Ultra-high resolution X-ray structures of two forms of human recombinant insulin at 100 K

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form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure.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 res...

What Gives an Insulin Hexamer Its Unique Shape and Stability? Role of Ten Confined Water Molecules

“…These water molecules, along with three His-10 residues per Zn 2+ ion, create an octahedral coordination shell around the ion (Figure a inset). − However, reports of tetrahedral arrangements (with three His-10 and one water) are also available in the literature. − In our simulations, the octahedral geometry is preserved throughout the 100 ns trajectory, indicating a very stable coordination geometry. Thus, we ask the question, how stable is this complex?…”

Serial macromolecular crystallography at ALBA Synchrotron Light Source

“…S7(a)-7(c) (see supporting information) show the ratio of temperature B-factor values for the S /C atoms of cysteines involved in the disulfide bonds calculated as described in the Materials and methods section (x2) for the lysozyme, proteinase K and insulin structures presented herein, and for several structures deposited in the PDB, i.e. 6qqe (Gotthard et al, 2019), 1qio (Weik et al, 2000), 1iee (Sauter et al, 2001) and 6ftr (Wiedorn et al, 2018) for lysozyme, 2pwa (unpublished results), 5kxu (Masuda et al, 2017) and 1ic6 (Betzel et al, 2001) for proteinase K, and 4f1g (Fa ´vero-Retto et al, 2013) and 5e7w (Lisgarten et al, 2017) for insulin. These PDBs were selected based on the relevance of the experimental conditions at which the crystal structures were collected, such as the temperature of data collection, the sample delivery method used and the X-ray radiation source.…”

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X

“…The intra-chain disulfide bond of Cys6-Cys11 of recombinant insulin was reported to affect the chain flexibility. 30 It has also been shown that the disulfide bond formation and disruption regulate the angiotensin release. 31 The transition of angiotensinogen under oxidative environment to its more active form with sulphydryl-bridged plays an important role in pregnancy hypertension and pre-eclampsia.…”