Elsevier

Biophysical Chemistry

Volumes 173–174, March–April 2013, Pages 1-7
Biophysical Chemistry

A T3R3 hexamer of the human insulin variant B28Asp

https://doi.org/10.1016/j.bpc.2013.01.003Get rights and content

Abstract

Insulin shows a complex equilibrium between monomers and hexamers, involving varying conformers and association states. We sought to perform a structural characterization of the fast-acting human insulin variant B28Asp (“aspart”). Small-angle X-ray scattering measurements reveal similar globular behavior in both the aspart and regular human insulin, with a Rg of 19 Å and a Dmax of approximately 50 Å, indicating similar mean quaternary assembly distribution. Crystallographic assays revealed a T3R3 assembly of the aspart insulin formed by the TR dimer in the asymmetric unit, with all the first 8 residues of the B chain in the R-state monomer in helical conformation and the participation of its B3Asn in the stabilization of the hexamer. Our data provide access to novel structural information on aspart insulin such as an aspart insulin dimer in solution, the aspart insulin in T conformation and a pure R-state conformer establishing a T3R3 assembly, providing further insight on the stepwise conformational transition and assembly of this fast-insulin.

Graphical abstract

Highlights

► The aspart insulin variant is a fast-acting due to improved monomerization. ► We have crystallized the aspart insulin in the T3R3 conformation. ► Hexamer stability is supported by B3Asn interaction. ► Existence of monomers, dimers and hexamer is supported by SAXS, ion-mobility spectrometry and crystallography.

Introduction

Insulin is a pancreatic hormone that regulates glucose homeostasis. The mature insulin molecule comprises two polypeptide chains A and B, linked by disulfide bonds. High-resolution and high-definition spectroscopic techniques, such as crystallography, nuclear magnetic resonance (NMR) and ion mobility spectroscopy (IMS), have long been used in the structural characterization of insulin [1], [2], [3], [4], [5], [6], [7], [8].

Insulin has been shown to assembly in the T and R conformation, in which the aminoacids B1 to B8 vary from an extended to an α-helical conformation respectively. Insulin has been reported to assembly into hexamers in the T6 (e.g., PDB ID 1MSO), T3R3f (e.g. PDB ID 1TRZ from single crystal, PDB entry 1FUB and 1FU21FUB1FU2 from powder diffraction), T3R3 (e.g. PDB ID 2QIU), and R6 (e.g., PDB ID 1EV6) conformers, where T states for “tense” conformer, R for “relaxed” and Rf states for “frayed,” in which the amino acids B1–B3 are in extended conformation and B4–B8 are in helical conformation equivalent to the R-conformation [9], [10], [11].

Mutational analysis of human insulin has shown that some point mutant can result in weakening of the hexameric form, resulting in increased propensity for dissociation into the active monomeric form and thus a quicker delivery of insulin [12], including the human insulin analog B28 Pro  Asp (aspart insulin), a therapeutic form of insulin for fast-acting, meal-time administration [12], [13], [14], [15], [16]. To date, the crystal structure of the aspart insulin has only been reported in a R6 assembly [13] and there is a lack for evidences of intermediate oligomeric species between hexamers and monomers. We sought to perform a structural characterization of the aspart insulin from the pharmaceutical formulation, since ultimately it would provide a deeper insight on their structure–function relationship [17]. In the present work we report the T3R3 hexamer of the aspart insulin, in which the R-monomer is shown with the whole segment comprising the first 8 residues of the B chain N-termini in α-helical conformation. Along with additional high-resolution spectroscopic data by ion mobility mass spectrometry and small-angle X-ray scattering (SAXS) characterizing the oligomeric distribution and conformation of the aspart insulin in solution, we discuss our findings in light of the weakening of monomer–monomer interaction which would favor the enhanced propensity for hexamer dissociation of the aspart human insulin variant.

Section snippets

Chemicals

Human insulin formulations at 100 U/mL were acquired direct from drug store and kept under refrigeration at 4 °C until use. In this work, we used the human insulin analog B28 Pro  Asp (Aspart insulin, NovoRapid Penfill Aspart Insulin, Novo Nordisk, Brazil, lot # XS62590) and wild-type, regular human insulin (Novolin® R; Novo Nordisk, Brazil, lot # TS62953). According to the manufacture's product leaflet, the composition of the aspart formulation is the following: insulin aspart (rDNA origin) 100 

Ion mobility mass spectrometry

In order to gain insight on the oligomeric distribution of aspart insulin in its pharmaceutical formulation we have conducted electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI–IMS–MS) measurements. The combination of electron spray ionization with ion mobility and mass spectrometry allows the separation and accurate measurement of molecular species in a polydisperse system by the decrease in spectral congestion, with the accurate evaluation of their molecular weight,

Discussion

We showed here that a T3R3 conformer can be achieved for the hexameric aspart insulin. These data indicate the conformational plasticity of the aspart insulin even at hexameric assembly, being able to convert from a full-R (R6) conformer to a structure with the T state (T3R3).

In the present SAXS measurements, we inferred similar mean oligomeric organization for both aspart and regular human insulin, with equivalent scattering profiles (Fig. 2) and structural parameters (Table 2). Both the

Acknowledgments

We would like to thank MS Marcela Rosa (INMETRO) for helpful assistance and Dr. Gustavo H. M. F. de Souza (Research Scientist, HRMS Bio Applications, Waters Corporation — Brazil) for helpful discussion on ESI–IMS–MS data analysis. This research was supported by the CAPES, CNPq, INCT-CNPq, FAPERJ, LNLS and Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (INMETRO). Funding agencies had no role in study design, data collection or analysis.

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