptor. The insulin receptor (IR) itself is a disulfide-linked (αβ)2 homodimer; each αβ monomer comprises from its N-terminus two homologous leucine-rich repeat domains (L1 and L2), separated by a cysteine-rich region (CR) comprised of eight disulphide-linked modules (6). These domains are followed by three fibronectin type III domains (FnIII-1, FnIII-2 and FnIII-3) one of which, FnIII-2, contains a 120-residue insert domain (ID) that includes the αβ cleavage site (6). C-terminal to FnIII-3 lies the transmembrane helix, and the intracellular juxtamembrane region, tyrosine kinase domain and C-terminal tail. The extracellular domains of the homodimer adopt a folded-over, Λ-shaped conformation (6), each “leg” of which consists of the L1-CR-L2 module of one receptor monomer packed against the linearly arranged FnIII-1, FnIII-2, and FnIII-3 domains of the alternate receptor monomer (Fig. 1A). The primary insulin-binding site is comprised of the central β-sheet (L1-β2) of the L1 of one IR monomer and the C-terminal segment of the α-chain (termed αCT) of the alternate monomer (5,7). Our ITC experiments (4,5) showed that the Site 1 component of the optimized Site 2 - Site 1 peptide S519 (SLEEEWAQVECEVYGRGCPS-GSLDESFYDWFERQLG, where the hyphen denotes the Site 2 - Site 1 junction (2)) bound to an isolated three-domain L1-CR-L2 construct of IR (IR485) (8) with Kd = 11 nM. Entropic considerations led us to conclude further that in so doing the Site 1 peptide was capable of displacing bound exogenous αCT peptide from the L1 surface. Such binding would thus disrupt the primary insulin binding site. We noted further that the Site 1 component of S519 has sequence similarity to the insulin receptor αCT segment (Fig. 1B). However, to date, no three-dimensional structural data exist to support these conclusions.

We describe here the crystal structure of the C-terminal 16-residue Site 1 component of S519 ("S519C16") in complex with the N-terminal L1-CR fragment IR310.T of human IR bound in turn to the Fv domain of the monoclonal antibody 83-7 (7,9). To increase the crystallization propensity of the ternary complex, the IR310.T / 83-7 Fv

complex was subjected to mild endoglycosidase H (EH) treatment prior to the addition of the peptide. Our crystal structure reveals that S519C16 binds to the central β-sheet of the L1 domain in a fashion closely similar to that of the αCT peptide in the apo-receptor (5), demonstrating that agonistic properties of S519 arise from a disruption of the primary insulin binding site.

RESULTS

Sample characterization. SDS-PAGE analysis of the EH-treated IR310.T / 83-7 Fv revealed a small drop in MW (~3 kDa) compared to that of the untreated complex, slightly less than the drop in MW (~5 kDa) upon similar EH-treatment of IR310.T in the absence of attached Fv (Fig. 2A). These data suggested that attachment of the Fv module resulted in a slight reduction in the accessibility of the endoglycosidase to the IR310.T N-linked glycan. The dissociation constant of the S519C16 peptide for the EH-treated IR310.T / Fv 83-7 complex was determined by isothermal titration calorimetry (ITC) to be Kd = 2.5±0.6 nM (Fig. 2B; see Experimental Procedures), similar to that reported (Kd = 2.6±0.7 nM) for S519C16 upon ITC against the IR485 construct (4), indicating that its relative affinity was not adversely affected by either removal of the L2 domain, partial glycosylation or Fv 83-7 complexation. Errors are standard errors of the mean in both instances.

Crystallographic phasing and refinement. The successful initial phasing of the diffraction data by molecular replacement (see Experimental Procedures) revealed that the IR310.T / 83-7 Fv complex was assembled in a fashion closely similar to that seen in the previous structures of which this complex is a subset, in particular, that of the intact IR ectodomain in complex with two copies each of 83-7 Fab and 83-14 Fab (10) (Fig. 3). (2mFobs-DFcalc) difference electron density maps calculated post-refinement of the molecular replacement solution revealed a helix-like tube of difference density lying over the hydrophobic trough of the central β-sheet of domain L1 of IR310.T. Protuberances compatible with side chains at the resolution of the data set extended from the feature in a pattern suggestive of an α-helix. Given the high affinity of the peptide for the


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IR310.T / Fv 83-7 complex, we interpreted this density to be associated with the S519C16 peptide (and indeed there was no other candidate for it from either the buffer or the remaining protein within the crystal). An initial polyalanine α-helix was placed objectively into the density feature using the "place-helix-here" tool within COOT (11). Various trials were performed with different origin points for the placed helix; these resulted in helices of very similar respective length (~21 residues) and disposition and with identical N-to-C directions. The extra length in all instances arose from the polyalanine α-helix C-terminus extending artefactually into unrelated density associated with a neighbouring crystallographic monomer. The seven C-terminal residues of the template α-helix were therefore deleted. Assignment of the sequence register was guided by the qualitative nature of the side-chain-related density features as well as by the amphipathic nature of a helix generated by the S519C16 sequence and its implied engagement with the overwhelmingly hydrophobic trough on the L1-β2 surface. In particular, we noted that the engagement was mediated predominantly by a five-residue motif, of which the first two and last two were presumed hydrophobic by virtue of their positioning within the L1-β2 trough. Within the S519C16 sequence (Fig. 1B), the only such motif is the Site 1-defining FYDWF quintuplet; the 14-mer template thus corresponding to the 14 central residues of S519C16, with the N- and C-terminal glycines then being left unmodelled. The alternative registers resulting from a shift of +/- one residue resulted in implausible interactions with the L1 domain. Crystallographic refinement of the structure inclusive of the peptide proceeded according to standard iterative building / refinement protocols. A number of residues within the IR310.T and 83-7 Fv moieties where left unmodelled due to poor or absent electron density (see Experimental Procedures). These residues all lay remote from the S519C16 binding site (Fig. 3) and corresponded to either (i) loop regions within IR310.T known to be of relatively high mobility (i.e., B-factor) in other structures of which IR310.T is a subset [e.g., PDB 2HR7 (8)] or (ii) polypeptide termini that arise as artificial truncations with respect to parent protein (i.e., human IR or 83-7 mAb). The final (2mFobs-DFcalc)

difference electron density for the peptide is presented in Fig. 4 and final refinement statistics in Table 1.

Detail of the S519C16 / IR L1-β2 interface. The manner in which S519C16 engages the IR L1-β2 surface is shown in Fig. 5A. S519C16 forms an amphipathic α-helix, its hydrophobic surface engaging the same trough on the L1-β2 surface as that engaged by the αCT peptide within the apo-form of the receptor (PDB entries 4XST and 4ZXB) (5,10,12) (Table 2). The periphery of this trough is formed by the side chains of IR residues Gln 34, Tyr60, Phe88, Phe89, Tyr91, Arg118, Glu120, Phe96, Phe64, Leu37 and Arg14 (in clockwise order), and its floor by the side chains of residues Leu36, Leu62 and Leu94. The side chain of S519C16 Phe273 lies close to IR Leu94 at one end of the trough, that of S519C16 Phe31 close to IR Leu62 near the middle of the trough and that of S519C16 Trp30 close to IR Leu36 at the opposite end of the trough. The side chains of S519C16 Ser26, Tyr28, Gln34 and Leu35 engage the periphery of the L1-β2 trough. None of the S519C16 side chains appear to be in van der Waals contact with the floor of the trough, as far as can be discerned at the current resolution. Notable further interactions include the parallel stacking of the side chain of S519C16 Phe27 against the side-chain guanidinium group of IR Arg118 and the perpendicular stacking of the side chain of S519C16 Phe31 against the side chains of IR Phe64 and Phe96. While the interaction of peptide residues C-terminal to Phe31 with L1-β2 is not extensive, we note that extension of the peptide beyond the FYXWF motif to at least Gln34 is required for maximum affinity to IR (13); these additional residues may assist in stabilizing the helical conformation of the peptide. Details of the peptide / IR interactions are presented in Table 2.

Comparison with the IR αCT / IR L1-β2 interface. Two structures exist that reveal the disposition of IR αCT on the surface of IR L1-β2 in the absence of bound ligand. The first is that of the intact IR ectodomain (PDB entry 4ZXB) and the second is that of the so-called insulin microreceptor (µIR, a complex of IR310.T plus exogenous IR αCT peptide; PDB entry 4XST). Direct superposition of the structure presented here with that of PDB entry


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4ZXB via their L1 domains results in a root-mean-square difference (r.m.s.d.) of 1.1 Å across the Cα atoms of residues 24-35 in the S519C16 helix and their respective counterparts in the IR αCT helix, (i.e., the Cα atoms of IR residues 698-709). The equivalent r.m.s.d. of S519C16 and the αCT component of a similarly-overlaid PDB entry 4XST is 2.2 Å. A more detailed comparison of the respective helical axes of S519C16 and that of IR αCT is given in Table 3 and illustrated in Fig. 5B. Of the set of L1-β2 residues involved in engaging αCT in the apo-receptor structure, almost all play a role here in engaging S519C16, with these residues displaying conserved side chain conformation across the two structures. Furthermore, the manner in which S519C16 residues Phe27, Phe31 and Leu35 engage the L1-β2 surface is similar to that in which IR αCT residues Phe701, Phe705 and Leu709 of engage the L1-β2 surface (Fig. 5C).

The total molecular surface area buried by S519C16 as it engages IR L1-β2 is 959 Å2, compared to 930 Å2 for the equivalent interaction between IR αCT and apo-IR L1-β2. The shape complementarity Sc (14) of the interface between S519C16 and IR L1-β2 is 0.80 compared to a shape complementarity Sc of 0.81 for the interface between IR apo-αCT and IR L1-β2 in PDB entry 4XST.

DISCUSSION

The interaction of S519C16 with the L1 surface of the insulin receptor aligns with our earlier suggestion that it may function by competition for binding with the native IR αCT segment (4) rather than by mimicking the interaction of insulin itself. Competitive displacement of the αCT segment from the L1 surface will dramatically reduce the receptor's affinity for insulin, as L1 itself has no measurable affinity for insulin (4). αCT displacement from the L1-β2 surface may destabilize the receptor as a whole, given that the αCT segments are coupled to each other via inter-α-chain disulfide bond(s) within the Cys682-Cys683-Cys685 motif and form a bridge across the pair of L1 domains (10,15).

We note that crystallization of an S519C16 / IR 310.T complex proved refractory prior to our use

of recombinantly-derived 83-7 Fv adjunct; the most likely reason for such failure being the relatively high level of surface glycosylation of IR310.T. The adjunct provides additional, non-glycosylated polypeptide surface to the IR310.T complex and in so doing likely increases its propensity for crystallization. As such, it acts as a “crystallization chaperone” (16). In addition, attachment of the Fv was found to maintain solubility of IR310.T post endoglycosidase treatment and thus increase protein purification yield. 83-7 Fv has the advantage over the (hybridoma-derived) 83-7 Fab employed in our prior crystallographic studies in that it lacks the (conformationally-flexible) hinge region between the Fab variable and constant domains, such hinge regions also having the propensity to decrease crystallizability. Use of Fv domains as crystallization adjuncts is well established in membrane protein crystallography [see, for example, (17)] but we are unaware of any attempt to use them within the context of receptor tyrosine kinase crystallization.

The manner in which the Site 2 component of S519 binds to the receptor remains enigmatic. However, the now-determined location of S519C16 on the L1-β2 surface places constraints on the location of the S519 Site 2 component in the context of the intact receptor. We have undertaken some tentative molecular modelling of the Site 2 component in order to gain insight into what role their characterizing disulfide loop might play in receptor binding (see Experimental Procedures). As may be anticipated, in the three classes of models that emerged from the modelling, the disulfide loop motif is directed towards the two-fold axis of the receptor (Fig. 6) and not towards the putative second binding site of insulin (i.e., the FnIII-1 / FnIII-2 junction) (7). We hence speculate that Site 2 peptides might function by disulfide exchange with either of the two inter-α-chain IR disulfide bonds (Cys524 or within the Cys682-Cys683-Cys685 triplet) as these are nearby, exposed and highly mobile. Such exchange is, of course, conjectural. We note, however, that disulfide exchange of insulin with its receptor has been reported (18,19), implying a labile disulfide bond in proximity to the insulin binding site.


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Finally, we note that the agonistic interaction of the S519 helix with the IR L1-β2 surface is reminiscent of that of two other regulatory helices relevant in therapeutic contexts. The first is the interaction of the helix of the MDM2 oncoprotein with the transactivation domain of the tumour suppressor protein p53 (20), and the second is the interaction of individual helices from the pro-apoptotic Bcl-2 family proteins with a groove on the surface of their pro-survival relatives (21). In the first case, the interaction of the MDM2 helix with a groove on the surface of p53 is mediated primarily by the side chains of a tryptophan residue and a phenylalanine residue in the same (i, i+4) helical spacing as is observed here for the S519C16 engagement with IR L1-β2. In the second case, the helix / groove interaction is more extensive and mediated by a set of smaller hydrophobic residues. In both cases, attempts have been made to develop mimetics (either small molecule or stabilized peptide) of the cognate helix, with reasonable success (22,23). These examples raise the question whether the S519C16 interaction with IR can be mimicked by non-peptide drug-like molecules. Such compounds may find application as agonists of IR in the context of diabetes or antagonists of both IR and IGF-1R in the context of cancer.

EXPERIMENTAL PROCEDURES

83-7 VH and VL domain cloning and expression. Codon and expression-optimised DNA corresponding to murine monoclonal antibody 83-7 variable heavy (VH) chain residues 1-118 (6,9,10) followed by the sequence SLVPRGSSSEQKLISEEDLN (thrombin cleavage site + c-myc tag) was synthesized and then cloned into the vector pCDNA3.1 by DNA2.0 (USA). Similarly, DNA encoding the 83-7 variable light (VL) chain residues 1-112 (6,9,10) followed by the sequence SSDYKD (FLAG tag) was synthesized and then cloned into the vector pJ201 (DNA2.0, USA). Both genes were then individually transferred into the BamH1/Xba1 sites (in frame with a secretion signal) of the plasmid pNCM02 (Takara Bio, Japan) for independent transformation into Brevibacillus choshinensis cells (Takara Bio, Japan). Isolated colonies of the transformed B. choshinensis cells were then screened by Western blot (antibodies

9E10 and M2, respectively) for over-expression of the expected domain. The highest-expressing colonies were then stored as glycerol stocks. For 1 L scale-up, each glycerol stock was used to inoculate 2 ml of 2SY broth containing 10 µg / ml neomycin sulfate (Sigma Aldrich, USA) (2SYnm), followed by incubation overnight at 30°C at 120 rpm. 0.2 ml of these respective cultures were then used to inoculate a further 20 ml of 2SYnm broth and, once sufficiently grown, 5 ml of this inoculum was used to inoculate a further 500 ml of 2SYnm broth in Tunair™ flasks (Sigma Aldrich, USA). Cultures were incubated for 72-96 hrs, with 1 ml samples taken at 24 hr intervals to monitor production via SDS-PAGE and Western blot. Optical density was monitored at 660 nm. Samples were centrifuged at 13,000 rpm for 5 min to pellet bacteria and to recover the supernatant containing the secreted product.

Assembly and purification of 83-7 Fv. 700 ml of conditioned medium containing the c-myc-tagged 83-7 VH domain was combined with 850 ml of conditioned medium containing FLAG-tagged 83-7 VL domain and incubated for 30 min at room temperature, followed by addition of 3M Tris HCl (pH 8.5) at the ratio of 5 ml / L of combined media. This process was estimated to give a slight excess of VL monomers in the VL / VH mixture. The pH-adjusted combined media was then run through a 9E10 Mini-Leak Low affinity column (Kem En Tec, Denmark) (24) and the desired 83-7 Fv eluted with c-myc peptide (decameric form) prepared in Tris-buffered saline plus azide (24.8 mM Tris-HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl plus 0.02% NaN3; TBSA). Fractions were combined with one tablet of cOmplete protease inhibitor cocktail (Roche, Switzerland). Superdex 200 10/300 size-exclusion chromatography (SEC; GE Healthcare Life Sciences) fractions examined by SDS-PAGE showed the presence of two bands of molecular weight 14 and 16 kDa respectively, indicating a correctly-formed Fv eluting at 22 kDa. The c-myc tag was then removed from the 83-7 VH domain of the 83-7 Fv as follows. The 83-7 Fv was diluted to ~ 4 mg / ml in TBSA and then combined with 10 mM CaCl2 and 0.5 U human thrombin per mg 83-7 Fv (Roche, Switzerland). The sample was incubated at 37°C for 4 hrs and the reaction stopped by the addition of 1 mM phenylmethylsulfonyl fluoride and by incubation


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on ice. The sample was then re-purified by means of a Superdex S75 column (GE Healthcare Life Sciences, USA). SEC and SDS-PAGE estimates of molecular weight agreed with the expected masses. IR binding activity of 83-7 Fv was confirmed by SEC with SDS-PAGE, with IR310.T and 83-7 Fv co-eluting as a single peak (data not shown).

Partial deglycosylation of IR310.T / 83-7 Fv complex. IR310.T (produced as described previously (7) and prepared in TBSA) was combined with a 1.25-fold molar ratio of the c-myc-tag-removed 83-7 Fv and then incubated with endoglycosidase H (EH; New England Biosciences catalogue no. P0702) in G5 buffer (as per the manufacturer's protocol) for approximately 30 h at 37°C at a ratio of 1 mg IR310.T / 10,000 U EH. Sample pH was immediately thereafter adjusted by addition of 1/20th volume of 3 M Tris-HCl, pH 8.5. The EH-treated sample was then purified by SEC using a Superdex S200 column (GE Healthcare) equilibrated with TBSA buffer to separate the desired form from aggregated complex and to remove EH and excess 83-7 Fv. The final complex was concentrated to 29 mg / ml in TBSA buffer. The extent of deglycosylation was assessed by SDS-PAGE (Fig. 2A).

Isothermal titration calorimetry. S519C16 peptide (synthesized by Genscript, USA, and prepared at a concentration of 60 µM in TBSA) was titrated against the IR310.T / 83-7 Fv complex (prepared at a concentration of 6 µM in TBSA) using a MicroCal iTC200 instrument (Malvern Instruments, UK). Experiments were conducted at 25°C in “Highest Quality” mode of the instrument. The volume of the sample placed in the cell was 0.30 mL and the titrant was injected in 2.52 µL volumes over 5.04 s at 3 min intervals, with the total number of injections being 16 (the first injection was 1.0 µL and judged unreliable). The sample contents were stirred at a speed of 750 rpm over the duration of the titration. S519C16 peptide was first injected into a solution of TBSA alone and the heat of dilution was found to be a constant, which was then subtracted from the subsequent titration data. All data were analysed using the MicroCal Analysis software incorporated within Origin 7 employing a single-site interaction model based on minimisation of χ2. Measurements were

conducted in triplicate and the resultant ITC-derived thermodynamic parameters averaged. A sample titration curve and its integration is shown in Fig. 2B.

ITC experiments, wherein 83-7 Fv was titrated against IR485 using protocols similar to those above, yielded Kd = 9.4±0.3 nM, comparable to the Kd ~ 40 nM for papain-released 83-7 Fab (6) upon titration against IR485 (data not shown).

Crystallization of the IR310.T / 83-7 Fv / S519C16 complex. 15 µl of the above deglycosylated IR310.T / 83-7 Fv complex was combined with 130 µl of S519C16 peptide prepared in 10 mM HEPES-NaOH (pH 7.5) to yield a final complex-to-peptide molar ratio of 1:3. The resultant mix was then subjected to a 576-condition robotic sparse-matrix sitting-drop screen (CSIRO Collaborative Crystallization Centre; Parkville, Australia) to identify hit crystallization conditions. A crystal of ~120 µm in size was obtained in a 1.75 M (NH4)2HPO4 condition. The crystal was transferred to a solution containing 1.75 M (NH4)2HPO4 + 30% glycerol immediately prior to loop mounting and cryo-cooling in liquid nitrogen. Diffraction data were collected at 100K at the MX2 beamline of the Australian Synchrotron (25) and then processed using XDS (build 2014118) (26). Data collection statistics are presented in Table 1. Attempts to improve the initial crystallization conditions via manual hanging drop vapour diffusion protocols were unsuccessful.

Structure determination and crystallographic refinement. Initial phases were obtained by molecular replacement using PHASER (27) with an IR310.T / 83-7 Fv entity being employed as a search object (excised as a subset of PDB entry 4OGA (28), which contains inter alia the IR310.T / 83-7 Fab complex). Phases were then improved by crystallographic refinement using autoBUSTER (ver. 2.10.2) (29). Difference electron density was apparent on the surface of the L1-β2 sheet of IR310.T in a location approximating that of the receptor αCT peptide (5,12) and was interpreted as the S519C16 peptide in an α-helical conformation. The direction of the peptide was deduced using the "place-helix-here" tool within COOT (11), allowing ready model building of the central 14 residues of the S519C16


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peptide in a helical conformation into B-factor-sharpened (2mFobs-DFcalc) difference electron density (5,12). The in-principle ±1-residue register ambiguity was resolved by assessing both the shape of the density side chains as well as the hydrophobic complementarity to the L1-β2 surface. Carbohydrate residues were identified at five of the six anticipated N-linked glycosylation sites (30) on IR310.T and these were then rebuilt independently of their conformation in PDB entry 4OGA. Refinement was carried out using PHENIX (ver. 1.10pre-2104-1692) (31) and finally autoBUSTER (ver. 2.10.2) (29), iterated with model building within COOT (ver. 0.8.1). Reference model restraints were included for IR310.T (to both the A and B chains of PDB entry 2HR7) and 83-7 Fv (variable heavy chain to PDB entry 1FNS, variable light chain to PDB entry 3MBX). Electron density associated with IR310.T residues 1-4, 151-153, 160-167, 174-177, 266-275 and 304 onwards, S519C16 residues 21 and 36, 83-7 variable heavy chain residues 117 onwards and 83-7 variable light chain residues 113 onwards was poor and did not allow these residues to be convincingly modelled and were omitted from the final model. Final refinement statistics are presented in Table 1.

Molecular modelling. Comparative modelling was performed using MODELLER (ver. 9.15) (32). First, a model of the S519C16 peptide bound to the IR ectodomain was created by superposing the structure determined here onto the recently-improved structure of the ectodomain (10) with the αCT peptide (residues 686-719) of one monomer being excluded to allow for accommodation of S519C16. Models of full-length S519 were then created separately by imposing secondary structural constraints on the S519 peptide (as predicted by PSIPRED (ver. 3.3) (33), namely, that S519 residues Leu2 to Val13 and Leu25 to Leu35 are α-helical in conformation) and linking the pair of cysteine residues (S519 Cys11 and Cys18) to form a disulfide bond. Finally, models of full-length S519 bound to the IR ectodomain were created by superimposing the C-terminal residues of the S519 models onto those of the S519C16 peptide in complex with the IR ectodomain. Models with overlap between S519 and the IR were rejected, reducing the 5,000 initial models to 901. The 901 models were clustered

using the MMTSB cluster.pl utility (34). The final models were subjected to molecular dynamics minimization with the YASARA (ver. 15.7.12) program (www.YASARA.org). Hydrogen atoms were added to fill missing valencies, water molecules included, and a short simulated annealing protocol applied, using the YAMBER3 force field.

ACKNOWLEDGMENTS

This work was supported by Australian National Health and Medical Research Council (NHMRC) Project Grants 1005896 and 1058233 and the Hazel and Pip Appel Fund (to MCL); and NHMRC Independent Research Institutes Infrastructure Support Scheme Grant 361646 and Victorian State Government Operational Infrastructure Support Grant (to the Walter and Eliza Hall Institute of Medical Research). Diffraction data were obtained at the Australian Synchrotron (beam line MX2). We thank Louis Lu and the fermentation group at CSIRO Materials Science and Engineering (Parkville, Australia) for large-scale mammalian cell culture. CFL and NAS acknowledge Australian Postgraduate Awards.

CONFLICT OF INTEREST

Part of MCL's research is funded by Sanofi (Germany).

AUTHOR CONTRIBUTIONS

MCL, CFL, BJS and CWW wrote the manuscript, MCL and BJS supervised research, CFL, NAS, MBM and JGM performed research, all authors reviewed the manuscript.

DATA DEPOSITION

The coordinates of the structure of IR310.T in complex with 83-7 Fv and S519C16 have been deposited in the Protein Data Bank (entry 5J3H).


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FOOTNOTES Footnote 1 The two surfaces of the insulin receptor that are engaged by insulin itself are also conventionally referred to as Site 1 and Site 2 in the literature. While this study reveals that the Site 1 mimetic peptide does indeed target Site 1 on the receptor, it does so by mimicking αCT rather than insulin. For the Site 2 peptides, it remains uncertain as to whether they bind to Site 2 on the receptor. To avoid any implied correspondence or confusion in the current manuscript, we will refer to the two insulin-binding surfaces on the receptor as the primary and secondary insulin binding sites, respectively, rather than use the Site 1 / Site 2 nomenclature for these receptor sites. Footnote 2 The abbreviations used are: αCT, the C-terminal region of the insulin receptor α-chain; CR, cysteine-rich region; EH, endoglycosidase H; FnIII-1, -2, -3, the first, second and third fibronectin type III domains; Fv, antibody variable domain; JM, juxtamembrane region; ID, insert domain; IGF-1R, type 1 insulin-like growth factor receptor; IR, insulin receptor; IR310.T, residues 1-310 of the human IR followed by seven residual linker and thrombin cleavage site residues; IR485 , the L1-CR-L2 construct of IR comprising residues 1-485; ITC, isothermal titration calorimetry; L1, first leucine-rich repeat domain; L1-β2, the central β-sheet of the L1 domain; L2, second leucine-rich repeat domain; S519C16, the 16 C-terminal residues of the peptide S519; S519N20, the 20 N-terminal residues of the peptide S519; SEC, size-exclusion chromatography; µIR, the L1-CR module (IR310.T) of IR in complex with the IR αCT segment; TBSA, Tris-buffered saline plus azide; TK, tyrosine kinase domain; VH, variable domain of an immunoglobulin heavy chain; VL, variable domain of an immunoglobulin light chain. Footnote 3 S519C16 residue numbering here and throughout is based on that of the full-length S519 (36-mer) peptide.


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FIGURE LEGENDS Figure 1 Structure of the IR ectodomain and of the insulin mimetic peptides. (A) The folded-over, Λ-shaped structure of the IR ectodomain. One monomer is in ribbon representation with domains labelled; the other in molecular surface, apart from its insert domain (ID), which is in ribbon. (B) The sequence similarity between IR αCT (residues 694-710) and S519C16 (residues 21-36) proposed to underly their competition for binding the three-domain IR L1-CR-L2 construct IR485 (4,8).

Figure 2 Sample characterization. (A) Representative non-reducing SDS-PAGE analysis of endoglycosidase-H digestion of the IR310.T / 83-7 Fv complex. Lanes 1 and 2, IR310.T; Lane 3, endo-H treated IR310.T / 83-7 Fv; Lanes 4 and 5, endo-H treated IR310.T in the absence of bound 83-7 Fv. MW, molecular weight markers (kDa). (B) Representative isothermal titration calorimetry data for the titration of the S519C16 peptide against the IR310.T / 83-7 Fv complex. Figure 3 Overlay of ribbon representations of the structure determined here with the structure of the equivalent modules within that of the intact IR ectodomain in complex with two copies of both 83-7 Fab and 83-14 Fab (PDB entry 4ZXB). Light cyan: L1 domain of IR310.T; black: CR domain of IR310.T; orange: VL domain of 83-7 Fv bound to IR310.T; green: VH domain of 83-7 Fv bound to IR310.T; tan: S519C16 bound to IR310.T; white: L1-CR and 83-7 variable domains within the ectodomain complex. Overlay is based on the L1-CR modules. The polypeptide termini of the IR310.T / 83-7 Fv complex that abut unmodelled residues of the complex are indicated by spheres (red for C-termini and blue for N-termini)—these are remote from the S519C16 moeity. Also absent from the model are the N- and C-terminal glycine residues of S519C16. Figure 4 Stereo images of the (2mFobs-DFcalc) difference electron density associated with the S519C16 peptide within the IR310.T complex, viewed (A) parallel and (B) anti-parallel to the direction of the L1-β2 strands. Figure 5 Interaction of the S519C16 helix with the IR L1-β2 surface. (A) Side-chain detail within the S519C16 / IR L1-β2 interface. The backbone of IR L1-β2 and of S519C16 is shown in tube representation and colored light cyan and tan, respectively, with the surface of L1-β2 shown in transparent light cyan. (B) Comparision of the disposition of S519C16 on the surface of IR L1-β2 compared to that of IR αCT in its apo- and insulin-complexed forms. (C) Comparision of the engagement of the side chains of S519C16 residues Phe27, Tyr28, Trp30, Phe31 and Gln34 with the IR L1-β2 surface compared to that of apo-IR αCT residues Phe701, Phe705 and Tyr708. Color scheme is as in (A) and (B). Figure 6 Representative models from the three respective clusters of putative locations for S519 in the context of the intact IR ectodomain. The ectodomain is shown in ribbon representation; the domain modules in one of the two legs of the Λ-shaped receptor is shown in light grey, while in the other leg, the L1 domain is shown in cyan and the FnIII domains in yellow, with the CR and L2 domains of that leg omitted for clarity. Sites of inter-α-chain disulfide are shown in green and labelled. The S519 peptide is shown in orange, with it disulfide bond shown in green and labelled with an asterisk.


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Table 1 X-ray data processing and refinement statistics

X-ray data processing Space group P41212 # molecules in asymmetric unit 1 Unit cell a, c (Å) 176.78, 86.19 Resolution (Å) 35.0 - 3.27 (3.39 - 3.27)a No. measurements 135463 (13325) No. unique reflections 21483 (2088) <I/σ(I)> 7.68 (1.0)

CC1/2 0.994 (0.484) Rmerge 0.234 (2.23) Completeness (%) 99.55 (98.91)

Refinement No. of reflections 21483b Rwork / Rfree 0.226 / 0.248c # protein atoms / glycan atoms 4074 / 179 <B> protein / <B> glycan (Å2) 123 / 132

σ bonds (Å) / σ angles (°) 0.010 / 1.2 Ramachandran plot (%) 94.7 / 4.9 / 0.4d

a Numbers in parentheses refer to the outer shell. b Cut-off: F > 0 σF c Free set comprises 5% of the diffraction data. R values refer to R<xpct> (35), as reported by autoBUSTER. d Ideal / allowed / outlier, computed using MolProbity (36) with PHENIX (ver. 1.10pre-2104-1692) (31).


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Table 2 Contacts between S519C16 and IR L1-β2

a

S519C16 residue L1-β2 residue Buried contact area (Å2)

Potential salt bridges / hydrogen bonds

Leu23 Asn90 32 Tyr91 42 Asp24 Arg118 48 Asp20 Oδ1 ⇔ Arg118 NΗ1 /NΗ2 Ser26 Phe89 55 Phe27 Phe89 65 Arg118 77 Glu120 31 Tyr28 Phe96 48 Glu120 42 Tyr 28 OΗ ⇔ Glu120 Oε1 / Oε2 Arg118 38 Lys121 36 Trp30 Gln34 40 Phe88 60 Phe31 Phe64 77 Phe88 46 Gln34 Arg14 41 Gln34 Oε1 ⇔ Arg 14 NΗ1 / NΗ2 Leu37 44 Leu35 Leu37 40 a Two residues are deemed to be in contact if any pair of their non-hydrogen atoms are within 3.8 Å of each other. Calculation was performed using the programs CONTACT and SC within the CCP4 suite (37).


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Table 3. Relative disposition of αCT and S519C16 helices on the L1-β2 surface a

S519C16 apo-IR apo-µIR S519C16 - 10° / 0.1 Å 7° / 1.3 Å apo-IR - 17° / 1.1 Å apo-µIR -

a The values reported in each cell of the Table are the angle and distance of closest approach between the respective axes of the αCT or S519C16 helices after structural overlay of the L1 domains. Angles and distances were computed with the "Axes/Planes/Centroid" tool within CHIMERA (38).


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Colin W. Ward and Michael C. LawrenceCallum F. Lawrence, Mai B. Margetts, John G. Menting, Nicholas A. Smith, Brian J. Smith,

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

published online June 8, 2016J. Biol. Chem.

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insulin mimetic peptide disrupts the primary binding site of the

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