Structure

Insulin is a small, 6000 dalton protein (polypeptide) consisting of an alpha and beta chain. The alpha chain has 21 amino acids, while the beta chains has 30 amino acids. These two chains are connected by two disulfide bridges which yield a protein with a total of 51 amino acids. The final product is a protein that has a 3D conformation which is highly conserved among vertebrate species (1).

However, the insulin hormone is not a protein that is directly synthesized through translation. It is actually derived from a polypeptide known as preproinsulin which has to undergo a series of alterations and modifications in order to obtain the insulin molecule. For example, in the beta cells of the pancreas, insulin mRNA is originally translated and produced as preproinsulin which is composed of 110 amino acids. This polypeptide is then passed on to the endoplasmic reticulum where the signal peptide consisting of 24 amino acids is cleaved by enzymatic activity during insertion into the organelle. The resulting peptide is known as proinsulin which consists of an amino-terminal B chain, a carboxy-terminal A chain, and a C peptide. This polypeptide is then passed onto the golgi apparatus where the middle section consisting of 33 amino acids (C peptide) is cleaved by endopeptidases. After this cleavage we are left with “mature insulin” consisting of an A and B chain and the C peptide molecule. Both of these peptides are then packaged into vesicles within the Golgi which bud off the Golgi body and accumulate in the cytoplasm. Here, the “mature insulin” molecule is modified to its active form by enzymes within the vesicles. These vesicles which contain active insulin are stored in the cytoplasm until the beta cell is stimulated causing the release of both insulin and the C peptide into the blood via exocytosis (7).

The primary structure of the protein consists of the two separate alpha and beta chains which both have a relatively linear conformation. The amino acids of both of these chains are connected by peptide bonds with an amino group (N-terminal) on one end of each chain and a carboxylic acid group (C-terminal) on the other end of each chain (2).

In the secondary structure of the hormone, hydrogen bonds form between the peptide bonds of the two chains. This causes the amino acids of the joined chains to coil to form short sections of alpha helix. The alpha chain of the secondary structure is fairly compact and contains two sections of alpha helix which are separated by a flat ribbon section. This formation allows the two helices to lay along side one another which brings the side chains of the helices in close proximity so that they can interact through Van der Waals forces. This also helps to stabilize the secondary structure of the hormone. Meanwhile, the beta chain consists of a single, larger section of alpha helix which appears to wrap around the alpha chain. It also has smaller glycine residues which allow it to fold into a V shape. This brings the C-terminal residues in Van der Waals conatact with the residues of the alpha helix portion of the beta chain which also helps to stabilize the structure. Overall, the secondary structure of insulin is mainly stabilized by hydrogen bonds which form a relatively compact and simple three-dimensional configuration (2).

In the tertiary structure of insulin, this three-dimensional configuration is further stabilized by disulphide bridges which form between the thiol (SH) groups on the cysteine residues. Due to the fact that there are only six cysteines present in this protein, only three disulfide bridges are able to form in the tertiary structure. Two of these bridges form between the alpha and beta chains of the hormone, while one bridge forms within the alpha chain itself. In any event, these three bridges are the primary means of stabilizing the tertiary structure and are a very effective means of keeping the protein intact. Meanwhile, there are also weaker bonds such as salt bridges and Van der Waal’s forces which also aid in stabilizing the tertiary structure of the hormone. Once bound together by these forces, the tertiary structure of insulin has a compact three-dimensional dimmer with several alpha helical domains. The exterior of this molecule is mainly polar, while the interior is mainly non-polar (2).

Finally, the insulin molecule can also take on a quaternary structure as a dimer or hexamer. For instance, in solution, insulin molecules have a tendency to form dimers as hydrophobic bonds can also occur between the residues of two insulin monomers (3). However, insulin is only active as a single unit or monomer so the dimer is unable to initiate a biological response. In the meantime, in the presence of zinc ions, insulin can also form into granules consisting of hexamers. These hexamers (granules) are formed by interactions between the hydrophobic surfaces of six insulin molecules which creates a very strong and stable structure. These hexamers can be very useful when it comes to storing insulin as the toroidal form of these hexamers is the form in which insulin is stored in beta cells before it is released into the blood stream (2).

In any event, even though the insulin molecule can form primary, secondary, tertiary, and quaternary structures, it is only active in one specific form. This is the monomer version of its tertiary structure. The reason for this is that the insulin receptor only recognizes insulin as a single molecule or monomer. Thus, it is not active as a dimer or hexamer (quaternary structures) which are actually the storage forms of the molecule. Similarly, it is also not active in its primary and secondary structures as the insulin receptor is also unable to recognize the insulin molecules in these forms. For instance, the protein has to be folded and compacted in the correct conformation before it can be recognized by the receptor. Consequently, the primary and secondary structures of the molecule do not yet have the correct conformation and are too unstable to be recognized by the insulin receptor (5).

Now that we know that the monomer form of insulin's tertiary structure is the only version of the molecule that is biologically active, we can also look at what portion of the molecule is acutally involved in the activation of the recpetor. In the case of insulin it has several surface residues including Ser-A12, Leu-A13, Glu-A17, His-B10, Glu-B13, and Leu-B17 which are all responsible for binding the hormone to the receptor. Together, these residues are collectively known as the "primary binding surface" and are most important to the function and biological activity of the insulin hormone. The reason for this is that this is the portion of the molecule that actually interacts with and binds to the receptor. Thus, it is through these residues that insulin is able to interact with the cells and initiate the appropriate biochemical response. In this way, without this "primary binding surface", insulin would not be a biologically active molecule (5).

At the same time, we also know that there is an active insulin receptor for the insulin hormone. This receptor is a transmembrane protein with tyrosine kinase activity. It has two alpha and two beta subunits which are linked together by disulfide bonds. The alpha chains are completely extracellular and house the insulin binding domains. In other words, this is the part of the receptor that recognizes and binds the insulin hormone. Meanwhile, the beta chains pass through the membrane and the intracellular portion has tyrosine kinase activity. Through these tyrosine kinase domains, the beta subunits of the receptor are able to phosphorylate themselves (autophosphorylation) after the hormone binds to the alpha subunits of the receptor. This activates the receptor and initiates the corresponding biochemical response to the hormone within the cell. For instance, once active, the insulin receptor begins to phosphorylate a number of other proteins and enzymes within the cell through action of the tyrosine kinase. This alters the activity of these enzymes causing them to activate specific biochemical pathways which generates the desired response to the hormone (4).

Gene coding sequence for preproinsulin in humans:

1 agccctccag gacaggctgc atcagaagag gccatcaagc agatcactgt ccttctgcca
61 tggccctgtg gatgcgcctc ctgcccctgc tggcgctgct ggccctctgg ggacctgacc
121 cagccgcagc ctttgtgaac caacacctgt gcggctcaca cctggtggaa gctctctacc
181 tagtgtgcgg ggaacgaggc ttcttctaca cacccaagac ccgccgggag gcagaggacc
241 tgcaggtggg gcaggtggag ctgggcgggg gccctggtgc aggcagcctg cagcccttgg
301 ccctggaggg gtccctgcag aagcgtggca ttgtggaaca atgctgtacc agcatctgct
361 ccctctacca gctggagaac tactgcaact agacgcagcc cgcaggcagc cccacacccg
421 ccgcctcctg caccgagaga gatggaataa agcccttgaa ccagcaaaa

Comparative analysis of the protein sequences of preproinsulin in homo sapiens (humans), rattus losea (rats), and felis catus (cats):

*From this analysis one can see that the amino acid sequences for insulin is highly conserved among vertebrate species.


References:

1) http://en.wikipedia.org/wiki/Insulin
2) http://www.biotopics.co.uk/as/insulinproteinstructure.html
3) http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin_struct.html
4) http://en.wikipedia.org/wiki/Insulin_receptor
5) http://images.google.ca/imgres?imgurl=http://www.betacell.org/images/CMS/structure-insulin-panel3-th.jpg&imgrefurl=http://www.betacell.org/content/articles/print.php%3Faid%3D8&h=536&w=500&sz=50&hl=en&start=2&tbnid=Na2M8_NNQxwKDM:&tbnh=132&tbnw=123&prev=/images%3Fq%3Dinsulin%2Breceptor%26svnum%3D10%26hl%3Den
6) http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/pancreas/insulin.html
7) http://www.betacell.org/content/articles/print.php?aid=1