Substrate specificity is critically important for enzyme catalysis. In the adrenaline-synthesizing enzyme PNMT (phenylethanolamine N-methyltransferase), minor changes in substituents can convert substrates into inhibitors. Here we report the crystal structures of six human PNMT complexes, including the first structure of the enzyme in complex with its physiological ligand R-noradrenaline. Determining this structure required rapid soak methods because of the tendency for noradrenaline to oxidize. Comparison of the PNMT-noradrenaline complex with the previously determined PNMT-p-octopamine complex demonstrates that these two substrates form almost equivalent interactions with the enzyme and show that p-octopamine is a valid model substrate for PNMT. The crystal structures illustrate the adaptability of the PNMT substrate binding site. in accepting multi-fused ring systems, such as substituted norbornene, as well as noradrenochrome, the oxidation product of noradrenaline. These results explain why only a subset of ligands recognized by PNMT are methylated by the enzyme; bulky substituents dictate the binding orientation of the ligand and can thereby place the acceptor amine too far from the donor methyl group for methylation to occur. We also show how the critical Glu(185) catalytic residue can be replaced by aspartic acid with a loss of only 10-fold in catalytic efficiency. This is because protein backbone movements place the Asp(185) carboxylate almost coincident with the carboxylate of Glu(185). Conversely, replacement of Glu(185) by glutamine reduces catalytic efficiency almost 300-fold, not only because of the loss of charge, but also because the variant residue does not adopt the same conformation as Glu(185).

Choline kinase (ChoK) catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine.Overexpression and increased activity of one of the human ChoK isoforms, ChoK alpha have been constitutively reported in malignant cells and tumour tissues.This suggests that the enzyme plays a relevant role in tumorigenesis.Some sets of in vitro and in vivo experiments confirmed that ChoK inhibition is one of the potential novel strateges for the development of new antiproliferative and anticancer drugs.The importance of ChoK for the regulation of cell proliferation has been studied by using an inhibitor hemicholinium-3 (HC-3), which was initially characterized as a lethal, respiratory paralytic agent.Most of the ChoK inhibitors introduced so far are the chemically modified derivatives based on the structure of HC-3.Crystal structures of HC-3 bound human ChoK alpha were determinded with and without ADP.In the crystal structures, HC-3 molecule was well accommodated between the N and C-terminal lobes of ChoK protein, and its one end was placed on the same binding site as a substrate choline, while the other end partially exposed to the solvent.The inhibitor molecule was stabilized mainly through the hydrophobic interactions contributed by the C-terminal lobe.These 3D information provides the first molecular detailed view concerning the mode of inhibitory action and expand our understanding of the factors governing selectivity.

Received 4 April 2007 Accepted 8 May 2007 The production of diffraction-quality crystals of Munc18c, a protein involved in regulating vesicular exocytosis in mammals, is reported. The diffraction resolution of Munc18c crystals was optimized by (i) cocrystallizing with a peptide fragment of the Munc18c functional binding partner syntaxin4, (ii) using nanolitre free-interface diffusion crystallization-screening chips and microlitre hanging-drop vapour diffusion and (iii) applying a post-crystallization dehydration treatment. Crystals belonging to the cubic space group P213, with unit-cell parameters a = b = c = 170.8 Å, = = = 90 , were generated that diffract to 3.7 Å resolution on a laboratory X-ray source.

Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins.

Shape complementarity is a fundamental principle of inhibitor design. Here we show that an enzyme for which the crystal structure has been determined (phenylethanolamine N-methyltransferase, PNMT) conceals a cryptic binding site. This site is revealed upon binding of inhibitors that are double the size of the physiological substrate. These large inhibitors are not predicted to bind in that they protrude through the accessible surface calculated from a PNMT/7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (SK&F 29661) crystal structure, yet they are potent inhibitors of PNMT. We determined structures of the enzyme complexed with large inhibitors and found that the volume of the active site increases by 140 A3 upon binding. Changes in active site size and shape are brought about by unfavorable side chain conformations and rigid body helix motions. The energetic cost is modest, estimated at 2-3 kcal/mol from mutational analyses. Our findings further underline the importance of protein flexibility in structure-based inhibitor design studies.

The production of diffraction-quality crystals of Munc18c, a protein involved in regulating vesicular exocytosis in mammals, is reported. The diffraction resolution of Munc18c crystals was optimized by (i) cocrystallizing with a peptide fragment of the Munc18c functional binding partner syntaxin4, (ii) using nanolitre free-interface diffusion crystallization-screening chips and microlitre hanging-drop vapour diffusion and (iii) applying a post-crystallization dehydration treatment. Crystals belonging to the cubic space group P 2 1 3, with unit-cell parameters a = b = c = 170.8 Å, α = β = γ = 90°, were generated that diffract to 3.7 Å resolution on a laboratory X-ray source.

The production of diffraction-quality crystals of Munc18c, a protein involved in regulating vesicular exocytosis in mammals, is reported. The diffraction resolution of Munc18c crystals was optimized by (i) cocrystallizing with a peptide fragment of the Munc18c functional binding partner syntaxin4, (ii) using nanolitre free-interface diffusion crystallization-screening chips and microlitre hanging-drop vapour diffusion and (iii) applying a post-crystallization dehydration treatment. Crystals belonging to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 170.8 A, alpha = beta = gamma = 90 degrees , were generated that diffract to 3.7 A resolution on a laboratory X-ray source.

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH- could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH- with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the alpha2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT Ki = 1.3 microM) is the most potent compound in this series and is quite selective for PNMT versus the alpha2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT Ki = 0.13 microM). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH- is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Temperature is an important parameter controlling protein crystal growth. A new temperature-screening system (Thermo-screen) is described consisting of a gradient thermocycler fitted with a special crystallization-plate adapter onto which a 192-well sitting-drop crystallization plate can be mounted (temperature range 277-372 K; maximum temperature gradient 20 K; interval precision 0.3 K). The system allows 16 different conditions to be monitored simultaneously over a range of 12 temperatures and is well suited to conduct wide (approximately 20 K) and fine (approximately 3 K) temperature-optimization screens. It can potentially aid in the determination of temperature phase diagrams and run more complex temperature-cycling experiments for seeding and crystal growth.

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N‐terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N‐terminal interaction and that are consistent with the N‐terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre‐assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c–SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.