A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets. MMPs2 are a family of structurally related zinc-binding proteolytic enzymes that digest extracellular matrix proteins and participate in tissue remodeling and signaling events (1). Currently, ϳ23 MMPs have been identified, comprising secreted and membrane-bound forms, and different family members share some common structural and functional domains and have varying degrees of substrate specificity. Abnormal expression and activation of MMPs has been implicated in the pathogenesis and pathological progression of several different human diseases that are centered in many different tissues in the periphery and central nervous system (2, 3). Initial clinical exploration of synthetic MMP inhibitors was focused on oncology indications, as preventing the breakdown of tissue matrices and barriers was viewed as a potential mechanism to limit tumor metastasis.Despite intensive efforts over many years to develop synthetic MMP inhibitors, only a single MMP inhibitor, Periostat, a tetracycline derivative used in periodontal disease, has progressed into regular clinical use (4). Of the ϳ50 other clinical trials conducted with active site MMP inhibitors, all have failed due to the onset of significant dose-limiting musculoskeletal toxicity ...

The X-ray crystallographic structure of Ac-(D)Phe-Pro-boroArg-OH [DuP714, Ki = 0.04 nM; Kettner, C., Mersinger, L., & Knabb, R. (1990) J. Biol. Chem. 265, 18289] complexed with human alpha-thrombin shows the boron atom covalently bonded to the side-chain oxygen of the active site serine, Ser195. The boron adopts a nearly tetrahedral geometry, and the boronic acid forms a set of interactions with the protein that mimic the tetrahedral transition state of serine proteases. Contributions of the arginine side chain to inhibitor affinity were examined by synthesis of the ornithine, lysine, homolysine, and amidine analogs of DuP714. The basic groups interact with backbone carbonyl groups, water molecules, and an aspartic acid side chain (Asp189) located in the thrombin S1 specificity pocket. The variation in inhibition constant by 3 orders of magnitude appears to reflect differences in the energetics of interactions made with thrombin and differences in ligand flexibility in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

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Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a = b = c = 182 +/- 0.5 A. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 A, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 A. Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu-57, -58, -59, and -61 are all replaced by Ala. The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541-544, 1991) as the model. The crystallographic R factor for the 6-2.2 A shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 A. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function.