The series of events that occur in the catalytic cycle of matrix metalloproteinases were modeled on the basis of X‐ray crystal structures of the active, uninhibited enzymes and of the same enzymes following the hydrolysis of a peptide substrate. After the peptide bond has been broken, both peptide fragments remain bound to the protein initially (see structure of the active‐site cavity of the enzyme MMP‐12 immediately after substrate hydrolysis).
By solving high-resolution crystal structures of a large number (14 in this case) of adducts of matrix metalloproteinase 12 (MMP12) with strong, nanomolar, inhibitors all derived from a single ligand scaffold, it is shown that the energetics of the ligand-protein interactions can be accounted for directly from the structures to a level of detail that allows us to rationalize for the differential binding affinity between pairs of closely related ligands. In each case, variations in binding affinities can be traced back to slight improvements or worsening of specific interactions with the protein of one or more ligand atoms. Isothermal calorimetry measurements show that the binding of this class of MMP inhibitors is largely enthalpy driven, but a favorable entropic contribution is always present. The binding enthalpy of acetohydroxamic acid (AHA), the prototype zinc-binding group in MMP drug discovery, has been also accurately measured. In principle, this research permits the planning of either improved inhibitors, or inhibitors with improved selectivity for one or another MMP. The present analysis is applicable to any drug target for which structural information on adducts with a series of homologous ligands can be obtained, while structural information obtained from in silico docking is probably not accurate enough for this type of study.
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.
Die Abfolge von Ereignissen, die im Katalysezyklus von Matrix‐Metalloproteinasen eintreten, wurde auf der Basis von Kristallstrukturen der aktiven, nicht inhibierten Enzyme und derselben Enzyme nach Hydrolyse eines Peptidsubstrats modelliert. Nach dem Bruch der Peptidbindung bleiben beide Peptidfragmente zunächst an das Protein gebunden (siehe Struktur der Kavität am aktiven Zentrum des Enzyms MMP‐12 direkt nach der Substrathydrolyse).
Nuclear receptors (NRs) regulate gene expression through DNA-and ligand-binding and thus represent crucial therapeutic targets. The ultraspiracle protein/ecdysone receptor (USP/EcR) complex binds to half-sites with a one base pair spaced inverted repeat (IR1), a palindromic DNA response element (RE) reminiscent of IRs observed for vertebrate steroid hormone receptors. Here we present the cryo electron microscopy structure of the USP/EcR complex bound to an IR1 RE which provides the first description of a full IR-bound NR complex. The structure reveals that even though the DNA is almost symmetric, the complex adopts a highly asymmetric architecture in which the ligand-binding domains (LBDs) are positioned 5 0 off-centred. Additional interactions of the USP LBD with the 5 0 -flanking sequence trigger transcription activity as monitored by transfection assays. The comparison with DR-bound NR complexes suggests that DNA is the major allosteric driver in inversely positioning the LBDs, which serve as the main binding-site for transcriptional regulators.
Proteolytic processing of HIV gp160 to produce gp120 and gp41 is performed by PC enzymes. This process is a prerequisite for the virus infectivity, since both gp120 and gp41 participate in the virus HIV-1 entry mechanism. The structure of the gp120/gp41 junction remains to be elucidated, and the structural features required for molecular recognition between HIV-1 gp160 and proteolytic enzymes have not been clarified. Furin is the best PC candidate for the gp160 proteolytic processing known to date. In previous studies on model peptides, we have shown the relevance of an N-terminal helix for the proper recognition of the gp160 processing site by furin. Here we analyze the effect of point mutations in peptides lacking a regular N-terminal helix. To this end, we present the structure-activity characterization of three peptide analogues of the HIV gp160 processing site that all present mutations in proline at positions P3 and/or P2', while sharing the same N-terminal sequence, containing helix-breaking D-amino acids. Conformational analysis of the peptides was carried out in solution by NMR techniques, and furin's efficiency in cleaving them was measured. Structural findings are presented and discussed in relation to the different exhibited activity.
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