Th The importance of TyrB26 to the 50 degree turn was examined by Zakova et al.  Initially, researchers assumed the residues B26-B30 on Chain B were not important for binding; their deletion resulted in no change in the hormone's affinity for the IR microreceptor.  However, while they do not meaningfully interact with the microreceptor, the properties of B26 are important for ensuring the crucial B26-turn occurs.

Insulin Interactions with the Insulin Receptor

Taylor Maurer '17 and Morgan Perrett '17


I. Introduction

Insulin, an important hormone in the endocrine system, regulates and maintains carbohydrate metabolism, promoting cell growth and function.  When Insulin interacts with the  , a part of the Insulin Receptor (IR), it triggers a phosphorylation cascade starting in the holoreceptor's tyrosine kinase domain.  This leads to the introduction of glucose into the cell.  Without Insulin, glucose is prevented from entering the cell; thus, Insulin's interaction with the IR regulates the intracellular concentration of glucose.  Until recently, the Insulin-IR binding mechanism was unknown.  We will uncover the newly discovered mechanisms between Insulin and its receptor by highlighting the interactions that solidify its binding.  

II. General Structure

Insulin is stored as a zinc-coordinated hexamer.  However, this hexamer dissociates into zinc-free monomers that are able to bind to the IR.  A single Insulin monomer has two chains -   and  - that are connected by three disulfide bonds (Figure 1), one of which is an intramolecular disulfide bond on Chain A.  Both of these chains are needed for Insulin to interact with its receptor.

Figure 1: Disulfide Bonds in an Insulin Monomer

The Insulin receptor is a heterotetramer consisting of multiple subunits.  Each IR monomer includes an alpha-subunit leucine-rich repeat domain (L1 Beta Two Sheet) combined with a cysteine-rich domain (CR, as well as an alpha-subunit C-terminal segment (alpha-CT.  These are located in the extracellular matrix and constitute the .   The supplementary image shows an additional leucine-rich repeat domain (L2) and the first, second, and third fibronectin type III domains, which, combined with the microreceptor, constitute the holoreceptor.  There are two isoforms of the Insulin receptor: IR-A and IR-B.  IR-A has an additional 12 amino acids on the C-terminal of the alpha-CT subunit.  The displayed protein is in the IR-A form.

The two extracellular subunits -L1 Beta Two Sheet and alpha-CT- bind a single Insulin monomer.  This results in a change in conformation in both the Insulin monomer and the IR's holoreceptor, which initiates the aforementioned phosphorylation cascade.

III. Insulin Conformation and Stability

Before Insulin can bind to the microreceptor, it must change conformation.  Insulin has two conformations: an active conformation used in binding and a free conformation.  If Insulin does not change conformation, there is a steric clash between the alpha-CT subunit and the B25-B30 residues.  The change between the two forms is mediated by two "hinge-like" rotations at the .  Specifically, the hormone rotates approximately 10 degrees around the  residue, followed by a 50 degree turn -known as the B26 turn because of B26's crucial role- around the  residue.  After both of the aforementioned rotations,  is anti-parallel to the first strand of the L1 Beta Two Sheet and perpendicular to the  Chain B alpha-helix .  Simultaneously, the alpha-CT helix extends to include residues 711-714, the alpha-CT helix between the L1 Beta Two Sheet and Insulin's Chain A.  Therefore, after the two "hinge-like" rotations, the 705-714 residues in the alpha-CT helix occupy the space previously occupied by B25-B30 residues in the free hormone.

The B26 turn is stabilized and maintained by involving TyrB26.  One hydrogen bond involves a water-mediated reaction between TyrB26 and the backbone of GlyB8, while in the other TyrB26 interacts with the backbone of PheB24.  The importance of these hydrogen bonds, and thus TyrB26's presence, to the 50 degree turn was examined by Zakova et al. (2014).  In order to demonstrate the importance of the TyrB26 side chain hydroxyl, they substituted Phenylalanine into position B26.  This mutation resulted in a 50% decrease in Insulin's binding affinity and highlights the importance of TyrB26's two hydrogen bonds to the backbone of GlyB8 and PheB24.  These hydrogen bonds are important for stabilizing and maintaining the rotations necessary for Insulin to assume the active conformation.

Furthermore, in order for the active form of Insulin to bind to its receptor, the Insulin monomer must be stable.  In particular, the stability of the N-terminal A chain alpha helix is crucial for the correct placement of many hormone receptor contacts.  Any mutations that cause a distortion of this helix will inhibit correct binding.  This helix is by the packing of ValA3, IleA2, as well as Chain A's intramolecular disulfide bond.

The importance of  to the stability of the N-terminal A chain alpha helix was determined using several amino acid substitutions.  Xu et al. (2002) found that when IleA2 is substituted with Alanine, the N-terminal A chain alpha helix undergoes segmental unfolding, which inhibits correct binding.  Furthermore, the importance of ValA3 was already well known due to naturally occurring mutations.  In response, Huang et al. (2007) studied ValA3's contribution to the stability of the Insulin molecule.  They first converted ValA3 to a smaller entity: alpha-aminobutyric acid (Aba, Figure 2).

Figure 2: Alpha-aminobutyric Acid (Aba) Bond-line Structure

Despite fitting nicely into the Chain A-Chain B  that ValA3 typically resides in, AbaA3 Insulin had decreased stability.  Aba's lack of ValA3's gamma methyl group created fewer opportunities for hydrophobic interactions in the mostly nonpolar crevice.  Next, Huang et al. determined how a polar moiety would affect stability by creating ThrA3 Insulin.  ThrA3 Insulin's N-terminal A chain alpha helix was also less stable than ValA3.  This shows the significance of nonpolar packing in the Chain A-Chain B crevice ValA3 resides in.

IV. Insulin Binding

How Insulin interacts with the IR is still an area of investigation.  Researchers currently use multiple techniques, including mutagenesis and photo-crosslinking to crystallized mini-receptors, to study this complicated molecule.  Due to the complexity of Insulin-IR binding, every crucial interaction used in the binding of Insulin to the IR could not be expanded upon here.  However, the following section highlights some of the interactions pivotal to the binding of Insulin to its receptor.

Many of the residues that play an essential role in Insulin-IR binding are found in Site 1, a grouping of residues defined by Zakova et al. (2014) to be responsible for effective IR binding.  After Insulin's two rotations,  - which contains GlyA1, IleA2, ValA3, GlnA5, TyrA19 on Chain A, and ValB12, LeuB11, PheB24, and PheB25 on Chain B- is exposed.  Even though the all of the exact interactions and conformations of these residues are unclear, it is certain they insert themselves between the alpha-CT subunit and the L1 Beta Two Sheet.  They can then interact with the microreceptor using  .  

Specifically, many of the interactions between Insulin and the IR occur when IR residues insert themselves in nonpolar pockets created by Site 1 residues.  For example, Phe714 (not available) in the alpha-CT subunit inserts itself into a  formed by GlyA1, IleA2, TyrA19LeuB11, and ValB12.  Hydrophobic interactions like this help hold the Insulin molecule and IR together.

Furthermore, the aromatic nature of some residues is of great importance.  Specifically, PheB25's side chain projects away from the L1 Beta Two Sheet, which allows for its insertion into a shallow pocket located in the alpha-CT  between Pro718 and Val715.  The aromatic portion of  is also crucial. Kristensen et al. (1996) found that the creation of LeuA19 Insulin reduced binding 1000 fold, while the creation of PheA19 Insulin only reduced binding affinity 4 to 5 fold.  This indicates TyrA19's aromatic ring is crucial for Insulin-IR interactions.  Another important aromatic residue is PheB24. Its aromatic ring projects into a hydrophobic pocket, where it can interact using  with residue Phe714, as well as B-chain residues ValB12, LeuB15, and TyrB26.

Non-aromatic residues are also crucial for Insulin binding.  For example, ValA3, which also plays a large role in stabilizing Insulin.  Photo-crosslinking studies by Huang et al. (2007) show that the orientation of the residue in the Chain A-Chain B crevice allows it to interact using van der waals interactions with   , encompassed in the alpha-CT subunit. 

V. Implications

Understanding the residues essential to Insulin-IR binding sheds light on the causes of certain diseases, such as Diabetes Mellitus.  As demonstrated above, many of Insulin's hydrophobic and aromatic residues must be maintained to retain proper binding and engagement with its receptor.  Insulin's reduced binding affinity is determental to the life of the cell and the organism.  These findings are an invaluble tool for the design of more effective Insulin analogs, as well as new drug therapies.

According to the National Center of Chronic Disease Prevention and Health Promotion, 29.1 million Americans suffer from Type 1 and Type 2 Diabetes Mellitus.  Fortunately, Insulin analogs can be administered by injection to lower elevated blood sugar levels in the body.  However, eliminating this life altering disease should remain the focus of healthcare professionals, reseachers and patients.  The research of the included authors contributes greatly to the understanding of this hormone and its receptor. With further research, a cure for Diabetes is on the horizon.

VI. References

Diabetes Latest. June 17, 2014. National Center of Chronic Disease Prevention and Health Promotion. December 16, 2015. <>

Stevan R. Hubbard. 1997. Crytstal structure of the activated insulin receptor tryosine 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.

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