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Abstract
The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and Rfree = 0.1466. The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6–Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S–S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported.Graphical abstractConformations of PheB25 and PheD25 in three insulin structures: implications for biological activity? Insulin residues PheB25 and PheD25 are considered to be important for insulin receptor binding and changes in biological activity occur when these residues are modified. In porcine insulin and Intergen (II) PheB25 adopts conformation B and PheD25 conformation D. However, unexpectedly PheB25 in Insugen (I) human recombinant insulin adopts two distinct conformations corresponding to B and D, Figure 1 and PheD25 adopts a single conformation corresponding to B not D, Figure 2. Conformations of this residue in the ultra-high resolution structure of Insugen (I) are therefore unique within this set. Figures were produced with Biovia, Discovery Studio 2016.
Highlights
A definitive account of the 1.5 Å resolution structure (PDB 4INS) of hexagonal porcine insulin, which differs in sequence by only one amino acid at B30 from human insulin ( Fig. 1), was published by Baker et al [1].Success in the use of pig insulin to control diabetes lies in its ability to mimic the activity of the human form, which is a consequence of near perfect structural isomorphism
To the best of our knowledge acetate has not been assigned to any other published insulin structure; further evidence for this assignment can be found in Additional file 1: Text S1 and Figure S3: (ii) a solvated propanol molecule has been assigned as described below in detail
Conclusions on the comparison between Insugen (I) and Intergen (II) structures Possible explanations for the observed bifurcation of chain C(3) Sγ6–Sγ11 disulphide are as follows: CysC6 is hydrogen bonded to a water molecule and there are several other waters modelled in this region which may be associated with greater conformational flexibility compared to CysA6
Summary
A definitive account of the 1.5 Å resolution structure (PDB 4INS) of hexagonal porcine insulin, which differs in sequence by only one amino acid at B30 (and D30) from human insulin ( Fig. 1), was published by Baker et al [1].Success in the use of pig insulin to control diabetes lies in its ability to mimic the activity of the human form, which is a consequence of near perfect structural isomorphism. The use of nonhuman forms of insulin to control diabetes is known to lead to both allergic reactions and other complications resulting from antibody production in some patients [2]. For this reason the use of recombinant forms of human insulin which have been developed is becoming more commonplace, on the assumption that their. It has been noted that the use of human recombinant insulin in combination with other drugs may blunt the signs and symptoms of hypoglycaemia [2] It has been reported [4] that several regions of the insulin molecule are closely related to its biological activity. Substitution of IleA2 by alanine results in segmental unfolding of the A1–A8 α-helix, lower thermodynamic stability and impaired F binding [5]; (c)
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