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

DOI: 10.1186/s13065-017-0296-y DOI Help

Authors: David R. Lisgarten (Canterbury Christ Church University) , Rex A. Palmer (Birkbeck College, London) , Carina M. C. Lobley (Diamond Light Source) , Claire E. Naylor (Molecular Dimensions Ltd) , Babur Z. Chowdhry (University of Greenwich (Medway Campus)) , Zakieh I. Al-Kurdi (The Jordanian Pharmaceutical Manufacturing Company (PLC)) , Adnan A. Badwan (The Jordanian Pharmaceutical Manufacturing Company (PLC)) , Brendan J. Howlin (University of Surrey) , Nicholas C. J. Gibbons (University of Middlesex) , José W. Saldanha (MRC National Institute for Medical Research) , John N. Lisgarten (University of Greenwich (Medway Campus)) , Ajit K. Basak (Birkbeck College, London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemistry Central Journal , VOL 11

State: Published (Approved)
Published: August 2017

Open Access Open Access

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.

Diamond Keywords: Diabetes

Subject Areas: Biology and Bio-materials, Medicine, Chemistry

Instruments: I02-Macromolecular Crystallography

Other Facilities: Photon Factory, Japan

Added On: 09/08/2017 14:25


Discipline Tags:

Non-Communicable Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)