Crystal Structure Of Insulin Research Articles

In Memoriam: Guy Dodson (1937–2012)

In Memoriam: Guy Dodson (1937–2012)

OR02-1 First crystal structure of insulin bound to its primary binding site on the insulin receptor

We present here the first crystal structure of insulin to be obtained in complex with its primary binding site (Site 1) on the insulin receptor (IR). While the three-dimensional (3D) structure of insulin was an early triumph of protein crystallography, there has to date been no prior 3D structural information regarding the manner in which insulin binds to IR. Given the close structural relationship between IR and the Type insulin-like growth factor receptor (IGF-1R), the mode of binding of insulin to Site 1 of its receptor immediately leads for a model for the way in which insulin-like growth factors bind to IGF-1R. Our structure reveals that the B-helix of insulin packs parallel to the IR a-chain C-terminal helix (aCT) which in turn lies on the surface of the central b-sheet of the first leucine-rich repeat (L1) domain of IR. Our structure indicates that the aCT helix mediates the displacement of the C-terminal segment of the insulin B-chain away core of the hormone, a critical structural transition that has long been predicted to occur in insulin upon receptor binding. The aCT helix is seen to be repositioned on the L1 surface with respect to its position in the apo-IR structure, indicating that not only is there induced fit of the hormone to the receptor, but also of the receptor to the hormone. Our structure indicates further that elements of the hexamer-forming surface of insulin would be directed towards Site 2 within an intact IR, but with significant steric overlap within the context of the known apo-receptor three-dimensional structure. This observation suggests a structural mechanism for signal transduction. These findings have direct implications for the design both of novel insulins and of small-molecule IGF-1R antagonists that could be directed towards Site 1.

OR02-2 Insulin receptor (InsR) and insulin-like growth factor-I receptor (IGF-GR) translocate to nucleus and regulate IGF-GR gene expression in breast cancer cells

We present here the first crystal structure of insulin to be obtained in complex with its primary binding site (Site 1) on the insulin receptor (IR). While the three-dimensional (3D) structure of insulin was an early triumph of protein crystallography, there has to date been no prior 3D structural information regarding the manner in which insulin binds to IR. Given the close structural relationship between IR and the Type insulin-like growth factor receptor (IGF-1R), the mode of binding of insulin to Site 1 of its receptor immediately leads for a model for the way in which insulin-like growth factors bind to IGF-1R. Our structure reveals that the B-helix of insulin packs parallel to the IR a-chain C-terminal helix (aCT) which in turn lies on the surface of the central b-sheet of the first leucine-rich repeat (L1) domain of IR. Our structure indicates that the aCT helix mediates the displacement of the C-terminal segment of the insulin B-chain away core of the hormone, a critical structural transition that has long been predicted to occur in insulin upon receptor binding. The aCT helix is seen to be repositioned on the L1 surface with respect to its position in the apo-IR structure, indicating that not only is there induced fit of the hormone to the receptor, but also of the receptor to the hormone. Our structure indicates further that elements of the hexamer-forming surface of insulin would be directed towards Site 2 within an intact IR, but with significant steric overlap within the context of the known apo-receptor three-dimensional structure. This observation suggests a structural mechanism for signal transduction. These findings have direct implications for the design both of novel insulins and of small-molecule IGF-1R antagonists that could be directed towards Site 1.

Insulin Assembly Damps Conformational Fluctuations: Raman Analysis of Amide I Linewidths in Native States and Fibrils

The crystal structure of insulin has been investigated in a variety of dimeric and hexameric assemblies. Interest in dynamics has been stimulated by conformational variability among crystal forms and evidence suggesting that the functional monomer undergoes a conformational change on receptor binding. Here, we employ Raman spectroscopy and Raman microscopy to investigate well-defined oligomeric species: monomeric and dimeric analogs in solution, native T6 and R6 hexamers in solution and corresponding polycrystalline samples. Remarkably, linewidths of Raman bands associated with the polypeptide backbone (amide I) exhibit progressive narrowing with successive self-assembly. Whereas dimerization damps fluctuations at an intermolecular β-sheet, deconvolution of the amide I band indicates that formation of hexamers stabilizes both helical and non-helical elements. Although the structure of a monomer in solution resembles a crystallographic protomer, its encagement in a native assembly damps main-chain fluctuations. Further narrowing of a β-sheet-specific amide I band is observed on reorganization of insulin in a cross-β fibril. Enhanced flexibility of the native insulin monomer is in accord with molecular dynamics simulations. Such conformational fluctuations may initiate formation of an amyloidogenic nucleus and enable induced fit on receptor binding.

Receptor binding redefined by a structural switch in a mutant human insulin

Crystal structures of insulin have been determined in various distinct forms, the relevance of which to receptor recognition has long been the subject of speculation. Recently the crystal structure of an inactive insulin analogue has been determined and, surprisingly, found to have a conformation identical to native insulin. On this basis Dodson and colleagues have suggested that the known insulin crystal structures reflect an inactive conformation, and that a change in conformation is required for activity--specifically, the carboxy terminal residues of the B-chain are proposed to separate from the amino terminal residues of the A-chain. Here we report the solution structure of an active insulin mutant, determined by two-dimensional NMR, which supports this hypothesis. In the mutant, the carboxy terminal beta-turn and beta-strand of the B-chain are destabilized and do not pack across the rest of the molecule. We suggest that analogous detachment of the carboxy terminal region of the B-chain occurs in native insulin on binding to its receptor. Our finding that partial unfolding of the B-chain exposes an alternative protein surface rationalizes the receptor-binding properties of a series of anomalous insulin analogues, including a mutant insulin associated with diabetes mellitus in man.

Transmission of conformational change in insulin.

Crystal structures of insulin contain molecules that are similar but not identical in conformation. Packed helices move relative to each other, these shifts being accommodated by motions of side-chain atoms arising from small changes in torsion angles. Such low-energy conformational adjustments can accommodate shifts of no more than approximately 1.5 A. This limits the extent to which conformational changes can be dissipated locally, causing their transmission over long distances.

The crystal structure of insulin: III. Evidence for a 2-fold axis in rhombohedral zinc insulin

The existence of non-space-group symmetry elements in rhombohedral 2 Zn insulin and 4 Zn insulin crystals has been investigated. A rotation function shows the existence of a 2-fold axis perpendicular to the crystallographic c -axis (3-fold) and making an angle of 44° with the a -axis. A translation function, and independent arguments from the Patterson series, show that this is a 2-fold axis, without translation parallel to its length. In 2 Zn insulin the 2-fold axes pass through or within 0·5 A of a 3-fold or, less probably, a 3-fold screw axis; in 4 Zn insulin they are about 1 A from it.

The crystal structure of insulin: II. An investigation of rhombohedral zinc insulin crystals and a report of other crystalline forms

X-Ray diffraction photographs have been taken of zinc-free cubic insulin and of monoclinic zinc insulin crystals, prepared from buffers containing phenol. The wet cubic crystals are very small rhombic dodecahedra, a = 76 Å, ρ = 1·09 approx. and space group, I23 or I213; there is one insulin monomer (mol. wt about 6000) in the asymmetric unit. Wet monoclinic crystals have a = 62·3, b = 61·8, c = 47·8 Å, β = 110·7°, ρ = 1·19 g cm−3 and air-dried crystals have a = 61·4, b = 53·4, c = 43·8 Å, β = 134·5°, ρ = 1·29 g cm−3; the space group is P21 and there are six insulin monomers in the asymmetric unit.

The crystal structure of insulin I. The investigation of air-dried insulin crystals.

The crystal structure of insulin is of interest not only in itself but as part of the wider problem of protein structure considered as a whole. The early X-ray work on protein fibres gave strong support to the original chemical theories of the existence of polypeptide chains as the essential backbone of the protein molecule. The comparatively small spacings of 3-4, 4∙5 and 10-11 A which are most prominent on a large section of X-ray photographs of the scleroproteins are easily interpretable in terms of a fundamental chain structure (Astbury and Woods 1933; Astbury and Sisson 1935). It is on the other hand difficult to reconcile a chain structure with certain of the physical properties of the globular proteins, particularly their crystalline form and their behaviour in the ultracentrifuge as approximately spherical masses of molecular weight some multiple of 35,000 (Svedberg 1937 a ). Yet there is much evidence, physical as well as chemical, that the two types of protein structure are closely interrelated. Certain of the natural protein fibres show X-ray reflexions corresponding to very long spacings superimposed on the simple β keratin type of fibre pattern. These long spacings—up to 300 A in feather keratin (Astbury and Marwick 1932), tendon and collagen (Wyckolf and Corey 1936)—are more of the order of magnitude of the spacings which define the crystal structure, i. e. intermolecular distances, in crystals of the soluble proteins. And further fibre forms can be obtained from the crystalline proteins, particularly relevant observations being those on edestin and excelsin (Astbury, Dickinson and Bailey 1935). Several attempts have been made on the theoretical side to reconcile these facts, and two views of protein structure in particular have been stated in some detail. Astbury has advocated a variety of straight chain structures for the different fibre proteins which may be coiled into spiral forms or folded in layers in the globular proteins (Astbury and others 1935), as also discussed by Pauling and Mirsky (1936), while D. M. Wrinch (1937 a ) has shown that the occurrence of an internal cyclizing process might lead to the development from polypeptide chains of beautifully symmetrical “cyclol” networks both open and space enclosing. But on the experimental side many more observations are necessary before even these quite different hypotheses can be distinguished, and one possible approach seems to be through a more complete X-ray study of the crystalline globular proteins.