FANCI Protein Binds to DNA and Interacts with FANCD2 to Recognize Branched Structures

In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200–1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001–1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.Fanconi anemia (FA)³ is a genetic disorder characterized by chromosome instability, predisposition to cancer, hypersensitivity to DNA cross-linking agents, developmental abnormalities, and bone marrow failure (1–9). There are at least 13 distinct FA complementation groups, each of which is associated with an identified gene (2, 9, 10). Eight of them are components of the FA core complex (FANC A, B, C, E, F, G, L, and M) that is epistatic to the monoubiquitination of both FANCI and FANCD2, a key event to initiate interstrand cross-link (ICL) repair (2, 9, 11). Downstream of or parallel to the FANCI and FANCD2 monoubiquitination are the proteins involved in double strand break repair and breast cancer susceptibility (i.e. FANCD1/BRCA2, FANCJ/BRIP1, and FANCN/PALB2) (2, 9).FANCI is the most recently identified FA gene (11–13). FANCI protein is believed to form a FANCI-FANCD2 (ID) complex with FANCD2, because they co-immunoprecipitate with each other from cell lysates and their stabilities are interdependent of each other (9, 11, 13). FANCI and FANCD2 are paralogs to each other, since they share sequence homology and co-evolve in the same species (11). Both FANCI and FANCD2 can be phosphorylated by ATR/ATM (ataxia telangiectasia and Rad3-related/ataxia telangiectasia-mutated) kinases under genotoxic stress (11, 14, 15). The phosphorylation of FANCI seems to function as a molecular switch to turn on the FA repair pathway (16). The monoubiquitination of FANCD2 at lysine 561 plays a critical role in cellular resistance to DNA cross-linking agents and is required for FANCD2 to form damage-induced foci with BRCA1, BRCA2, RAD51, FANCJ, FANCN, and γ-H2AX on chromatin during S phase of the cell cycle (17–25). In response to DNA damage or replication stress, FANCI is also monoubiquitinated at lysine 523 and recruited to the DNA repair nuclear foci (11, 13). The monoubiquitination of both FANCI and FANCD2 depends on the FA core complex (11, 13, 26), and the ubiquitination of FANCI relies on the FANCD2 monoubiquitination (2, 11). In an in vitro minimally reconstituted system, FANCI enhances FANCD2 monoubiquitination and increases its specificity toward the in vivo ubiquitination site (27).FANCI is a leucine-rich peptide (14.8% of leucine residues) with limited sequence information to indicate which processes it might be involved in. Besides the monoubiquitination site Lys⁵²³ and the putative nuclear localization signals (Fig. 1A), FANCI contains both ARM (armadillo) repeats and a conserved C-terminal EDGE motif as FANCD2 does (11, 28). The EDGE sequence in FANCD2 is not required for monoubiquitination but is required for mitomycin C (MMC) sensitivity (28). The ARM repeats form α-α superhelix folds and are involved in mediating protein-protein interactions (11, 29). In addition, FANCI, at its N terminus, contains a leucine zipper domain (aa 130–151) that could be involved in mediating protein-protein or protein-DNA interactions (Fig. 1A) (30–33). FANCD2, the paralog of FANCI, was reported to bind to double strand DNA ends and Holliday junctions (34).Open in a separate windowFIGURE 1.Purified human FANCI binds to DNA promiscuously. A, schematic diagram of predicted FANCI motifs and mutagenesis strategy to define the DNA binding domain. The ranges of numbers indicate how FANCI was truncated (e.g. 801–1328 represents FANCI-(801–1328)). NLS, predicted nuclear localization signal (aa 779–795 and 1323–1328); K523, lysine 523, the monoubiquitination site. The leucine zipper (orange bars, aa 130–151), ARM repeats (green bars), and EDGE motif (blue bars) are indicated. Red bars with a slash indicate the point mutations shown on the left. B, SDS-PAGE of the purified proteins stained with Coomassie Brilliant Blue R-250. R1285Q and D1301A are two point mutants of FANCI. All FANCI variants are tagged by hexahistidine. FANCD2 is in its native form. Protein markers in kilodaltons are indicated. C, titration of WT-FANCI for the DNA binding activity. Diagrams of the DNA substrates are shown at the top of each set of reactions. *, ³²P-labeled 5′-end. HJ, Holliday junction. Concentrations of FANCI were 0, 20, 40, 60, and 80 nm (ascending triangles). The substrate concentration was 1 nm. Protein-DNA complex is indicated by an arrow. D, supershift assay. 1 nm of ssDNA was incubated with PBS (lane 1), 80 nm FANCI alone (lane 2), and 80 nm FANCI preincubated with a specific FANCI antibody (lane 3) in the condition described under “Experimental Procedures.”In order to delineate the function of FANCI protein, we purified the recombinant FANCI from the baculovirus expression system. In this study, we report the DNA binding activity of FANCI. Unlike FANCD2, FANCI binds to different DNA structures, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), 5′-tailed, 3′-tailed, splayed arm, 5′-flap, 3′-flap, static fork, and Holliday junction with preference toward branched structures in the presence of FANCD2. Our data suggest that the dynamic DNA binding activity of FANCI and the preferential recognition of branched structures by the ID complex are likely to be the mechanisms to initiate downstream repair events.     

The Nutrigenetics of Hyperhomocysteinemia: QUANTITATIVE PROTEOMICS REVEALS DIFFERENCES IN THE METHIONINE CYCLE ENZYMES OF GENE-INDUCED VERSUS DIET-INDUCED HYPERHOMOCYSTEINEMIA*

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine β-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p ≤ 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS^+/− mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS^+/− genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.Homocysteine (Hcy)1 is a thiol-containing amino acid that is produced in every cell of the body as an intermediate of the methionine cycle (Fig. 1, Reactions 1–5) (1). Once formed, the catabolism of homocysteine occurs via three enzymatic pathways. 1) Hcy is remethylated back to methionine using vitamin B₁₂-dependent methionine synthase (Fig. 1, Reaction 4) and/or 2) betaine-homocysteine methyltransferase (BHMT) (Fig. 1, Reaction 5), and 3) Hcy is converted to cysteine via the transsulfuration pathway using CBS and γ-cystathionase (Fig. 1, Reactions 6 and 7). Under normal conditions ∼40–50% of the Hcy that is produced in the liver is remethylated, ∼40–50% is converted to cysteine, and a small amount is exported (1–3). However, when Hcy production is increased (i.e. increased dietary methionine/protein intake) or when Hcy catabolism is decreased (i.e. CBS deficiency or B vitamin deficiencies), excess Hcy is exported into the extracellular space, resulting in hyperhomocysteinemia (1–5).Open in a separate windowFig. 1.Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.Homocystinuria was first described in the 1960s by Carson et al. (6): they observed 10 pediatric patients with severely elevated levels of Hcy in the urine and hypermethioninemia. Normal concentrations of plasma total homocysteine (tHcy) range from 5 to 12 μm (7); however, in homocystinuria, tHcy levels can exceed 100 μm. Homocystinuric patients present with mental retardation, abnormal bone growth, fine hair, malar flush, and dislocation of the lens of the eye, and most die from premature cardiovascular disease (6, 8). Autopsy findings indicate widespread thromboembolism, arteriosclerosis, and fatty livers (6, 8). Mudd et al. (9, 10) identified the cause of homocystinuria as a defect in the enzyme cystathionine β-synthase. A recent study of newborn infants in Denmark estimated the birth prevalence for CBS heterozygosity to be about 1:20,000 (11).Plasma tHcy concentrations are also directly correlated with dietary methionine/protein intake (12, 13). Guttormsen et al. (13) demonstrated that a protein-rich meal affected tHcy for at least 8–24 h. When normal subjects were fed a low protein-containing breakfast (12–15 g), plasma methionine levels increased slightly after 2 h (22.5–27.5 μm), but tHcy levels did not change significantly. However, when these same subjects were fed a high protein meal (52 g), plasma methionine levels peaked after 4 h (38 μm), and tHcy rose steadily until a maximum level was reached 8 h postmeal (7.6 versus 8.5 μm) (13). Thus, the following questions can be raised. How does the hepatic proteome respond to a hyperhomocysteinemic diet, and are the changes that accompany such a diet the same as or different from those that may be observed in gene-induced hyperhomocysteinemia?Because hyperhomocysteinemia is a strong independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease, most of the current research has focused on the mechanisms involved in Hcy-induced endothelial dysfunction (14–24). The results of those studies have concluded that Hcy induces intracellular oxidative stress by generating ROS, which in turn lead to decreased bioavailable nitric oxide (NO), altered gene expression, increased endoplasmic reticulum stress, and activation of cholesterol biosynthesis. Also, several studies have examined the association between hyperhomocysteinemia and alcoholic liver disease, but few have looked at the effect of Hcy on the non-alcoholic liver even though fatty liver is a constant finding in homocystinuria (6, 8), and the liver is the major source of circulating Hcy (4, 5, 10). We hypothesize that 1) the liver proteome will respond to hyperhomocysteinemia by altering the expression of proteins involved in methionine/homocysteine metabolism and antioxidant defense and that 2) the set of proteins that are expressed when hyperhomocysteinemia is induced by CBS deficiency will differ from those expressed as a result of a high methionine diet. In the present study, we use a well established mouse model of CBS deficiency to study the early changes in the liver proteome that accompany hyperhomocysteinemia (25).     

Photoreduction of the Folate Cofactor in Members of the Photolyase Family

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH⁻* to the DNA lesion and electron back-transfer to semireduced FADH^o after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.DNA photolyases and cryptochromes (cry)² form a large family of related flavoproteins with DNA repair activity and photoreceptor function, respectively. Members of this protein family were identified in all kingdoms of life and can be grouped in at least nine subclades (1). DNA photolyases repair cytotoxic and mutagenic DNA lesions that are formed during exposure of DNA to UV-B. These DNA lesions are cyclobutane pyrimidine dimers (CPDs) or pyrimidine-pyrimidone (6-4) photoproducts. According to their substrate specificity, DNA photolyases are designated as CPD photolyases or (6-4) photolyases (2). The repair of both types of DNA lesions by photolyase requires the catalytic fully reduced and anionic flavin cofactor FADH⁻ that, when photoexcited, injects an electron directly into the DNA lesion (1) as shown in Fig. 1A (electron transfer pathway 1). During extraction from the cell and purification under aerobic conditions the flavin cofactor is usually oxidized to the semireduced and eventually to the fully oxidized form. Reduction of these flavin species to FADH⁻ in vitro can be achieved by illumination of the enzyme in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol. This process is named photoactivation (1). Photoactivation in vitro requires photoexcitation of the flavin and a triad of redox-active residues in the protein moiety that is highly conserved in DNA photolyases (3, 4) as shown in Fig. 1A (electron transfer pathway 2). These residues are generally tryptophans that allow transport of an electron from the protein surface to the U-shaped flavin cofactor, which is buried within the C-terminal α-helical domain (5–9). Whether the same mechanism is used by photolyase to photoreduce FAD in vivo is a matter of debate (10). Photoreduction of the flavin cofactor was also observed in cryptochrome blue/UV-A photoreceptors. However, instead of fully reduced flavin, semireduced flavin species (either anionic flavin semiquinone radical or neutral semiquinone radical) accumulate. This form of the photoreceptor is considered as the signaling state (11–14).Open in a separate windowFIGURE 1.Electron transfer pathways in cry3 and structures of folates. A, indicated are the distances of the tryptophans in the tryptophan triad (Trp-356, -409, -432) of Trp-432 to FADH⁻ and of FADH⁻ to the 5,10-methenylTHF (MTHF) cofactor in cry3. Shown are also the two established routes of electrons from FADH⁻ to the DNA lesion (Route 1) and within the tryptophan triad to FAD (Route 2). The third electron transfer pathway from FADH⁻ to 5,10-methenylTHF (Route 3) is the subject of this study. B, chemical structures of folates and their molecular masses. Folypolyglutamate molecules have a pteridin and a p-aminobenzoate moiety linked with a glutamate chain with a variable number of glutamic acids. The various THF species differ in their oxidation state of the C1 unit that is attached at the N-5 or N-10 position or form a bridge between both.A recently discovered subclade of the DNA photolyase/cryptochrome family are DASH cryptochromes, which have members in plants, bacteria, and aquatic animals (6, 15–17). Because DASH cryptochromes were found to lack repair activity for CPDs in double-stranded DNA, they were considered as cryptochrome-type photoreceptors (6, 16). However, it was recently shown that DASH cryptochromes repair CPDs in single-stranded DNA (18) and loop structures of double-stranded DNA (19) and, thus, belong to the CPD photolyase group. In contrast to conventional CPD photolyases, DASH cryptochromes are unable to flip the CPD lesion out of the DNA duplex (7).Besides the flavin cofactor that is essential for enzymatic activity, DNA photolyases and most likely all cryptochromes contain a second chromophore (1). Like the catalytic flavin, the second chromophore is non-covalently attached to the protein moiety. The majority of DNA photolyases and, as far as studied, the cryptochromes including the DASH-type like cry3 from Arabidopsis thaliana contain polyglutamated 5,10-methenyltetrahydrofolate (5,10-methenylTHF) as the second chromophore (1, 12, 17, 20, 21) (see Fig. 1B for folate structures). Several organisms like the cyanobacterium Anacystis nidulans (Synechococcus elongatus) produce deazariboflavins (7,8-didemethyl-8-hydroxy-5-deazariboflavin) and utilize them as second cofactor (22). In photolyases of thermophilic bacteria and Archaea of the genus Sulfolobus, FMN and FAD, respectively, were found as second cofactors (23, 24). The sole function of the second cofactors demonstrated at present is transfer of excitation energy to the catalytic flavin cofactor via a Förster-type mechanism. The crystal structures of DNA photolyases and DASH cryptochromes revealed that the second chromophores are located in a cleft between the N-terminal α/β domain and the C-terminal α domain (7–9). The centroid distances between the catalytic FAD and the second chomophore are in the range of 15–18 Å. The close distances and the angles between the transition dipole moments of the two cofactors are favorable for efficient energy transfer. Indeed, energy transfer efficiencies are about 70% for Escherichia coli photolyase (25), close to 100% for A. nidulans photolyase (26), and between 78% (dark-adapted) and 87% (light-adapted) for Arabidopsis cry3 (27). Although the second cofactors are not essential for catalysis (28, 29), they increase the efficiency of repair and possibly of photoactivation by having higher extinction coefficients than FADH⁻ in the near UV and blue region (30). The spectral overlap between 5,10-methenylTHF emission and the absorption of the different flavin redox states is on the order FADH^o > FAD_ox > FADH⁻ (31).Illumination in vitro of photolyase that contains fully oxidized or semireduced flavin results in light-induced absorbance changes. The decrease in absorption in the 450–470-nm region reflects a decrease in the amount of fully oxidized FAD concomitant with transient increase in absorption above 500 nm, which indicates the formation of a neutral semiquinone radical. Excitation of the 5,10-methenylTHF antenna chromophore at its absorption peak at 380 nm causes a likewise photoreduction of the catalytic FAD (1, 27, 28, 30, 31). However, irreversible bleaching of the 380-nm peak is observed under high irradiance UV-A or camera flash illumination (28, 30). This irreversible bleaching goes along with release of the folate cofactor from the protein moiety (30) and was named photodecomposition of 5,10-methenylTHF (28). However, the identity of the formed folate species remained unknown (30). In our previous spectroscopic characterization of Arabidopsis cry3, a similar bleaching of the 380-nm peak was observed (27).Here we show that a third electron transfer pathway exists in photolyase and DASH cryptochome, where the 5,10-methenylTHF cofactor is photoreduced to 5,10-methyleneTHF. Thus, bleaching at 380 nm does not simply reflect destruction but is a specific chemical conversion of the second chromophore.     

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

The FAD-dependent choline oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of choline oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the k_cat/K_m and k_cat values were observed as compared with wild-type choline oxidase at pH 10 and 25 °C, with no significant effect on k_cat/K_O using choline as substrate. Both the k_cat/K_m and k_cat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both ^D(k_cat/K_m) and ^Dk_cat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the ^Dk_red values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by choline oxidase.A number of enzymes, including dehydrogenases (1–3), monooxygenases (4–7), halogenases (8–11), and oxidases (7, 12, 13), employ flavin cofactors (FAD or FMN) for their catalytic processes. About a tenth of all flavoproteins have been shown to contain a covalently attached cofactor, which may be linked at the C8M position via histidyl, tyrosyl, or cysteinyl side chains or at the C6M position via a cysteinyl side chain (14). Glucooligosaccharide oxidase (15, 16), hexose oxidase (17), and berberine bridge enzyme (18, 19) are examples of flavoproteins (FAD as cofactor) with both linkages present in one flavin molecule. The covalent linkages in flavin-dependent enzymes have been shown to stabilize protein structure (20–22), prevent loss of loosely bound flavin cofactors (23), modulate the redox potential of the flavin microenvironment (20, 23–27), facilitate electron transfer reactions (28), and contribute to substrate binding as in the case of the cysteinyl linkage (20). However, no study has implicated a mechanistic role of the flavin covalent linkages in enzymatic reactions in which a hydride ion is transferred by quantum mechanical tunneling.The discovery of quantum mechanical tunneling in enzymatic reactions, in which hydrogen atoms, protons, and hydride ions are transferred, has attracted considerable interest in enzyme studies geared toward understanding the mechanisms underlying the several orders of magnitudes in the rate enhancements of protein-catalyzed reactions compared with non-enzymatic ones. Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29, 30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31–33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35, 36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37). This leaves choline oxidase as the only characterized enzyme with a covalently attached flavin cofactor (12, 38), where the oxidation of its substrate occurs unequivocally by quantum mechanical tunneling.Choline oxidase from Arthrobacter globiformis catalyzes the two-step FAD-dependent oxidation of the primary alcohol substrate choline to glycine betaine with betaine aldehyde, which is predominantly bound to the enzyme and forms a gem-diol species, as intermediate (Scheme 1). Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism against stress conditions, thus making genetic engineering of relevant plants of economic interest (39–45), and the biosynthetic pathway for the osmolyte is a potential drug target in human microbial infections of clinical interest (46–48). The first oxidation step catalyzed by choline oxidase involves the transfer of a hydride ion from a deprotonated choline to the protein-bound flavin followed by reaction of the anionic flavin hydroquinone with molecular oxygen to regenerate the oxidized FAD (for a recent review see Ref. 50). The gem-diol choline, i.e. hydrated betaine aldehyde, is the substrate for the second oxidation step (49), suggesting that the reaction may follow a similar mechanism. The isoalloxazine ring of the flavin cofactor, which is buried within the protein, is physically constrained through a covalent linkage via the C(8) methyl of the flavin and the N(3) atom of the histidine side chain at position 99 (Fig. 1) (12). Also contributing to the physical constrain are the proximity of Ile-103 to the pyrimidine ring and the interactions of the backbone atoms of residues His-99 through Ile-103 with the isoalloxazine ring. The rigid positioning of the isoalloxazine ring could only permit a solvent-excluded cavity of ∼125 ų adjacent to the re face of the FAD to accommodate a 93-ų choline molecule in the substrate binding domain (12). Mechanistic data thus far obtained on choline oxidase, coupled with the crystal structure of the wild-type enzyme resolved to 1.86 Å, are consistent with a quantum tunneling mechanism for the hydride ion transfer occurring within a highly preorganized enzyme-substrate complex (Scheme 2) (12, 34, 50). Exploitation of the tunneling mechanism requires minimal independent movement of the hydride ion donor and acceptor, with the only dynamic motions permitted being the ones that promote the hydride transfer reaction.Open in a separate windowSCHEME 1.Two-step, four-electron oxidation of choline catalyzed by choline oxidase.Open in a separate windowFIGURE 1.x-ray crystal structure of the active site of wild-type choline oxidase resolved to 1.86 Å (PDB 2jbv). Note the significant distortion of the flavin ring at the C(4a) atom, which is due to the presence of a C(4a) adduct (69).Open in a separate windowSCHEME 2.The hydride ion transfer reaction from the α-carbon of the activated choline alkoxide species to the N(5) atom of the isoalloxazine ring of the enzyme-bound flavin in choline oxidase.In the present study, the contribution of the physically constrained flavin isoalloxazine ring to the reaction catalyzed by choline oxidase has been investigated in a variant enzyme in which the histidine residue at position 99 was replaced with an asparagine. The results suggest that, although not being required per se, the covalent linkage in choline oxidase contributes to the hydride tunneling reaction by either preventing independent movement or contributing to the optimal positioning of the flavin acting as hydride ion acceptor with respect to the alkoxide species acting as a donor. However, the covalent linkage is not required for the reaction.     

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains ^4–5FnI and ³FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes ³FnIII and disrupts the observed ^4–5FnI-³FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.Fibronectin (FN),4 a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2–4). Although interactions between domains that are close in primary sequence have been demonstrated (3, 5), studies of multidomain fragments generally assume a beads-on-string model (2). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5–8), sites of protein interactions (9), and sites that control cellular activity (10, 11). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12–14); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15–17) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18) has also not been straightforward (2), possibly because of domain stabilization through nearest-neighbor effects (19, 20).A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10, 21) and breast carcinoma cells using the Boyden chamber (22). MSF comprises FN domains ¹FnI to ⁹FnI, a truncated ¹FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to ¹FnI-⁹FnI (Fn70kDa) displays the same activity (10). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains ⁶FnI-⁹FnI) (23) and partial activity to a shorter fragment spanning domains ^7–9FnI (24). Two IGD tripeptides of domains ⁷FnI and ⁹FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10, 24, 25); however, similar IGD tripeptides outside the GBD, on domains ³FnI and ⁵FnI, appear to have little effect (10, 23). Full-length adult FN does not affect cell migration in similar assays (10, 23); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement.Open in a separate windowFIGURE 1.Motogenic activity of FN fragments. a, schematic representation of the FN domain structure (top) and enlargement of the FN N terminus (bottom). Type I domains are shown as pentagons; type II domains as hexagons; and type III domains as ovals. b, comparison of motogenic activity versus protein concentration of wild-type Fn70kDa and Fn100kDa fragments. Error bars are derived from duplicate experiments, and a gray band denotes migration activity of media without additives. c, similar comparisons for mutant Fn100kDa fragments. d, analytical size exclusion chromatography of large FN fragments. The trace of UV absorbance at 280 nm versus elution volume shown here indicates a larger hydrodynamic radius for Fn100kDa R222A compared with the wild type, consistent with our model (Fig. 6a).Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26), as well as amphibians, birds, and mammals (27), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (^1–3FnIII), suggesting that these domains are responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains ^4–5FnI and ³FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences.     

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu²⁺, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1)³ cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1–10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11–16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These “as-isolated” SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between Cys-57 and Cys-146 (20). The significance of these results is that even WT-like mutants, which exhibit a nearly normal backbone structure (21–23), may be vulnerable to destabilizing influences in vivo. Our group and others subsequently showed that the mutant SOD1 proteins share a susceptibility to increased hydrophobicity under conditions that reduce disulfide bonds and/or chelate metal ions (5) and that similar hydrophobic species exist in tissue lysates from mutant SOD1 transgenic mice (4–6). One consequence of such hydrophobic exposure could be the facilitation of abnormal interactions between the mutant enzymes and other cellular constituents (e.g. chaperones, mitochondrial components, or other targets), which might influence pathways leading to motor neuron death (15, 16, 24–27).Accumulating evidence suggests that metal deficiency of SOD1 is an important factor that can influence SOD1 aggregation or neurotoxicity (4, 28–33), but the metal-deficient states of SOD1 that are most relevant to ALS remain unclear. Zinc-deficient, copper-replete SOD1 species, which can be produced in vitro by adding copper to SOD1 that has been stripped of its metal ions at acidic pH, were shown to be toxic to motor neurons in culture (28). However, it has not been shown that zinc-deficient, copper-replete SOD1 is produced in vivo as a consequence of ALS mutations, and loading of copper into SOD1 by the copper chaperone for SOD1 (CCS) is not required for toxicity (34, 35). Furthermore, the MBR mutants have a disrupted copper site and have been found to be severely deficient in both zinc and copper (17, 30), yet expression of these SOD1s still produces motor neuron disease (1, 2, 30, 34, 36, 37).When recombinant human SOD1 was overexpressed in insect cells, we instead observed zinc-replete but copper-deficient species for most WT-like mutants, probably because the capacity of the copper-loading mechanism was exceeded (17). These preparations indicate that zinc can be efficiently incorporated into many WT-like mutants in vivo, and much of it is retained after purification. Furthermore, these copper-deficient biologically metallated proteins may be useful reagents to assess the influence of copper binding upon other properties of SOD1 mutants that may be relevant to their neurotoxicity.We previously observed that reduction of the Cys-57—Cys-146 disulfide bond facilitates the ability of metal chelators to alter the electrophoretic mobility and to increase the hydrophobicity of SOD1 mutants (5). This is consistent with the known properties of this linkage to stabilize the dimeric interface, to orient Arg-143 via a hydrogen bond from the carbonyl oxygen of Cys-57 to Arg-143-NH₂, and to prevent metal ion loss (38–40). However, it remains unclear whether the Cys-57—Cys-146 bond is required to prevent abnormal SOD1 hydrophobic exposure or whether the aberrant conformational change primarily results from metal ion loss. Ablation of the disulfide bond by the experimental (non-ALS) mutants C57S and C146S provides useful reagents to test the relative influence of the disulfide bond and copper binding upon SOD1 properties.In this study we sought to correlate the consequences of copper deficiency, copper and zinc deficiency, and disulfide reduction upon the hydrodynamic behavior and surface hydrophobicity of WT and representative mutant SOD1 enzymes (Fig. 1A). We quantitated the metal contents of as-isolated SOD1 proteins, detected changes in conformation or metal occupancy using native PAGE to assess their electrophoretic mobility, a measure of global conformational change, and correlated these changes to hydrophobic exposure using 1-anilinonaphthalene-8-sulfonic acid (ANS), which is very sensitive to local conformational changes. ANS is a small amphipathic dye (Fig. 1B) that has been used as a sensitive probe to detect hydrophobic pockets on protein surfaces (41–44). Free ANS exhibits only weak fluorescence that is maximal near 520 nm, but when ANS binds to a hydrophobic site in a partially or fully folded protein, the fluorescence peak increases in amplitude and shifts to a shorter wavelength (42). ANS also has an anionic sulfonate group that can interact with cationic groups (e.g. Arg or Lys residues) through ion-pair formation which may be further strengthened by hydrophobic interactions (43–46).Open in a separate windowFIGURE 1.A, WT SOD1 structure showing the position of the C57-C146 intrasubunit disulfide bond (S–S, yellow), bound copper and zinc ions, and ALS mutant residues. The residues altered in A4V, G85R, G93A, D124V, and S134N SOD1s are indicated as green spheres. The backbone of the β-barrel core and the loops is shown in a rainbow color, from blue at the amino terminus to red at the carboxyl terminus. The figure was generated using PyMOL (84) and PDB entry 1HL5 (22). B, chemical structure of ANS fluorophore.To evaluate further the importance of metal ion binding, we measured spectral changes related to the binding of cobalt and copper to the same SOD1 proteins. We observed that as-isolated WT-like mutants containing zinc could interact with copper ions to produce an electrophoretic mobility and decreased hydrophobicity resembling that of the fully metalated holo-WT SOD1. In contrast, we saw no evidence for copper binding to MBR mutants in a manner that alters their hydrodynamic properties or their hydrophobicity. Our data suggest that binding of both copper and zinc are important determinants of SOD1 conformation and that perturbation of such binding may be relevant to the ALS disease process.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).