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Qing-xin Hua Bin Xu Kun Huang Shi-Quan Hu Satoe Nakagawa Wenhua Jia Shuhua Wang Jonathan Whittaker Panayotis G. Katsoyannis Michael A. Weiss 《The Journal of biological chemistry》2009,284(21):14586-14596
A central tenet of molecular biology holds that the function of a protein
is mediated by its structure. An inactive ground-state conformation may
nonetheless be enjoined by the interplay of competing biological constraints.
A model is provided by insulin, well characterized at atomic resolution by
x-ray crystallography. Here, we demonstrate that the activity of the hormone
is enhanced by stereospecific unfolding of a conserved structural element. A
bifunctional β-strand mediates both self-assembly (within β-cell
storage vesicles) and receptor binding (in the bloodstream). This strand is
anchored by an invariant side chain (PheB24); its substitution by
Ala leads to an unstable but native-like analog of low activity. Substitution
by d-Ala is equally destabilizing, and yet the protein diastereomer
exhibits enhanced activity with segmental unfolding of the β-strand.
Corresponding photoactivable derivatives (containing l- or
d-para-azido-Phe) cross-link to the insulin receptor with
higher d-specific efficiency. Aberrant exposure of hydrophobic
surfaces in the analogs is associated with accelerated fibrillation, a form of
aggregation-coupled misfolding associated with cellular toxicity. Conservation
of PheB24, enforced by its dual role in native self-assembly and
induced fit, thus highlights the implicit role of misfolding as an
evolutionary constraint. Whereas classical crystal structures of insulin
depict its storage form, signaling requires engagement of a detachable arm at
an extended receptor interface. Because this active conformation resembles an
amyloidogenic intermediate, we envisage that induced fit and self-assembly
represent complementary molecular adaptations to potential proteotoxicity. The
cryptic threat of misfolding poses a universal constraint in the evolution of
polypeptide sequences.How insulin binds to the insulin receptor
(IR)2 is not well
understood despite decades of investigation. The hormone is a globular protein
containing two chains, A (21 residues) and B (30 residues)
(Fig. 1A). In
pancreatic β-cells, insulin is stored as Zn2+-stabilized
hexamers (Fig. 1B),
which form microcrystal-line arrays within specialized secretory granules
(1). The hexamers dissociate
upon secretion into the portal circulation, enabling the hormone to function
as a zinc-free monomer. The monomer is proposed to undergo a change in
conformation upon receptor binding
(2). In this study, we
investigated a site of conformational change in the B-chain
(PheB24) (arrow in Fig.
1A). In classical crystal structures, this invariant
aromatic side chain (tawny in Fig.
1B) anchors an antiparallel β-sheet at the dimer
interface (blue in Fig.
1C). Total chemical synthesis is exploited to enable
comparison of corresponding d- and l-amino acid
substitutions at this site, an approach designated “chiral
mutagenesis”
(3-5).
In the accompanying article, the consequences of this conformational change
are investigated by photomapping of the receptor-binding surface
(6). Together, these studies
redefine the interrelation of structure and activity in a protein central to
the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the
B-chain (upper) and A-chain (lower) with disulfide bridges
as indicated. The arrow indicates invariant PheB24. The
B24-B28 β-strand is highlighted in blue. B, crystal structure of
the T6 zinc insulin hexamer (Protein Data Bank code 4INS): ribbon
model (left) and space-filling model (right). The B24-B28
β-strand is shown in blue, and the side chain of
PheB24 is highlighted in tawny. The B-chain is otherwise
dark gray; the A-chain, light gray; and zinc ions,
magenta. Also shown at the left are the side chains of
HisB10 at the axial zinc-binding sites. C, cylinder model
of the insulin dimer showing the B24-B26 antiparallel β-sheet
(blue) anchored by the B24 side chain (tawny circle). The A-
and B-chains are shown in light and dark gray, respectively.
The protomer at the left is shown in the R-state, in which the central
α-helix of the B-chain is elongated (B3-B19 in the frayed Rf
protomer of T3Rf3 hexamers and B1-B19 in the
R protomer of R6 hexamers). The three types of zinc insulin
hexamers share similar B24-B26 antiparallel β-sheets as conserved
dimerization elements.The structure of an insulin monomer in solution resembles a
crystallographic protomer (Fig.
2A)
(7-9).
The A-chain contains an N-terminal α-helix, non-canonical turn, and
second helix; the B-chain contains an N-terminal segment, central
α-helix, and C-terminal β-strand. The β-strand is maintained
in an isolated monomer wherein the side chain of PheB24
(tawny in Fig.
2A), packing against the central α-helix of the
B-chain, provides a “plug” to seal a crevice in the hydrophobic
core (Fig. 2B).
Anomalies encountered in previous studies of insulin analogs suggest that
PheB24 functions as a conformational switch
(4,
7,
10-14).
Whereas l-amino acid substitutions at B24 generally impair activity
(even by such similar residues as l-Tyr)
(15), a seeming paradox is
posed by the enhanced activities of nonstandard analogs containing
d-amino acids (10-12).
Open in a separate windowaAffinities are given relative to wild-type insulin (100%).bLymphocytes are human, and hepatocytes are rat; CHO designates Chinese
hamster ovary.cStandard deviations are not provided in this reference.Open in a separate windowFIGURE 2.Role of PheB24 in an insulin monomer. A, shown
is a cylinder model of insulin as a T-state protomer. The C-terminal B-chain
β-strand is shown in blue, and the PheB24 side chain
is shown in tawny. The black portion of the N-terminal
A-chain α-helix (labeled buried) indicates a hidden
receptor-binding surface (IleA2 and ValA3). B,
the schematic representation of insulin highlights the proposed role of the
PheB24 side chain as a plug that inserts into a crevice at the edge
of the hydrophobic core. C and D, whereas substitution of
PheB24 by l-Ala (C) would only partially fill
the B24-related crevice, its substitution by d-Ala (D)
would be associated with a marked packing defect. An alternative conformation,
designated the R-state, is observed in zinc insulin hexamers at high ionic
strength (74) and upon binding
of small cyclic alcohols (75)
but has not been observed in an insulin monomer.Why do d-amino acid substitutions at B24 enhance the activity of
insulin? In this study, we describe the structure and function of insulin
analogs containing l-Ala or d-Ala at B24
(Fig. 2, C and
D). Our studies were conducted within an engineered
monomer (DKP-insulin, an insulin analog containing three substitutions in the
B-chain: AspB10, LysB28, and ProB29) to
circumvent effects of self-assembly
(16). Whereas the inactive
l-analog retains a native-like structure, the active
d-analog exhibits segmental unfolding of the B-chain. Studies of
corresponding analogs containing either l- or
d-photoactivable probes
(l-para-azido-PheB24 or
d-para-azido-PheB24 (l- or
d-PapB24), obtained from photostable
para-amino-Phe (Pmp) precursors
(17)) demonstrate specific
cross-linking to the IR. Although photo-contacts map in each case to the
N-terminal domain of the receptor α-subunit (the L1 β-helix),
higher cross-linking efficiency is achieved by the d-probe.
Together, this and the following study
(6) provide evidence that
insulin deploys a detachable arm that inserts between domains of the IR.Induced fit of insulin illuminates by its scope general principles at the
intersection of protein structure and cell biology. Protein evolution is
enjoined by multiple layers of biological selection. The pathway of insulin
biosynthesis, for example, successively requires (a) specific
disulfide pairing (in the endoplasmic reticulum), (b) subcellular
targeting and prohormone processing (in the trans-Golgi network),
(c) zinc-mediated protein assembly and microcrystallization (in
secretory granules), and (d) exocytosis and rapid disassembly of
insulin hexamers (in the portal circulation), in turn enabling binding of the
monomeric hormone to target tissues
(1). Each step imposes
structural constraints, which may be at odds. This study demonstrates that
stereospecific pre-detachment of a receptor-binding arm enhances biological
activity but impairs disulfide pairing and renders the hormone susceptible to
aggregation-coupled misfolding
(18). Whereas the classical
globular structure of insulin and its self-assembly prevent proteotoxicity
(3,
19), partial unfolding enables
receptor engagement. We envisage that a choreography of conformational change
has evolved as an adaptative response to the universal threat of toxic protein
misfolding. 相似文献
TABLE 1
Previous studies of insulin analogsAnalog | Affinitya | Assayb | Ref. |
---|---|---|---|
% | |||
d-PheB24-insulin | 180 | Lymphocytes | 10 |
l-AlaB24-insulin | 1 | Hepatocytes | 68 |
l-AlaB24-insulin | 3 | Lymphocytes | 69 |
d-PheB24-insulin | 140 ± 9 | Hepatocytes | 11 |
l-AlaB24-insulin | 1.0 ± 0.1 | Hepatocytes | 11 |
d-AlaB24-insulin | 150 ± 9 | Hepatocytes | 11 |
GlyB24-insulin | 78 ± 11 | Hepatocytes | 11 |
DKP-insulin | 200c | CHO cells | 12 |
d-PheB24-DKP-insulin | 180 | CHO cells | 12 |
l-AlaB24-DKP-insulin | 7 | CHO cells | 12 |
GlyB24-DKP-insulin | 50 | CHO cells | 12 |