Introduction Insulin is a hormone released by pancreatic befa cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amine acids into liver, adipose tissue and muscle and promotes the storage of these nutrients in the form of glycogen, lipids and protein respectively. Failure to uptake and store nutrients results in diabetes. Type-] diabetes is characterized by the inability to synthesize insulin, whereas in type-2 diabetes the body becomes resistant to the effects of insulin, presumably because of defects in the insulin signaling pathway.Proteolytic cleavage Pro-Insulin InsulinIn 1954, Frederick Sanger determined primary structure of Insulin enurwe sna a A-chain is formed of 21 amino acid residue, while B-chain is formed of 30 amino acid residue. The A-chain has N-terminal glycine (GLY) and a C-terminal Asparagine (Asn), while the B-chain has an N-terminal phenylalanine (Phe) and a C terminal Alanine (Ala). Two disulphide bonds (-S-S-) present between two chains lie between cysteine amino acids located at 7th and 20th position of A- chain and 7th and 19th position of B-chain. A third disulfide bond also occurs in the A-chain between cysteine (Cys) amino acids at 6th and 11th position.Insulin is stored as a zinc-coordinated hexamer. However, this hexamer dissociates into zinc-free monomers that are able to bind to the insulin receptor (IR).When Insulin interacts with the microreceptor, 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 vanulatne tha intrarallilay sancanteatian af aliunnen‘The insulin receptor is a member of the ligand-activated receptor and tyrosine kinase family of transmembrane signaling proteins that collectively are fundamentally important regulators of cell differentiation, growth, and metabolism. The insulin receptor has a number of unique physiological and biochemical properties that distinguish it from other members of this large well-studied receptor family. The main physiological role of the insulin receptor appears to be metabolic regulation, whereas all other receptor tyrosine kinases are engaged in regulating cell growth and/or differentiation. Receptor tyrosine kinases are allosterically regulated by their cognate ligands and function as dimers. In all cases but the insulin receptor (and 2 closely related receptors), these dimers are noncovalent, but insulin receptors are covalently maintained as functional dimers by disulfide bonds. The initial response to the ligand is receptor autophosphorylation for all receptor tyrosine kinases. In most cases, this results in receptor association of effector molecules that have unique recognition domains for phosphotyrosine residues and whose binding to these results in a biological response. For the insulin receptor, this does not occur; rather, it phosphorylates a large substrate protein that, in turn, engages effector molecules.Insulin bound to receptor sites ‘The insulin receptor consists of two chains tocated on the outer face of the plasma membrane and two chains that traverse the membrane and protrude on the cytosolic face. Bind- ing of insulin to the a chains triggers autophos- phorylation of Tyr residues in the earboxyl-terminal domain of the B subunits, which allows the tyrosine kinase domain to catalyze phosphorylation of other target proteins. ‘The assubunit 'scomposed of 728 amino acids and has the insulin binding domain, ‘The @-subunit has £620 amino aeids and 3 compartmental domains: extracellular, trans- membrane, and cytosolic. Cytosolic domain has 3 clusters of tyrosine residues that can be phosphorylated on insulin binding Intracellular Cytosol “— insulin effectsOUTSIDE OF CELL Receptor. 2292202009 jn \Eeeeeses) PI3-kinase Ras-MAPK pathway @© Winen the insulin receptor © IRS-1 activates Pi 3-kinase, © PIP, binds a protein @ Akt catalyzes phorphoryiation binds insulin, the activated which catalyzes the addition Kinase called Akt, which is of key proteins, leading to an receptor phosphorylates of phosphate group tothe activated by other protein increase in glycogen synthase the IRS-1 protein. IRS-1 can _ membrane lipid PIP,, thereby kinases. activity and recruitment of the load to recruitmont of GRB2, converting it to PIP. PTEN glucose transporter, GLUTA, to activating the Ras pathway. can convert PIP, back to PIP, the membraneInsulin comprises two chains (A and B) containing three o-helices (residues A1-A8, A12-A18 and B9-B19) constrained by one intra- and two interchain disulphide bondsThe insulin receptor is - a disulphide-linked (%B)2 homodimer; the extracellular portion of each xP protomer contains six domains (L1, CR, L2, FnIlI-1, FnIlI-2 and FnIII-3) and an insert domain (ID) within FnIII-2” The a-chain component of the ID is terminated by a segment termed “CT, spanning residues 704-719 @ «CT segment 704-719 — Disulphide bond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 B20-B27 Beta turn. Specifically, the hormone rotates approximately 10 degrees around the Gly B20 residue, followed by a 50 degree turn -known as. the B26 turn because of B26's crucial role- around the Phe B24 residue. After both of the aforementioned rotations, B20-B27 is anti- parallel to the first strand of the Li Beta Two Sheet and perpendicular to the Chainn B alpha helix . Simultaneously, the alpha-CT helix extends to include residues 711-714, repositioning 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 resid 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 Tyr826. One hydrogen bond involves a water-mediated reaction between TyrB26 and the backbone of Gly&8, while in the other TyrB26 interacts with the backbone of Pho 24. 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 Pheh24. These hydrogen bonds are important for stabilizing and maintaining the rotations necessary for Insulin to assume the active conformation. 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.Two surfaces of insulin are understood to interact with the insulin receptor’ . The first consists predomi- nantly of hormone-dimerizing residues and contacts the primary binding site on the receptor (site 1; dissociation constant (Ka) ~6.4nM) comprising the #CT segment from one insulin receptor a-chain and the central B-sheet of L1 (L1-P2) of the other o-chain within the insulin receptor dimer The second consists predominantly of hormone-hexamerizing residues and is proposed to interact with a secondary insulin receptor site (site 2; Ky ~400nM) at the junction of FnIII-1 and Fnlll-2 of the insulin receptor o-chain opposite to that contributing L1 to site 1many UL Wie restuues Ural play an essenual rule ne Wisumar wiUMYy ae 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 Gly‘, lleA2, ValA3, GinA5, TyrA19 on Chain A, and , 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. 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 in the alpha-CT subunit inserts itself into a formed by Gy‘, lleA2, TyrA19, LeuB11, and 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-4IR 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 = 12, 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.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 mare effective Inculin analnes ac well ac new drug theranies