Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.     

Structural basis for the catalysis and substrate specificity of homoserine kinase

Homoserine kinase (HSK), the fourth enzyme in the aspartate pathway of amino acid biosynthesis, catalyzes the phosphorylation of L-homoserine (Hse) to L-homoserine phosphate, an intermediate in the production of L-threonine, L-isoleucine, and in higher plants, L-methionine. The high-resolution structures of Methanococcus jannaschii HSK ternary complexes with its amino acid substrate and ATP analogues have been determined by X-ray crystallography. These structures reveal the structural determinants of the tight and highly specific binding of Hse, which is coupled with local conformational changes that enforce the sequestration of the substrate. The delta-hydroxyl group of bound Hse is only 3.4 A away from the gamma-phosphate of the bound nucleotide, poised for the in-line attack at the gamma-phosphorus. The bound nucleotides are flexible at the triphosphate tail. Nevertheless, a Mg(2+) was located in one of the complexes that binds between the beta- and gamma-phosphates of the nucleotide with good ligand geometry and is coordinated by the side chain of Glu130. No strong nucleophile (base) can be located near the phosphoryl acceptor hydroxyl group. Therefore, we propose that the catalytic mechanism of HSK does not involve a catalytic base for activating the phosphoryl acceptor hydroxyl but instead is mediated via a transition state stabilization mechanism.     

Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3

Murine Atg8L/Apg8L has significant homology with the other known mammalian Atg8 homologs, LC3, GABARAP and GATE-16. However, it is unclear whether murine Atg8L modification is mediated by human Atg4B, Atg7 and Atg3. Expression of Atg8L in HEK293 cells led to cleavage of its C-terminus. In vitro, the C-terminus of Atg8L was cleaved by human Atg4B, but not human Atg4A or Atg4C. Atg8L-I formed an E1-substrate intermediate with Atg7(C572S), and an E2-substrate intermediate with Atg3(C264S). A modified form of Atg8L was detected in the pelletable fraction in the presence of lysosomal protease inhibitors under nutrient-rich conditions. Cyan fluorescent protein (CFP)-Atg8L colocalized with yellow fluorescent protein (YFP)-LC3 in HeLa cells in the presence of the inhibitors. However, little accumulation of the modified form of Atg8L was observed under conditions of starvation. These results indicate that Atg8L is the fourth modifier of mammalian Atg8 conjugation.     

Structural and functional basis for substrate specificity and catalysis of levan fructotransferase

Levan is β-2,6-linked polymeric fructose and serves as reserve carbohydrate in some plants and microorganisms. Mobilization of fructose is usually mediated by enzymes such as glycoside hydrolase (GH), typically releasing a monosaccharide as a product. The enzyme levan fructotransferase (LFTase) of the GH32 family catalyzes an intramolecular fructosyl transfer reaction and results in production of cyclic difructose dianhydride, thus exhibiting a novel substrate specificity. The mechanism by which LFTase carries out these functions via the structural fold conserved in the GH32 family is unknown. Here, we report the crystal structure of LFTase from Arthrobacter ureafaciens in apo form, as well as in complexes with sucrose and levanbiose, a difructosacchride with a β-2,6-glycosidic linkage. Despite the similarity of its two-domain structure to members of the GH32 family, LFTase contains an active site that accommodates a difructosaccharide using the -1 and -2 subsites. This feature is unique among GH32 proteins and is facilitated by small side chain residues in the loop region of a catalytic β-propeller N-domain, which is conserved in the LFTase family. An additional oligosaccharide-binding site was also characterized in the β-sandwich C-domain, supporting its role in carbohydrate recognition. Together with functional analysis, our data provide a molecular basis for the catalytic mechanism of LFTase and suggest functional variations from other GH32 family proteins, notwithstanding the conserved structural elements.     

Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase

N-Carbamoyl-d-amino acid amidohydrolase is an industrial biocatalyst to hydrolyze N-carbamoyl-d-amino acids for producing valuable d-amino acids. The crystal structure of N-carbamoyl-d-amino acid amidohydrolase in the unliganded form exhibits a alpha-beta-beta-alpha fold. To investigate the roles of Cys172, Asn173, Arg175, and Arg176 in catalysis, C172A, C172S, N173A, R175A, R176A, R175K, and R176K mutants were constructed and expressed, respectively. All mutants showed similar CD spectra and had hardly any detectable activity except for R173A that retained 5% of relative activity. N173A had a decreased value in kcat or Km, whereas R175K or R176K showed high Km and very low kcat values. Crystal structures of C172A and C172S in its free form and in complex form with a substrate, along with N173A and R175A, have been determined. Analysis of these structures shows that the overall structure maintains its four-layer architecture and that there is limited conformational change within the binding pocket except for R175A. In the substrate-bound structure, side chains of Glu47, Lys127, and C172S cluster together toward the carbamoyl moiety of the substrate, and those of Asn173, Arg175, and Arg176 interact with the carboxyl group. These results collectively suggest that a Cys172-Glu47-Lys127 catalytic triad is involved in the hydrolysis of the carbamoyl moiety and that Arg175 and Arg176 are crucial in binding to the carboxyl moiety, hence demonstrating substrate specificity. The common (Glu/Asp)-Lys-Cys triad observed among N-carbamoyl-d-amino acid amidohydrolase, NitFhit, and another carbamoylase suggests a conserved and robust platform during evolution, enabling it to catalyze the reactions toward a specific nitrile or amide efficiently.     

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme.     

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.     

Structural basis for catalysis by onconase

Onconase® (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5′-AMP complex at 1.65 Å resolution and a wild-type ONC-d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k_cat/K_M profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10²-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k_cat/K_M value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.     

The structural basis for specificity in lipoxygenase catalysis

Many intriguing facets of lipoxygenase (LOX) catalysis are open to a detailed structural analysis. Polyunsaturated fatty acids with two to six double bonds are oxygenated precisely on a particular carbon, typically forming a single chiral fatty acid hydroperoxide product. Molecular oxygen is not bound or liganded during catalysis, yet it is directed precisely to one position and one stereo configuration on the reacting fatty acid. The transformations proceed upon exposure of substrate to enzyme in the presence of O₂ (RH + O₂ → ROOH), so it has proved challenging to capture the precise mode of substrate binding in the LOX active site. Beginning with crystal structures with bound inhibitors or surrogate substrates, and most recently arachidonic acid bound under anaerobic conditions, a picture is consolidating of catalysis in a U‐shaped fatty acid binding channel in which individual LOX enzymes use distinct amino acids to control the head‐to‐tail orientation of the fatty acid and register of the selected pentadiene opposite the non‐heme iron, suitably positioned for the initial stereoselective hydrogen abstraction and subsequent reaction with O₂. Drawing on the crystal structures available currently, this review features the roles of the N‐terminal β‐barrel (C2‐like, or PLAT domain) in substrate acquisition and sensitivity to cellular calcium, and the α‐helical catalytic domain in fatty acid binding and reactions with O₂ that produce hydroperoxide products with regio and stereospecificity. LOX structures combine to explain how similar enzymes with conserved catalytic machinery differ in product, but not substrate, specificities.     

Structural basis for catalysis at the membrane-water interface

The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments – the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Dual roles of Atg8-PE deconjugation by Atg4 in autophagy

Modification of target molecules by ubiquitin or ubiquitin-like (Ubl) proteins is generally reversible. Little is known, however, about the physiological function of the reverse reaction, deconjugation. Atg8 is a unique Ubl protein whose conjugation target is the lipid phosphatidylethanolamine (PE). Atg8 functions in the formation of double-membrane autophagosomes, a central step in the well-conserved intracellular degradation pathway of macroautophagy (hereafter autophagy). Here we show that the deconjugation of Atg8-PE by the cysteine protease Atg4 plays dual roles in the formation of autophagosomes. During the early stage of autophagosome formation, deconjugation releases Atg8 from non-autophagosomal membranes to maintain a proper supply of Atg8. At a later stage, the release of Atg8 from intermediate autophagosomal membranes facilitates the maturation of these structures into fusion-capable autophagosomes. These results provide new insights into the functions of Atg8-PE and its deconjugation.     

Structural basis of the drug-binding specificity of human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of drugs, thereby restricting their free, active concentrations. The problem of overcoming the binding affinity of lead compounds for HSA represents a major challenge in drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of drugs and small-molecule toxins reveals the precise architecture of the two primary drug-binding sites on the protein, identifying residues that are key determinants of binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary binding sites for drugs distributed across the protein have also been identified. The binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of drug site 1. These results clarify the interpretation of accumulated drug binding data and provide a valuable template for design efforts to modulate the interaction with HSA.     

Structural basis for the mechanistic understanding of human CD38-controlled multiple catalysis

The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD(+)) has been proposed to go through an oxocarbenium ion-like transition state. Because of the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD(+) to synthesize two calcium-mobilizing molecules. By using NAD(+) and a surrogate substrate, NGD(+), we captured and determined crystal structures of the enzyme complexed with an intermediate, a substrate, and a product along the reaction pathway. Our results showed that the intermediate is stabilized by polar interactions with the catalytic residue Glu(226) rather than by a covalent linkage. The polar interactions between Glu(226) and the substrate 2',3'-OH groups are essential for initiating catalysis. Ser(193) was demonstrated to have a regulative role during catalysis and is likely to be involved in intermediate stabilization. In addition, a product inhibition effect by ADP-ribose (through the reorientation of the product) or GDP-ribose (through the formation of a covalently linked GDP-ribose dimer) was observed. These structural data provide insights into the understanding of multiple catalysis and clues for drug design.     

Structural basis of substrate-binding specificity of human arylamine N-acetyltransferases

The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9-A resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.     

Structural basis and specificity of human otubain 1-mediated deubiquitination

OTUB (otubain) 1 is a human deubiquitinating enzyme that is implicated in mediating lymphocyte antigen responsiveness, but whose molecular function is generally not well defined. A structural analysis of OTUB1 shows differences in accessibility to the active site and in surface properties of the substrate-binding regions when compared with its close homologue, OTUB2, suggesting variations in regulatory mechanisms and substrate specificity. Biochemical analysis reveals that OTUB1 has a preference for cleaving Lys(48)-linked polyubiquitin chains over Lys(63)-linked polyubiquitin chains, and it is capable of cleaving NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8), but not SUMO (small ubiquitin-related modifier) 1/2/3 and ISG15 (interferon-stimulated gene 15) conjugates. A functional comparison of OTUB1 and OTUB2 indicated a differential reactivity towards ubiquitin-based active-site probes carrying a vinyl methyl ester, a 2-chloroethyl or a 2-bromoethyl group at the C-terminus. Mutational analysis suggested that a narrow P1' site, as observed in OTUB1, correlates with its ability to preferentially cleave Lys(48)-linked ubiquitin chains. Analysis of cellular interaction partners of OTUB1 by co-immunoprecipitation and MS/MS (tandem mass spectrometry) experiments demonstrated that FUS [fusion involved in t(12;6) in malignant liposarcoma; also known as TLS (translocation in liposarcoma) or CHOP (CCAAT/enhancer-binding protein homologous protein)] and RACK1 [receptor for activated kinase 1; also known as GNB2L1 (guanine-nucleotide-binding protein beta polypeptide 2-like 1)] are part of OTUB1-containing complexes, pointing towards a molecular function of this deubiquitinating enzyme in RNA processing and cell adhesion/morphology.     

Structural basis for the positional specificity of lipoxygenases

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of mammalian lipoxygenases but, unfortunately, the structural reasons for lipoxygenase specificity are not well understood. Although there are no direct structural data on lipoxygenase/substrate interaction, experiments with modified fatty acid substrates and mutagenesis studies suggest that for 12- and 15-lipoxygenases, arachidonic acid slides into the substrate-binding pocket with its methyl end ahead. For arachidonate 5- and/or 8-lipoxygenation two alternative models for the enzyme/substrate interaction have been developed: 1) The orientation-determined model and 2) the space-determined model. This review explores the experimental data available on the mechanistic reasons for lipoxygenase specificity and concludes that each of the above-mentioned hypotheses may be valid for arachidonate 5-lipoxygenation under certain circumstances.     

Structural basis for specificity in the poxvirus topoisomerase

Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding alpha helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections.     

Structural basis for the design of selective phosphodiesterase 4B inhibitors

Phosphodiesterase-4B (PDE4B) regulates the pro-inflammatory Toll Receptor –Tumor Necrosis Factor α (TNFα) pathway in monocytes, macrophages and microglial cells. As such, it is an important, although under-exploited molecular target for anti-inflammatory drugs. This is due in part to the difficulty of developing selective PDE4B inhibitors as the amino acid sequence of the PDE4 active site is identical in all PDE4 subtypes (PDE4A-D). We show that highly selective PDE4B inhibitors can be designed by exploiting sequence differences outside the active site. Specifically, PDE4B selectivity can be achieved by capture of a C-terminal regulatory helix, now termed CR3 (Control Region 3), across the active site in a conformation that closes access by cAMP. PDE4B selectivity is driven by a single amino acid polymorphism in CR3 (Leu674 in PDE4B1 versus Gln594 in PDE4D). The reciprocal mutations in PDE4B and PDE4D cause a 70–80 fold shift in selectivity. Our structural studies show that CR3 is flexible and can adopt multiple orientations and multiple registries in the closed conformation. The new co-crystal structure with bound ligand provides a guide map for the design of PDE4B selective anti-inflammatory drugs.     

Structural basis of Atg8 activation by a homodimeric E1, Atg7

E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.