Inorg. Chem. 2016, 55, 10727-10740

Copper Coordination Features of Human Islet Amyloid Polypeptide: The Type 2 Diabetes Peptide

 

Carolina Sánchez-López†, Rodrigo Cortés-Mejía‡, Marco C. Miotto§, Andres Binolfi§, Claudio O. Fernández§, Jorge M. del Campo‡, Liliana Quintanar†*

†Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), Mexico City, Mexico, ‡Departamento de Física y Química Teórica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico, §Max Planck Laboratory for Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC) and Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario (IIDEFAR, UNR-CONICET), Universidad Nacional de Rosario, Ocampo y Esmeralda, S2002LRK Rosario, Argentina.

 

Abstract

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits found in pancreatic β-cells of patients with type 2 diabetes (T2D). Copper ions have an inhibitory effect in the amyloid aggregation of hIAPP and they may play a role in the etiology of T2D. However, deeper knowledge of the structural details of the copper-hIAPP interaction is required to understand the molecular mechanisms involved. Here, we performed a spectroscopic study of Cu(II) binding to hIAPP and several variants, using electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electronic absorption and circular dichroism (CD) in the UV-vis region in combination with Born-Oppenheimer Molecular Dynamics (BOMD) and density functional theory (DFT) geometry optimizations. We find that Cu(II) binds to the imidazole N1 of His18, the deprotonated amides of Ser19 and Ser20, and an oxygen-based ligand provided by Ser20, either via its hydroxyl group or its backbone carbonyl, while Asn22 might also play a role as an axial ligand. Ser20 plays a crucial role in stabilizing Cu(II) coordination towards the C-terminal, providing a potential link between the S20G mutation associated to early onset of T2D, its impact in Cu binding properties, and hIAPP amyloid aggregation. Our study defines the nature of the coordination environment in the Cu(II)-hIAPP complex, revealing that the amino acid residues involved in metal ion binding are also key residues for the formation of β-sheet structures and amyloid fibrils. Cu(II) binding to hIAPP may lead to the co-existence of more than one coordination mode, which in turn could favor different sets of Cu-induced conformational ensembles. Cu-induced hIAPP conformers would display a higher energetic barrier to form amyloid fibrils, hence explaining the inhibitory effect of Cu ions in hIAPP aggregation. Overall, this study provides further structural insights into the bioinorganic chemistry of T2D.

 

Supplementary

Type 2 diabetes (T2D) is a degenerative metabolic disease that affects over 400 million people worldwide [1]. T2D patients display elevated blood glucose levels, insulin resistance, and abnormal insulin secretion [2]. One of the hallmarks of T2D is the presence of amyloid deposits of islet amyloid polypeptide (IAPP) or amylin in the islets of Langerhans of pancreatic b-cells [3, 4], occurring in approximately 90% of T2D patients [5]. IAPP is a 37-residue peptide that is stored with insulin in secretory granules [6]. This peptide is released from β-cells in response to stimuli, such as insulin itself [7]. Although its physiological role is not fully understood, it has been proposed that IAPP is related to glucose metabolism, regulation of food intake and body weight [8, 9].

IAPP is an intrinsically disordered peptide, i.e., a peptide without a defined secondary structure, termed “random coil”[10]. This property provides the peptide with a wide conformational landscape (Figure 1A). The sequence of human IAPP (hIAPP) includes several hydrophobic residues, which make this peptide amyloidogenic, i.e. it has a high propensity to aggregate and form amyloid fibrils [11]. The formation of an amyloid fibril is a downhill process from the thermodynamic point of view (Figure 1). Several factors, such as ligand binding, might influence the conformational landscape of an intrinsically disordered peptide (Figure 1B). In this work, we have shown that copper ions can bind to hIAPP, and have an impact in its propensity to form amyloid fibrils.

 

 

Figure 1. Proposed conformational landscape of Metal free hIAPP (A) and Cu(II)-bound hIAPP (B).

 

The impact of metal ions in the amyloid aggregation of hIAPP has become a topic of great interest in the past few years, as metal ions have been implicated in protein aggregation processes associated to other degenerative diseases, such as Alzheimer´s and Parkinson´s diseases [12, 13]. In particular, a recent study shows that serum Cu and Zn levels are increased in T2D patients [14], while a meta-analysis study concludes that it is the non-ceruloplasmin bound Cu fraction the one that is increased in the serum of T2D patients [15]. Ceruloplasmin is a Cu-dependent enzyme that contains 90% of the Cu found in human blood. The presence of increased levels of Cu ions in serum that are not bound to this enzyme reflects an overall loss of Cu homeostasis. Indeed, a dysregulation of Cu homeostasis has been proposed to play a role in T2D [16].

In the test tube, it has been demonstrated that metal ions, such as Cu(II) and Zn(II), can inhibit the amyloid aggregation of hIAPP, by binding to His18 [17-20]. The imidazole ring of histidine residues is a preferred ligand for Cu(II) ions in metallo-proteins. hIAPP contains only one His at position 18, intercalating between hydrophobic residues in the region 15-22. In this study, we have performed a detailed spectroscopic study of Cu(II) binding to hIAPP, in order to define the coordination features of the monomeric Cu-peptide complex that is formed. This knowledge aids our understanding of how Cu(II) ion binding to His18 impacts the amyloid aggregation of hIAPP.

To study Cu(II) binding to hIAPP, we used different spectroscopic techniques to evaluate Cu(II) binding to the peptides, including electronic absorption spectroscopy, circular dichroism (CD), electron paramagnetic resonance (EPR) [21], and nuclear magnetic resonance (NMR); in combination with computational tools such as Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations. We first used a peptide fragment encompassing residues 15-22, which contains His18, the anchoring site for Cu(II) ions. We first demonstrated that the region 18-22 contains the minimal sequence required to reproduce Cu(II) binding to longer peptides, i.e., the only amino acids that are needed to bind Cu(II) ions are: HSSNN, His-Ser-Ser-Asn-Asn. Then, we evaluated the role of each potential ligand in the sequence HSSNN in Cu(II) coordination. From Figure 2, it is clear that, when the backbone amide groups associated to Ser19 and Ser20 are replaced, the spectroscopic features of Cu(II) bound to hIAPP change drastically (Figure 2A), indicating that these amide groups participate in Cu(II) coordination. Moreover, substitution of the OH group in Ser20 also impacts significantly the CD spectrum of the complex, while substitution of the OH group in Ser19 has no effect (Figure 2B). These results indicated that the OH group of Ser20 participates in Cu(II) coordination, while that of Ser19 is not engaged in metal ion binding. Together, with other spectroscopic and theoretical results, we conclude that the molecular structure shown in Figure 2C is the one that best describes the monomeric Cu(II)-hIAPP complex.

 

Figure 2. Circular Dichroism spectra of Cu(II) complexes with hIAPP (18-22) (black), hIAPP (18-22, S19Sar) (light blue), hIAPP (18-22, S20Sar) (dark blue), hIAPP (18-22, S19A) (orange) and hIAPP(18-22, S20A) (red), with 1.0 equivalent of Cu(II) and pH 7.5. The schemes in (A) and (B) represent the variants with sarcosine and alanine, respectively; and highlight the moieties in the peptide that are being evaluated for their role in Cu coordination. (C) Proposed coordination mode for the Cu(II)-hIAPP complex based on spectroscopic results.

 

Additionaly, BOMD simulations also showed that other two potential Cu(II) binding modes might be plausible, as shown in Figure 1B. Moreover, for a given Cu-hIAPP complex, BOMD simulations demonstrate that the complex displays considerable conformational freedom on the regions of the peptide where the amino acid chains are not engaged in metal ion binding; while there is little flexibility for the amino acids His18-Ser19-Ser20 that are involved in Cu(II) coordination (Figure 3).

 

 

Figure 3. Ensemble of conformers for the Cu(II)-hIAPP complex, as obtained from BOMD simulations. There is little variation on the structure of the amino acid residues involved in metal coordination, while a large flexibility is observed for the amino acids that are not engaged in Cu(II) binding, such as asparagines 21 and 22.

 

Our study defines the molecular features of Cu(II) binding to hIAPP, and demonstrates that the amino acids involved in metal ion coordination are also key residues for the formation of b-sheet structured species that are precursors to amyloid fibrils. Hence, our molecular description of the monomeric Cu(II)-hIAPP complex sheds light into the mechanism of the inhibitory effect of Cu ions in the amyloid aggregation of hIAPP. Cu(II) binding to hIAPP would favor a set of Cu-induced conformations, where the amino acids engaged in metal ion binding would not be available to form b-sheet structures and amyloid fibrils. Hence, Cu-induced hIAPP conformers would display a higher energetic barrier to form amyloid fibrils (Figure 1B), which explains the inhibitory effect of Cu ions in hIAPP amyloid aggregation. Overall, out study provides structural insights into the inhibitory effect of Cu(II) ions in hIAPP amyloid aggregation, contributing to the understanding of the bio-inorganic chemistry behind T2D.

 

Acknowledgements. This research was funded by the National Council for Science and Technology in Mexico (CONACYT) through Grant No. 221134 and graduate fellowships. The support from DGTIC-UNAM Project SC16-1-IR-12 for the use of their computational resources and DGAPA-UNAM Grant IA-104516 are also acknowledged.

 

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Corresponding Author:

Prof. Liliana Quintanar

Department of Chemistry,

Cinvestav (Center for Research and Advanced Studies)

Av. Instituto Politécnico Nacional No. 2508,

Col. San Pedro Zacatenco, Gustavo A. Madero.

Mexico City, MX.

Email: lilianaq@cinvestav.mx