Team:Unesp Brazil/Model/Bioinformatics
iGEM Unesp Brazil
Model
1. Introduction
The hormone Insulin is essential for the endocrine system, regulating glucose metabolism and promoting cell growth and function.
In resume, when insulin interacts with a part of the Insulin Receptor (IR), the micro-receptor, a phosphorylation cascade starts on the holoreceptor of tyrosine kinase domain.
This action promotes the introduction of glucose into the cell, in this way, the interaction between insulin and its receptor regulates the concentration of glucose into the cell.
1.1 Insulin-Receptor
The hormone Insulin is essential for the endocrine system, regulating glucose metabolism and promoting cell growth and function.
In resume, when insulin interacts with a part of the Insulin Receptor (IR), the micro-receptor, a phosphorylation cascade starts on the holoreceptor of tyrosine kinase domain.
This action promotes the introduction of glucose into the cell, in this way, the interaction between insulin and its receptor regulates the concentration of glucose into the cell.
2. Insulin Conformation
2.1 First Analysis (Tyr26B, Phe24B, Gly20B)
The conformation of Insulin must change before the hormone can bind to the micro-receptor.
The difference between the active form of insulin (used in binding) and a free conformation is mediated by rotations at the B20-B27 Beta Turn.
Specifically, the residue GlyB20 is involved, as the hormone rotates approximately 10 degrees around it, followed by a 50 degree turn around the PheB24 residue.
One of these important rotations, the B26 turn, is maintained by the residue TyrB26 mediated by two hydrogen bonds. One H-bond occurs between TyrB26 and the backbone of GlyB8 by a water-mediated reaction, while in the other TyrB26 interacts with PheB24.
The nucleotide sequence of our insulin-analog (SCI-57) with penetratin, necessary for the transformation of the microorganisms in our project, was used for the prediction of a structure model for the produced insulin. In this sense, the Insulin-Analogue produced by our project (IA) has almost 100% of similarity with the model predicted by Swiss Model, being an Insulin with one single chain, Figure 4b.
Objective 1: as the first point of analysis, we aimed to study if the insulin produced by our project would have the essential residues needed for the correct change of conformation of insulin, necessary for the interaction between Insulin and its receptor.
As noticed from Figure 4, the essential residues Tyr, Phe, Gly located in the positions Tyr26B, Phe24B, Gly20B in wild-type insulin can be also found in similar regions in the IA structure (Tyr76A, Phe74A, Gly70A).
This result is interesting as the cited residues are essential for the right changes of conformation and rotations in the insulin molecule, necessary for its interaction with the receptor.
2.2 Insulin Conformation - Second Analysis (ValA3, IleA2)
In addition to the first important residues described in the 2.1 section, it is known that for the active form of Insulin to bind to its receptor, the Insulin monomer must be stable.
The N-terminal A chain alpha-helix stability is fundamental for the right placement of many hormone receptor contacts. As a result, mutations that leave to a distortion of this helix will inhibit correct binding. This important helix is stabilized by the packing of ValA3, IleA2, also by intramolecular disulfide bond of Chain A. In this sense, in our second analysis we tried to verify if our IA would also have ValA3, IleA2 in similar positions as in wild-type insulin.
3. Insulin binding to its receptor
It is reported that the interaction of insulin to the IR is complex and is still an area of investigation, being studied by the use of different techniques as photo-crosslinking and crystallized mini-receptors.
Since it is not possible to explain every single interaction between insulin and IR, our objective in this section is to highlight some of the main aspects crucial for the binding of insulin to its receptor.
In this aspect, Zakova et al. (2014) defined essential residues responsible for effective insulin-IR binding. Following the two insulin’s important rotations, described in section 2, the residues GlyA1, IleA2, ValA3, GlnA5, TyrA19 on Chain A and ValB12, LeuB11, PheB24, and PheB25 from Chain B are exposed, Figure 6. This exposure makes the residues free to interact with the micro-receptor using Van der Waals interactions.
Regarding the position and presence of those crucial residues, we can observe that IA’s have all of them in similar positions in its one chain structure when compared to wild-type insulin residues, Figure 7.
A short video was made to show in 360° the positions and similarity of essential Gly, Ile, Val, Gln, Tyr on and Val, Leu, Phe, and Phe residues in wild-type insulin and IA.
References
Diabetes Latest. June 17, 2014. National Center of Chronic Disease Prevention and Health Promotion. December 16, 2015.
Stevan R. Hubbard. 1997. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. The EMBO Journal 16: 5573-5581.
Kun Huang, Shu Jin Chan, Qing-xin Hua, et al. 2007. The A-chain of Insulin Contacts the Insert domain of the Insulin Receptor. The Journal of Biological Chemistry 282.48: 35337-35349.
Lucie Kosinova, Vaclav Veverka, Pavlina Novotna, et al. 2014. Insight into the Structural and Biological Relevance of the T/R Transistion of the N-Terminus of the B-Chain in Human Insulin. The American Chemical Society 53: 3392-3402.
Claus Kristensen, Thomas Kjeldsen, Finn c. Wiberg, et al. 1996. Alanine Scanning Mutagenesis of Insulin. The Journal of Biological Chemistry 272.20: 12978-12983.
John G. Menting, Jonathon Whittatker, Mai B. Margetts, et al. 2013. How Insulin engages its primary binding site on the Insulin receptor. Nature 493.7431: 241-245.
John G. Menting, Yanwu Yang, Shu Jin Chan, et al. 2014. Protective hinge in Insulin opens to enable its receptor engagement. Proceedings of the National Academy of Sciences of the United States of America E3595 - E3404.
Bin Xu, Qing-xin Hua, Satoe G. Nakagwa. 2002. Chiral mutagenesis of Insulin's hidden receptor-binding surface: structure of an Allo-Isoleucine A2 analouge. Journal of Molecular Biology 316.3: 435-441.
Lenka Zakova, Emilia Klevikova, Martin Lepsik, et al. 2014. Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex. Acta Crystallographica D70: 1001-1007.