Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft ("entropy reservoir") as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies.isothermal titration calorimetry | metallopeptidase | peptide binding | X-ray crystallography T he endogenous opioid system, composed of opioid peptides and their receptors, modulates a large number of physiological processes, such as endocrine and immune function, gastrointestinal motility, respiration, reward, stress, complex social behavior (e.g., sexual activity), vulnerability to drug addiction, and most notably the procession and transmission of pain stimuli (nociception) (1, 2). Two major types of endogenous opioid peptides are those containing enkephalin sequences at the N terminus (TyrGly-Gly-Phe-Met/Leu) (3) and, more recently identified, endomorphins 1 and 2 (Tyr-Pro-Trp/Phe-Phe-NH 2 ) (4, 5). Knowledge and control over synthesis and degradation pathways of this class of molecules is prerequisite for the development of new therapies that target pertinent physiological processes.Dipeptidyl peptidase III (DPP III), also known as enkephalinase B, is an enkephalin-degrading enzyme that cleaves dipeptides sequentially from the N termini of substrates (6). All DPP IIIs described thus far contain the unique zinc-binding motif HEXXGH characteristic of metallopeptidase family M49 (7). Enzymes from several human and animal tissues, as well as from lower eukaryotes, were purified and biochemically characterized (8, 9). DPP III is largely found as a cytosolic protein, although membrane association in rat brain and Drosophila melanogaster has been described (10, 11). The 3D structure of the yeast ortholog has recently been determined, revealing a unique protein fold with two lobes forming a wide-open substrate-binding cleft (12). The lack of structural information on peptide complexes, however, left the question of substrate specificity largely unanswered.DPP III purified from monkey brain microsomes is strongly inhibited by the neuropeptide spinorphin (Leu-Val-Val-Tyr-ProTrp-Thr), an endogenous factor isolated from bovine spinal cord that also inhibits other enkephalin-degrading enzymes, such as neutral endopeptidase (NEP, neprilysin), aminopeptidase, and angiotensin-converting enzyme (13). Because of a different mode of action compared with morphine, spinorp...

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. Target 2035 aims to develop a pharmacological modulator for every protein in the human proteome to fill this gap.

Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.