Principles of Protein Structure Assignment 1996

Please enter:

Your name
Your e-mail adress
Your 4-letter PDB code

Insulin Sequences

Because insulin is essential for normal growth and metabolic regulation in higher organisms it is ubiquitous in the animal kingdom. To date, insulin sequences from over 80 different organisms have been determined. In addition, insulin-like material has been detected immunologically in several primitive species, indicating that the insulin fold is an ancient structural motif. It appears that all of these molecules have evolved from a common ancestral protein through gene duplication and diversification.

In this assessment exercise we will be retrieving and comparing insulin sequences of

the different vertebrate classes
the different mammalian orders
the strange mammalian sub-order Hystricomorpha (South-American rodents)
primitive organisms

Obtaining a Sequence from a Database

We have already looked at porcine insulin in the first section of the assignment. Now we will retrieve the porcine proinsulin sequence from an appropriate database on the WWW (Refer to the Introduction page of this assignment for help on sequence databases); you should start an additional window on your Web-browser to do this, retaining the Assignment page in the original (for example, Netscape's New Web Browser or New Window option, under File on the menu bar).

(Hint: the Swiss-Prot accession number for this sequence is P01315, and the ID is INS_PIG)

The sequence starts with the B-chain residues and ends with the A-chain, but you will note that it is longer than 51 amino acids. This is because insulin is synthesised as a larger molecule in which the B-chain C-terminus (B30) is connected to the A-chain N-terminus (A1) by a connecting sequence of about 30 residues- this is called proinsulin.

Discussion Question

Why is insulin made as proinsulin?

The connecting sequence is flanked on either side by a pair of basic amino acids. Proteolytic cleavage of proinsulin at these dibasic sequences liberates the insulin molecule and free C-peptide, which are released from the B-cells in equimolar quantities, into the bloodstream.

Q11) Which one of the following statements is false?

a) The dibasic processing sequence is Arg-Arg at the 'BC' junction.
b) The dibasic processing sequence is Lys-Arg at the 'CA' junction.
c) The C-peptide residues are numbered 33-61.
d) The C-peptide residues are numbered 31-66.

Comparing Insulins from Different Taxa

We will be comparing insulins from several taxa (units of classification). Figure 2 illustrates a simplified evolutionary tree based on the Grobben classification of the animal kingdom above the Coelentrates (jellyfish, sea anemones, etc) into two evolutionary lines.

The Vertebrate Classes

                           Pre                              B                                C                             A

                 1   5   10   15   20       1   5   10   15   20   25   30  1   5   10   15   20   25   30   35   1   5   10   15   20
                 |   |    |    |    |       |   |    |    |    |    |    |  |   |    |    |    |    |    |    |   |   |    |    |    | 
HUMAN            MALWMRLLPLLALLALWGPDPAAA   FVNQHLCGSHLVEALYLVCGERGFFYTPKT  RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR   GIVEQCCTSICSLYQLENYCN
CHUM SALMON      MAFWLQAASLLVLLALSPGVDA     AAAQHLCGSHLVDALYLVCGEKGFFYTPK   RDVDPLIGFLSPKSAKENEEYPFKDQTEMMVKR     GIVEQCCHKPCNIFDLQNYCN
CHICKEN          MALWIRSLPLLALLVFSGPGTSYA   AANQHLCGSHLVEALYLVCGERGFFYSPKA  RRDVEQPLVSSPLRGEAGVLPFQQEEYEKVKR      GIVEQCCHNTCSLYQLENYCN
AFR. CLAWED FROG MALWMQCLPLVLVLFFSTPNTEA    LVNQHLCGSHLVEALYLVCGDRGFFYYPKV  KRDMEQALVSGPQDNELDGMQLQPQEYQKMKR      GIVEQCCHSTCSLFQLESYCN
AMER. ALLIGATOR                             AANQRLCGSHLVDALYLVCGERGFFYSPKG                                        GIVEQCCHNTCSLYQLENYCN

Q12) Which one of the following statements is false?

a) The cysteine and glycine residues essential for formation of the insulin fold exist in all of these molecules.
b) The residues buried in the formation of dimers are conserved in all of these molecules
c) The residues buried in the formation of hexamers are conserved in all of these molecules
d) The cysteine and leucine residues buried in the core of the molecule are conserved in all of these molecules

Q13) Comparing the four connecting (C) peptide sequences given, which one of the following statements is false ?

a) There is a higher degree of variance in the connecting peptides than in the A and B chains of these molecules
b) The terminal basic dipeptides are conserved in all four sequences
c) The sequences do not contain any cysteine, tryptophan or histidine residues, and only the occassional tyrosine or phenylalanine residue.
d) The connecting peptide sequences are all of the same length.

Some Mammalian Orders

                           Pre                              B                                C                             A

                 1   5   10   15   20       1   5   10   15   20   25   30  1   5   10   15   20   25   30   35   1   5   10   15   20
                 |   |    |    |    |       |   |    |    |    |    |    |  |   |    |    |    |    |    |    |   |   |    |    |    | 
HUMAN            MALWMRLLPLLALLALWGPDPAAA   FVNQHLCGSHLVEALYLVCGERGFFYTPKT  RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR   GIVEQCCTSICSLYQLENYCN
DOG              MALWMRLLPLLALLALWAPAPTRA   FVNQHLCGSHLVEALYLVCGERGFFYTPKA  RREVEDLQVRDVELAGAPGEGGLQPLALEGALQKR   GIVEQCCTSICSLYQLENYCN
MOUSE (2)        MALLVHFLPLLALLALWEPKPTQA   FVKQHLCGPHLVEALYLVCGERGFFYTPKS  RREVEDPQVEQLELGGSPGDLQTLALEVARQKR     GIVDQCCTSICSLYQLENYCN
RABBIT           MASLAALLPLLALLVLCRLDPAQA   FVNQHLCGSHLVEALYLVCGERGFFYTPKS  RREVEELQVGQAELGGGPGAGGLQPSALELALQKR   GIVEQCCTSICSLYQLENYCN
BOVINE           MALWTRLRPLLALLALWPPPPARA   FVNQHLCGSHLVEALYLVCGERGFFYTPKA  RREVEGPQVGALELAGGPGAGGLEGPPQKR        GIVEQCCASVCSLYQLENYCN

Studies of the biological activities and sequences of insulins from different animals have suggested certain largely invariant residues which may be involved in receptor binding. The 'classical' receptor binding region of the insulin molecule has been postulated to consist of the residues A1 Gly, A5 Gln, A19 Tyr and A21 Asn in the A chain and B24 Phe, B25 Phe, B26 Tyr, B12 Val and B16 Tyr in the B chain.

Q14) Which one of the following statements is false?

a) The data are consistent with a model in which hydrophobic residues such as B24 Phe, B25 Phe, B26 Tyr, B12 Val and B16 Tyr are involved in receptor binding in a manner similar to their involvement in dimerization.
b) A1 Gly, , A4 Glu, A19 Tyr and A21 which play no direct part in dimerization could be on the periphery of the receptor-binding region.
c) All of these residues are on the surface of the insulin monomer.
d) If residues B24,B25 and B26 were involved in both dimerization and receptor binding, then binding of insulin to its receptor might be inhibited at high concentrations.

Q15) The rate of amino acid substitution for most insulins has been in the order of 1 x 10-9 per locus per year (this unit is called a Pauling). Which one of the following statements is false ?

a) The only significant variation in the B chain is at the carboxy-terminus
b) The only significant variation in the A chain is in the A9-A12 turn between the intrachain disulphide
c) There is a degree of conservation in the first half of the connecting peptides
d) The high degree of homology in the connecting peptides reflects the conservation of stable secondary structure which must have functional significance.

The Hystricomorphs

Click here for information on rodent classification.

                           Pre                              B                                C                             A

                 1   5   10   15   20       1   5   10   15   20   25   30  1   5   10   15   20   25   30   35   1   5   10   15   20
                 |   |    |    |    |       |   |    |    |    |    |    |  |   |    |    |    |    |    |    |   |   |    |    |    | 
HUMAN            MALWMRLLPLLALLALWGPDPAAA   FVNQHLCGSHLVEALYLVCGERGFFYTPKT  RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR   GIVEQCCTSICSLYQLENYCN
CASIRAGUA                                   YVGQRLCGSQLVDTLYSVCKHRGFYRPSE                                        GIVDQCCTNICSRNQLLTYCN
CHINCHILLA                                  FVNKHLCGSHLVDALYLVCGDRGFFYTPMA                                        GIVDQCCTSICTLYQLENYCN
COYPU                                       YVSQRLCGSQLVDTLYSVCRHRG_FYRPND                                        GIVDQCCTNICSRNQLMSYCND
CUIS                                        FFNRHLCGSNLVDALYVVCKDKGFFSRPD                                         GIVDQCCTRICTSYQLRNYCN         
DEGU             MAPWMHLLTVLALLALWGPNSVQA   YSSQHLCGSNLVEALYMTCGRSGFYRPHD   RRELEDLQVEQAELGLEAGGLQPSALEMILQKR     GIVDQCCNNICTFNQLQNYCNVP
GUINEA PIG       MALWMHLLTVLALLALWGPNTGQA   FVSRHLCGSNLVETLYSVCQDDGFFYIPKD  RRELEDPQVEQTELGMGLGAGGLQPLALEMALQKR   GIVDQCCTGTCTRHQLQSYCN
PORCUPINE                                   FVNQHLCGSHLVEALYLVCGNDGFFYRPKA                                        GIVDQCCTGVCSLYQLQNYCN

In the South American hystricomorphs, the rate of accepted mutation is fifty times higher than in other mammalian insulins, Most substitutions are on the surfaces involved in dimer and hexamer formation;

Q16) Which one of the following statements about the hystricomorph insulin sequences is false?

a) The sequences have divergence not only from the mammallian lineage but also amongst themselves.
b) All of these sequences (except chinchilla and porcupine) have an Asn or Gln residue at B10, indicating that they probably do not form zinc-hexamers
c) The presence of the charged polar residue Arg at B26 in coypu, casiragua and degu insulins would weaken the potential for dimerization and make the proposed hydrophobic interaction with the insulin receptor less favourable.
d) Conservation of the cysteine and glycine residues crucial for the insulin fold indicate that all these molecules could adopt a structure similar to that of porcine insulin

Some Primitive Insulins?

The primitive jawless hagfish (Myxine glutinosa)is a member of the class Agnatha of the phylum Chordata, which diverged from the main line of vertebrate evolution over 500 million years ago. The hagfish occupies a key branch point on the evolutionary tree, separating the vertebrates from the invertebrates.

Amphioxus (Branchiostoma califoriensis) is a primitive cephalochordate that occupies a key position in chordate development as a possible extant relative of the invertebrate progenitor from which the vertebrates emerged. The sequence of amphioxus pre-pro insulin-like peptide contains features characteristic of both insulin and the related protein insulin-like growth factor 1 (containing an octapeptide extension at the end of the A region known as the D-region). This indicates that IGF-1 emerged at a very early stage in vertebrate evolution from an ancestral insulin-type gene.

The sponge (Geodia cydonium) belongs to the phylum Profirea which are first identifiable from the Paleozoic era (about 1.5 billion years ago)

                           Pre                              B                                C                             A

                 1   5   10   15   20       1   5   10   15   20   25   30  1   5   10   15   20   25   30   35   1   5   10   15   20
                 |   |    |    |    |       |   |    |    |    |    |    |  |   |    |    |    |    |    |    |   |   |    |    |    | 
HUMAN            MALWMRLLPLLALLALWGPDPAAA   FVNQHLCGSHLVEALYLVCGERGFFYTPKT  RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR   GIVEQCCTSICSLYQLENYCN
SPONGE           MALWILLPLLALLILWGPDPAQA    FVNQHLCGSHLVEALYILVCGERGFFYTPMS RREVEDPQVGQVELGAGPGAGSEQTLALEVARQ     ARIVQQCTSGICSLYQENYCN
ATLANTIC HAGFISH MALSPFLAAVIPLVLLLSRAPPSADT RTTGHLCGKDLVNALYIACGVRGFFYDPTKM KRDTGALAAFLPLAYAEDNESQDDESIGINEVLKSKR GIVEQCCHKRCSIYDLENYCN
AMPHIOXUS        MNLSSVYVLASLAVVCLLVKE      TQAEYLCGSTLADVLSFVCGNRGYNSQP    RRSVSKRAIDFISEQQAKDYMGAMPHIRRRR       GLVEECCYNVCDYSQLESYCN

Q17) Which one of the following statements is false?

a) The B-chain helix of sponge "insulin" is probably distorted owing to the existence of an extra residue between the terminal cysteines, compared to other insulins.
b)The highly conserved intrachain disulphide of insulin is absent in sponge "insulin".
c) The atlantic hagfish and amphioxus probably do not store their insulins as 2-zinc hexamers.
d) Conservation of the FFY B-chain beta-strand sequence in all three primitive insulins implies that they all dimerize and that they probably all bind their receptors in a similar way to the more "advanced" insulins.

Index to Assignments

Last updated 11th Apr '96