Combining in Silico Tools and NMR Data To Validate Protein−Ligand Structural Models:  Application to Matrix Metalloproteinases

Abstract

A combination of in silico tools and experimental NMR data is proposed for relatively fast determination of protein-ligand structural models and demonstrated from known inhibitors of matrix metalloproteinases (MMP). The 15N 1H heteronuclear single quantum coherence (HSQC) spectral assignment and the 3D structure, either X-ray or NMR, are needed. In this method, the HSQC spectrum with or without the ligand is used to determine the interaction region of the ligand. Docking calculations are then performed to obtain a set of structural models. From the latter, the nuclear Overhauser effects (NOEs) between the ligand and the protein can be predicted. Guided by these predictions, a number of NOEs can be detected and assigned through a HSQC NOESY experiment. These data are used as structural restraints to reject/refine the initial structural models through further in silico work. For a test protein (MMP-12, human macrophage metalloelastase), a final structure of a protein-ligand adduct was obtained that matches well with the full structural determination. A number of structural predictions were then made for adducts of a similar protein (MMP-1, human fibroblast collagenase) with the same and different ligands. The quality of the final results depended on the type and number of experimental NOEs, but in all cases, a well-defined ligand conformation in the protein binding site was obtained. This protocol is proposed as a viable alternative to the many approaches described in the literature.

Examination of Matrix Metalloproteinase-1 in Solution

“…Although detailed structural characterization of the bound-state conformation is often possible, much more difficult is the analysis of the conformations sampled by multidomain proteins before the interaction. However, analysis of the conformational space experienced by the free protein is useful not only to investigate the mechanism of binding, but also to determine the role of the different domains in the identification of substrates or partners, to predict new possible substrates or partners, and to investigate natural and new mechanisms of inhibition (3,22,24,29,35,(57)(58)(59)(60).…”

Role of Sulfonamide Group in Matrix Metalloproteinase Inhibitors

“…Scozzafava et al [37,38] presumed that the sulfonamide group, together with the ZBG, coordinated the zinc ion within the MMP active site. Since the secondary sulfonamide moiety of such compounds would also possess good metal-coordinating properties, they expect a bidentate binding, in which both the sulfonyl and hydroxamate moieties participate in the interaction with the zinc ion (Fig.…”

Studies of metabolite–protein interactions: A review

“…These methods may involve the direct examination of binding that occurs between proteins and low mass drugs, hormones and their metabolites, or may involve an examination of the free concentrations of these molecules [9–12]. The approaches that are used for this purpose can be divided into three categories: in vitro , in vivo and in silico techniques [9,11–46]. …”

“…This method can allow for accurate prediction of ligand-binding proteins and enable the development of a database for these peptide sequences selected for binding to different metabolites [9,45]. These in silico methods can be combined with in vitro analysis to optimize the structural characterization of metabolite-protein interactions, as demonstrated in NMR experiments [46]. …”