Insulin conformational changes under high pressure in structural studies and molecular dynamics simulations
Graphical abstract
Introduction
Insulin (ins) is a small polypeptide hormone that regulates glucose level in higher organisms [1]. Clinical relevance of insulin misfolding and its high tendency to aggregate into amyloid fibrils justify the fact that it is often considered as a model protein in the study of misfolding. Although insulin exists in several oligomeric forms, it is monomeric insulin that is responsible for its biological activity. The most stable oligomer is formed by six insulin molecules and this hexameric state serves as its storage unit. High temperature, low pH or contact with hydrophobic media promote partial destabilization of insulin and a series of structural changes, which result in the formation of the ordered aggregates [2]. High hydrostatic pressure due to its effect on the energy of the system that only involves change of the volume contribution to Gibbs free energy has also proved to be a very powerful tool in the study of misfolding and aggregation [3]. Previous studies showed that insulin is indeed susceptible to fibrillation under high pressure and according to Piccirilli et al. low degree assemblies are more susceptible to pressurization than oligomers [4]. Insulin is a 5800 Da molecule composed of two chains (A-chain of 21 residues; B-chain of 30 residues) and has three α-helices, two in the A chain (A2-A8 and A13-A19) and one in the B chain (B9–B19). Three disulfide bonds take part in protein stability and functionality: two inter-chain bridges are formed between A7-B7 and A20-B19. One intra-chain disulfide bond binds cysteines A6 and A11. The hydrophobic core that is essential for correct folding of insulin [5] is formed by the residues Gly8, Leu11, Val12, Leu15, Phe24, Tyr26 of chain B and two sidechains of the residues located in chain A: Ile2 and Val3.
The first crystal structure of insulin determined in 1969 [5] was followed by other structures of insulin as well as its analogs in the storage form [6]. The latter form of insulin refers to a Zn stabilized hexamer, which is not active and provides the organism with the hormone when required. Native insulin can be crystallized in cubic, monoclinic and rhombohedral space group. The cubic form of insulin crystallizes in the absence of Zn ions (space group I213, a = 78.9 Å). These crystals have much larger solvent content (64%) in comparison to the Zn form (only 35% solvent content). Large solvent content and high symmetry of crystals are advantageous for high pressure crystallographic experiments, thus the cubic insulin crystals provide a good model system for exploring structural changes under high pressure conditions and were selected for our structural studies, and as a model in MD simulation.
Even though there are increasing data regarding insulin folding, assembly and dynamics the precise study of the structural basis for this phenomenon is still missing. It was reported that insulin aggregation must proceed through a bulky, non-native monomer [7], which quickly forms larger oligomers. Therefore, our analyses of insulin structure determined for crystals under high hydrostatic pressure is followed by an investigation of the mechanical properties of the most sensitive to pressure fragments performed with MD simulations. Considering the physiological and therapeutic importance of insulin [8] and the fact that high hydrostatic pressure was proved to evoke aggregation-prone intermediate states in insulin molecule [9] our studies aim in structural investigation of conformations present at the beginning of the aggregation process, which are crucial for the formation of non-native insulin conformations.
Section snippets
Chemicals
Liofilizated protein was purchased from Sigma (I6634, Sigma-Aldrich Co, St. Louis, Mo) and used without purification. All the other chemical reagents used were of analytical grade.
Crystallization
Crystals were grown at room temperature using the hanging drop vapor diffusion method according to Yu & Caspar [5]. Protein was dissolved in 0.01 M Na2HPO4, 0.1 M Na2EDTA, pH 10 to the final concentration of 10 mg/ml. A crystallization drop containing 3 μl of the protein and 2 μl of reservoir solution was equilibrated
Results and discussion
The symmetry of cubic space group I213 is preserved by compressed crystals. Diffraction data at ambient and high pressure were collected to maximum resolution 1.65 Å and 2.0–2.15 Å, respectively. Details of the data collection and refinement are summarized in Table 1. The noticeable loss of the diffraction was observed for insulin crystals compressed to a pressure above 220 MPa. The compressibility β of the unit cell parameter (Fig. 2) was calculated as β = 1/Vu(∂Vu/∂p). An increase of
Conclusions
In this studies, we presented structural models of insulin obtained under high pressure in the scope of 0–200 MPa, which serve as a starting point to discussion about insulin conformational stability. Aggregation of insulin is an important issue as it inactivates its therapeutic role. The comparison of structural data determined under high pressure with results obtained at ambient pressure condition together with molecular dynamics simulations indicates that terminal fragments of chain B are
Authors contributions
K.K. and K.L. initiated the study and directed the project. K.K. conducted crystallographic studies and computational analyses of structures. A.M. performed molecular dynamics simulations. K.K., K.L. and A.M. prepared the manuscript, which was revised and approved by all authors.
Acknowledgments
The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). Authors would like to thank Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Sciences for financial support of presented studies.
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