Abstract

Introduction

In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that 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, and 3.³ 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).

In earlier studies (4, 5), we provided isothermal titration calorimetry (ITC)⁴ 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) comprising eight disulfide-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 that includes the αβ cleavage site (6). C-terminal to FnIII-3 lie the transmembrane helix, 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 comprises 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 a K_d of 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.

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FIGURE 1.

Structures of the IR ectodomain and the insulin mimetic peptides. A, the folded over, Λ-shaped structure of the IR ectodomain. One monomer is in ribbon representation with domains labeled; the other is 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 underlie their competition for binding the three-domain IR L1-CR-L2 construct IR485 (4, 8).

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 aporeceptor (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 molecular mass (~3 kDa) compared with that of the untreated complex, slightly less than the drop in molecular mass (~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, K_d, of the S519C16 peptide for the EH-treated IR310.T·Fv 83-7 complex was determined by ITC to be 2.5 ± 0.6 nm (Fig. 2B; see “Experimental Procedures”), which is similar to that reported (K_d = 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 S.E. in both instances.

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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, endoglycosidase H-treated IR310.T·83-7 Fv; lanes 4 and 5, endoglycosidase H-treated IR310.T in the absence of bound 83-7 Fv. MW, molecular mass markers (kDa). B, representative isothermal titration calorimetry data for the titration of the S519C16 peptide against the IR310.T·83-7 Fv complex.

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). (2mF_obs − DF_calc) difference electron density maps calculated after 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 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 artifactually into unrelated density associated with a neighboring 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 5-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 unmodeled. The alternative registers resulting from a shift of ±1 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 were left unmodeled 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. Protein Data Bank code 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 (2mF_obs − DF_calc) difference electron density for the peptide is presented in Fig. 4, and final refinement statistics are presented in Table 1.

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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 (Protein Data Bank code 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. The overlay is based on the L1-CR modules. The polypeptide termini of the IR310.T·83-7 Fv complex that abut unmodeled residues of the complex are indicated by spheres (red for C termini and blue for N termini); these are remote from the S519C16 moiety. Also absent from the model are the N- and C-terminal glycine residues of S519C16.

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FIGURE 4.

Stereoimages of the (2mF_obs − DF_calc) difference electron density associated with the S519C16 peptide within the IR310.T complex viewed parallel (A) and antiparallel (B) to the direction of the L1-β₂ strands.

TABLE 1

X-ray data processing and refinement statistics

X-ray data processing
Space group	P41212
No. 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	135,463 (13,325)
No. unique reflections	21,483 (2,088)
I/σ(I)	7.68 (1.0)
CC1/2b	0.994 (0.484)
Rmerge	0.234 (2.23)
Completeness (%)	99.55 (98.91)
Refinement
No. of reflections	21,483c
Rwork/Rfree	0.226/0.248d
No. protein atoms/glycan atoms	4,074/179
B protein/B glycan (Å2)	123/132
σbonds (Å)/σangles (°)	0.010/1.2
Ramachandran plot (%)	94.7/4.9/0.4e

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^a Numbers in parentheses refer to the outer shell.

^b CC_1/2 is the Pearson correlation coefficient between two independently merged halves of the data set (35).

^c Cutoff: F > 0 σF.

^d Free set comprises 5% of the diffraction data. R values refer to R^xpct (36) as reported by autoBUSTER.

^e Ideal/allowed/outlier computed using MolProbity (37) with PHENIX (version 1.10pre-2104-1692) (31).

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 (Protein Data Bank codes 4XST and 4ZXB) (5, 10, 12) (Table 2). The periphery of this trough is formed by the side chains of IR residues Gln³⁴, Tyr⁶⁰, Phe⁸⁸, Phe⁸⁹, Tyr⁹¹, Arg¹¹⁸, Glu¹²⁰, Phe⁹⁶, Phe⁶⁴, Leu³⁷, and Arg¹⁴ (in clockwise order), and its floor is formed by the side chains of residues Leu³⁶, Leu⁶², and Leu⁹⁴. The side chain of S519C16 Phe^{27 5} lies close to IR Leu⁹⁴ at one end of the trough, that of S519C16 Phe³¹ lies close to IR Leu⁶² near the middle of the trough, and that of S519C16 Trp³⁰ lies 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. Although 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.

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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, comparison of the disposition of S519C16 on the surface of IR L1-β₂ compared with that of IR αCT in its apo and insulin-complexed forms. C, comparison of the engagement of the side chains of S519C16 residues Phe²⁷, Tyr²⁸, Trp³⁰, Phe³¹, and Gln³⁴ with the IR L1-β₂ surface compared with that of apo-IR αCT residues Phe⁷⁰¹, Phe⁷⁰⁵, and Tyr⁷⁰⁸. The color scheme is as in A and B.

TABLE 2

Contacts between S519C16 and IR L1-β₂

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 (38).

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	Tyr28 OΗ Glu120 Oϵ1/Oϵ2
	Arg118	38
	Lys121	36
Trp30	Gln34	40
	Phe88	60
Phe31	Phe64	77
	Phe88	46
Gln34	Arg14	41	Gln34 Oϵ1 Arg14 NΗ1/NΗ2
	Leu37	44
Leu35	Leu37	40

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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 (Protein Data Bank code 4ZXB), and the second is that of the so-called insulin microreceptor (μIR; a complex of IR310.T plus exogenous IR αCT peptide; Protein Data Bank code 4XST). Direct superposition of the structure presented here with that of Protein Data Bank code 4ZXB via their L1 domains results in a root mean square difference 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 root mean square difference of S519C16 and the αCT component of a similarly overlaid Protein Data Bank code 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 aporeceptor 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 engage the L1-β₂ surface (Fig. 5C).

TABLE 3

Relative disposition of αCT and S519C16 helices on the L1-β₂ surface

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 (39).

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

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The total molecular surface area buried by S519C16 as it engages IR L1-β₂ is 959 Ų compared with 930 Ų for the equivalent interaction between IR αCT and apo-IR L1-β₂. The shape complementarity, S_c (14), of the interface between S519C16 and IR L1-β₂ is 0.80 compared with a shape complementarity, S_c, of 0.81 for the interface between IR apo-αCT and IR L1-β₂ in Protein Data Bank code 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 affinity of the receptor 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 is 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 after endoglycosidase treatment and thus increase protein purification yield. 83-7 Fv has the advantage over the (hybridoma-derived) 83-7 Fab used 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 have the propensity to decrease crystallizability. Use of Fv domains as crystallization adjuncts is well established in membrane protein crystallography (for example, see Ref. 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 modeling of the Site 2 component 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 modeling, the disulfide loop motif is directed toward the 2-fold axis of the receptor (Fig. 6) and not toward 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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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 gray, whereas in the other leg, the L1 domain is shown in cyan and the FnIII domains are shown 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 labeled. The S519 peptide is shown in orange with it disulfide bond shown in green and labeled with an asterisk.

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 tumor suppressor protein p53 (20), and the second is the interaction of individual helices from the proapoptotic Bcl-2 family proteins with a groove on the surface of their prosurvival 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 druglike molecules. Such compounds may find application as agonists of IR in the context of diabetes or antagonists of both IR and type 1 insulin-like growth factor receptor in the context of cancer.

Experimental Procedures

Author Contributions

M. C. L., C. F. L., B. J. S., and C. W. W. wrote the manuscript. M. C. L. and B. J. S. supervised research. C. F. L., N. A. S., M. B. M., and J. G. M. performed research. All authors reviewed the manuscript.

Acknowledgments

References