The polypeptide backbone fold in the solution structure of murine epidermal growth factor has been determined by nuclear magnetic resonance spectroscopy and distance geometry calculations. The results are based on nearly complete sequence-specific resonance assignments and on 333 distance and dihedral-angle constraints; these were determined from nuclear Overhauser effect measurements, identification of hydrogen-bonded amide protons, the known locations of disulfide bonds, and backbone vicinal spin-spin coupling constants. The polypeptide chain of the protein is arranged into two distinct domains. The structures of these domains were determined independently in separate calculations and then combined to obtain an overall view of the protein. The backbone fold thus determined includes the regular backbone structure elements that were previously identified using different techniques for the analysis of the nuclear magnetic resonance data. The distance geometry calculations also provided additional details about the conformations of bends and loops and about the twists of the ,8-sheets.Epidermal growth factor (EGF) is a small single-chain protein containing 53 amino acids and three disulfide bonds (1-3). EGF and the homologous a-type transforming growth factor (TGF-a) appear to play an important role in the molecular mechanisms controlling mammalian cell growth, oncogenesis (4, 5), and wound healing (6). For this reason, there is much interest in structure-function studies of EGF and EGF-like molecules. Several laboratories have cloned genes for human EGF (7-9), and others have synthesized either polypeptide fragments of EGF (10-12) or the entire EGF protein (13). With this technology, it may be possible to design and engineer EGF-like molecules with desirable biological activities, such as growth factor antagonists or agonists. Knowledge of the three-dimensional structure will be indispensable for understanding the structural basis of EGF function. No crystal structure of EGF or of a homologous growth factor is presently available. This paper presents an initial description of the three-dimensional structure of murine EGF (mEGF) in aqueous solution determined by 1H NMR.Previous NMR studies of murine (14-18), rat (19), and human (20) EGF include two papers on the use of NMR techniques for obtaining complete sequence-specific proton resonance assignments and determination of the three-dimensional structure in solution (21-23). So far the principal elements of regular backbone structure have been identified for both mEGF (18) and human EGF (20). In the work described here, the structure determination of mEGF was continued by collection of a more extensive set of experimental NMR constraints and the use of distance geometry calculations for the structural analysis of the NMR data.
METHODSExperimental. mEGF (type al) from male submaxillary glands was purified as described (24, 25) and characterized as homogeneous by both C18 reversed-phase and Pharmacia Mono-Q anion-exchange HPLC. These homogeneity checks were ...