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PDBsum entry 1a7f
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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A structural switch in a mutant insulin exposes key residues for receptor binding.
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Authors
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S.Ludvigsen,
H.B.Olsen,
N.C.Kaarsholm.
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Ref.
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J Mol Biol, 1998,
279,
1-7.
[DOI no: ]
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PubMed id
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Abstract
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Despite years of effort to clarify the structural basis of insulin receptor
binding no clear consensus has emerged. It is generally believed that insulin
receptor binding is accompanied by some degree of conformational change in the
carboxy-terminal of the insulin B-chain. In particular, while most substitutions
for PheB24 lead to inactive species, glycine or D-amino acids are well tolerated
in this position. Here we assess the conformation change by solving the solution
structure of the biologically active (GluB16, GlyB24, desB30)-insulin mutant.
The structure in aqueous solution at pH 8 reveals a subtle, albeit well-defined
rearrangement of the C-terminal decapeptide involving a perturbation of the
B20-23 turn, which allows the PheB25 residue to occupy the position normally
taken up by PheB24 in native insulin. The new protein surface exposed
rationalizes the receptor binding properties of a series of insulin analogs. We
suggest that the structural switch is forced by the structure of the underlying
core of species invariant residues and that an analogous rearrangement of the
C-terminal of the B-chain occurs in native insulin on binding to its receptor.
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Figure 1.
Figure 1. X-ray structure (Baker et al., 1988) of the insu-
lin hexamer showing the symmetrical arrangement of
three dimers around a vertical 3-fold axis. The dimer
in front is shown schematically with helices and
b-strands, whereas the other two are in surface rep-
resentation, orange and green, respectively. The mono-
mer is composed of two polypeptide chains, the
A-chain (A1 to A21), which folds into a helix-loop-
helix motif, and the B-chain (B1 to B30) containing an
N-terminal arm, a central helix and a C-terminal
b-strand (flat arrow). B-Chain residues contribute to
the interface between monomers in the dimer including
an antiparallel b-sheet formed by the B-chain C termini
and side-chains of the central helices. The interface
between dimers involves a distinct set of protein-pro-
tein contacts and is mainly composed of residues in
the N-terminal part of the B-chain with contributions
from residues in the C-terminal A-chain helix and the
B-chain helix, i.e. B1, B2 and B4 from one dimer inter-
act with residues A13, A14, B1 and B16 to B20 of the
opposing dimer (see Baker et al., 1988, for details).
Three cystine bridges A6.A11, A7.B7, and A20.B19
are shown in yellow in stick representation. Figure of
molecule was produced using Insight (Molecular Simu-
lations Inc.).
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Figure 3.
Figure 3. Representative solution structures of: a,
(GluB16, GlyB24, desB30)-insulin at pH 8.0; and b,
HisB16-insulin (PDB code 1HLS) at pH 2.4 in aqueous solution
[Ludvigsen et al 1994] together with the corresponding surface
representations (c and d). The helical stretches (in a and b)
are shown in blue, and selected side-chains are annotated. In b,
the side-chains of B24, B25 and B26 are arranged as is observed
in crystal structures of wild-type insulin. In a, PheB25 has
moved into the position usually occupied by PheB24. This
structural change is quantitatively characterized by a value of
0.42(±0.17) Å for the mean displacement of the C^α
atom of PheB25 between the average and ensemble structures of
the (GluB16, GlyB24, desB30)-mutant. Furthermore, when the
helical regions (A2 to A9, A14 to A20 and B9 to B19) are used to
align the ensemble of structures for each mutant, a displacement
of only 1 Å is obtained between the average positions of
the PheB25 C^α atom of the (GluB16, GlyB24, desB30)-insulin and
PheB24 C^α atom of HisB16-insulin. Accordingly, the average
values of torsion angles, χ^1 and χ^2, are
50.3(±4.2)° and −96.8(±5.6)°,
respectively, for the PheB25 residue in the present mutant as
compared to values of 53.5(±5.3)° and
−79.2(±4.0)°, respectively, for the equivalent
PheB24 residue in the HisB16 mutant structure. TyrB26 remains
approximately in its original position. Overall, this change
perturbs the turn following the central B-chain helix and bends
the usual extended structure of the C-terminal strand. The
surface shown in d is rotated a little around the vertical axis
compared to b to display the side-chains of B24 and B26 which
are clearly differently exposed as compared to c. Side-chains of
interest and importance for receptor binding are color-coded and
annotated with sequence position in c and d. In a and c the bend
of the C-terminal residues and the accompanying reorientation
result in the exposure of a different surface compared to b and
d, i.e. in particular ValA3 and TyrB26 become more exposed.
Mutant insulins were constructed by oligonucleotide-directed
mutagenesis, fermented in yeast, and purified as described
[Markussen et al 1987 and Brange et al 1988]. Mutants are
expressed as single-chain miniproinsulin precursors,
PheB1...LysB29-Ala-Ala-Lys-GlyA1...AsnA21. The connecting
peptide is cleaved off using lysyl endopeptidase (Achromobacter
protease I, EC 3.4.21.50; Wako Inc., Osaka, Japan). The removal
of ThrB30 has no effect on the biological potency. Figures of
molecules were produced using Insight (Molecular Simulations
Inc.) (c and d) and MOLSCRIPT [Kraulis 1991] combined with
RASTER3D [Bacon and Anderson 1988 and Merritt and Murphy 1994]
(a and b).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
279,
1-7)
copyright 1998.
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