Ipglycermide Ce-2, a lariat-like cyclic peptide, was discovered and identified by Yu et al. [1] as a potent inhibitor of cofactor-independent phosphoglycerate mutase (iPGM), which facilitates intramolecular transfer of the phosphoryl group through a phosphoserine interm ediate. As the only PGM found in nematodes, this presents a highly selective anthelmintic drug target since the silencing of iPGM in C. elegans and B. malayi causes nematode death. The co-crystal structure of iPGM with Ce-2d, a truncated analogue of Ce-2 with retained potency, revealed the cyclic peptide structure was highly compact when bound to the hinge domain of the protein (PDB code 5KGN). Importantly, the iPGM-bound form of Ce-2d is stabilized by an interal hydrogen bond network mediated by its Asp6 residue and a number of water-mediated hydrogen bond interactions with both the phosphatase and transferase domains of iPGM. Conformational studies using 2D NOESY experiments presented a near lack of NOE interactions between the macrocyclic core and the appendant linear chain suggesting the structure has an expected greater flexibility in solution as compared to its crystalline form. Additionally, an unexpected minor isomer was observed in a 4:1 ratio with the major isomer. These d ata compelled us to determine the 3D structure of free Ce-2d in solution for comparsion of the preferred solution conformations with the co-crystal structure confirmation. The Ce-2d analogue has a well dispersed 1D 1H NMR spectrum possessing the requisite number of backbone amide-proton (NH) peaks expected for the major isomer. As such, a complete assignment of 1H and 13C NMR peaks of the major conformer was obtained using a suite of 1D and 2D NMR experiments confirming the proposed structure of Ce-2d. An observed second set of NH peaks with low signal intensity led to the hypothesis of a minor population of a Ce-2d conformer and prompted the collection of extensive NOE data for Ce-2d to calculate 3D structures by NMR using the Xplor-NIH program. A 3D structural ensemble of Ce-2d was determined with the ten lowest-energy structures having a pairwise backbone RMSD of 1.5 Å. In addition, variable temperature (VT) NMR experiments were conducted with coalescence observed between corre sponding peaks of the major and minor conformers resulting in a single set of broadened peaks at 60 °C. VT NMR was also used to determine amide temperature coefficients, a parameter sensitive to hydrogen bonding in peptides and proteins providing qualitative structural information. Moreover, we determined the solution conformations of two additional analogues, Ce-2d D6G and Ce-2g, to posit the mechanistic underpinnings of the Ce-2d-iPGM interaction deriving from Asp6 and the appendant linear chain, respectively. Collectively, the NMR data suggest the structure of free Ce-2d primarily exists in an extended, dynamic conformational ensemble that undergoes a conformational change upon or after binding to iPGM.
