High Precision NMR Structure and Function of the RING-H2 Finger Domain of EL5, a Rice Protein Whose Expression Is Increased upon Exposure to Pathogen-derived Oligosaccharides*

Shizue KatohDagger , Cui HongDagger , Yuki TsunodaDagger , Katsuyoshi MurataDagger , Ryota Takai§, Eiichi MinamiDagger , Toshimasa YamazakiDagger , and Etsuko KatohDagger

From the Dagger  Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan and the § Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan

Received for publication, October 15, 2002, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta -sheets (beta 1, Ala147-Phe149; beta 2, Gly156-His158), one alpha -helix (Cys161-Leu166), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha -helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.

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Upon sensing the invasion of microorganisms, plants evoke a variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defense-related genes. In some cases, the level of protein accumulation and the rapidity of gene induction in the host plant are correlated to the degree of its disease resistance. Therefore, it might be possible to control disease resistance by modifying the regulatory factors for the expression of defense-related genes. Such regulatory factors could be elements of signal transduction pathways leading from the recognition of invading pathogens to the activation of defense-related genes. Most of the defense responses are reproducible in suspension-cultured cells treated with specific substances called elicitor (1). Chitin fragments (N-acetylchitooligosaccharides) can act as elicitors (2), which induce the transient expression of several "early responsive" genes, such as EL5 (3). EL5 is a RING finger protein, which is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III), and RING-H2 finger domain (domain IV) followed by the C-terminal region with highly diverse amino acid sequences (4). Although some ATL family genes resemble EL5 in being induced in early stages of the defense responses (5), their biochemical function is obscure. Recently, it was shown that the fusion protein of EL5 with maltose-binding protein (MBP)1 was polyubiquitinated by incubation with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). Apparently, EL5 acted as a ubiquitin ligase (E3) and catalyzed the transfer of ubiquitin to the MBP moiety. Further, it was shown that a rice E2, OsUBC5b was induced by elicitor (6). Although these results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system, their molecular function has not been clarified at the atomic level.

The RING finger motif, which is known from many functionally distinct proteins, was first identified in the product of the human gene RING1 (Really Interesting New Gene 1) that is located proximal to the major histocompatibility region on chromosome 6 (7). The RING finger motif is defined by the consensus sequence Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-(Cys/His)-X2-Cys-X4-48-Cys-X2-Cys, in which X can be any amino acid. It binds two zinc atoms with its Cys and His residues in a unique "cross-brace" arrangement. The invariable spacing between the second and third pair of Cys/His residues indicates conservation of the distance between the two zinc-binding sites (8). The RING finger motif is widely distributed among proteins that play major roles in cell growth and differentiation (9). Certain types of RING finger domains seem to be required for multimerization, while others are elements of proteins involved in ubiquitination (10). Ubiqutination usually results in the formation of a bond between the C terminus of ubiquitin (Gly76) and the epsilon -amino group of a substrate Lys residue. This reaction requires the sequential action of three enzymes: (i) an activating enzyme (E1) that forms a thiol ester with the carbonyl group of Gly76 in ubiquitin, (ii) a conjugating enzyme (E2) that transiently carries the ubiquitin as a thiol ester, and (iii) a ligase (E3) that transfers the activated ubiquitin from the E2 to the substrate Lys residue. The efficiency and high selectivity of ubiquitination reactions depend on the accuracy of E3 action. All known E3s utilized either of catalytic domains, the RING finger domain or the HECT domain (11). The structures of RING finger domains have been determined by NMR (12, 13) and x-ray (14, 15). However, the relationship between molecular function and high order structure has not yet been established.

To elucidate the properties of EL5 at the atomic level, we determined the three-dimensional structure of its RING-H2 finger domain in solution by NMR spectroscopy. Furthermore, we characterized its functional properties by an ubiquitination assay in vitro and by NMR titration experiments. Our results illuminate the function of not only EL5 but also the ATL family proteins in general.

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Cloning and Purification of Recombinant Proteins for NMR Experiments-- EL5 RING-H2 finger domains and OsUBC5b were cloned as fusion proteins with thioredoxin (Trx) and His tag. The fusion proteins were constructed in the vector pET32a (Novagen). EL5(129-181) and EL5(96-181) DNA fragments from EL5 cDNA (AB045120) were amplified by polymerase chain reaction (PCR). OsUBC5b was amplified by PCR from OsUBC5b cDNA (AB074412). Trx-EL5(129-181) was constructed using the primers 5'-CATGCCATGGACGACGGCGTCGAGTG-3' and 5'-CGAATTCTTACACGACGACGGTGAGGC-3'. Trx-EL5-(96-181) was constructed using the primers 5'-CATGCCATGGGGGTCGACCCGGAGGTG-3' and 5'-CGGGATCCTTACACGACGACGGTGAGGC-3'. The purified PCR products were digested with NcoI and EcoRI or BamHI and were ligated into pET32a. Trx-OsUBC5b was constructed with the primers 5'-TTCCATGGCGTCCAGCGGATCCTCAAG-3' and 5'-AACTCGAGCTAGCCCATAGCATATTTCTGGGT-3'. The purified PCR product was digested with NcoI and XhoI and was ligated into pET32a.

All of the clones were transformed into Escherichia coli BL21(DE3) cells. The bacteria were grown at 37 °C in Luria Bertani for non-labeled protein and in M9 minimal medium for uniformly 15N- and 15N/13C-labeled protein, with 50 µg/ml ampicillin. Protein expression was induced by addition of isopropyl-beta -D-thiogalactopyranoside (IPTG) at a concentration of 1 mM. For EL5(129-181) and EL5(96-181), ZnSO4 (final concentration 100 µM) was added at the same time. After 3 h the bacteria were harvested by centrifugation, and the pellets were frozen.

Trx-EL5(129-181) and Trx-EL5(96-181) were purified by a similar procedure. Frozen pellets were thawed on ice and resuspended in 50 mM Tris-HCl buffer (pH 7.4), 50 mM NaCl, 50 µM ZnSO4, and 2.5 mM beta -mercaptoethanol, before lysis by sonication. Insoluble material remaining after lysis was removed by centrifugation at 27,000 × g for 30 min. The supernatant was loaded onto a 5-ml Ni-chelating column (Amersham Biosciences). After washing the column with buffer (50 mM NaCl, 50 mM Tris-HCl buffer (pH7.4), 20 µM ZnSO4, 1 mM beta -mercaptoethanol, and 15 mM imidazole), protein was eluted in buffer using 400 mM imidazole. Peak fractions were concentrated and applied to a HiLoad Superdex 75pg 26/60 column (Amersham Biosciences) that had been equilibrated in buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 µM ZnSO4, 2.5 mM beta -mercaptoethanol). Trx tag was removed by cleavage with 75 units of enterokinase (Invitrogen) per 31.5 mg of protein at 37 °C for 16 h. The solution was applied to a HiLoad Superdex 75pg 26/60 column. The peak fraction was 5-fold diluted with 50 mM Tris-HCl, 20 µM ZnSO4, and 2.5 mM beta -mercaptoethanol and was applied to a 5-ml HiTrap Q column (Amersham Biosciences). Peak fractions were dialyzed against NMR buffer (100 mM NaCl, 20 mM Tris-HCl buffer, pH 7.0, 20 µM ZnSO4, 1 mM dithiothreitol), and were concentrated using a Centricon spin dialysis tube (3-kDa cutoff; Amicon, Inc.).

For purification of Trx-OsUBC5b, frozen pellets were thawed on ice, resuspended in 20 mM phosphate buffer (pH 7.4) with 500 mM NaCl, and lysed by sonication. Insoluble material remaining after lysis was removed by centrifugation at 27,000 × g for 30 min. The supernatant was loaded onto a 5-ml Ni-chelating column. After washing the column with buffer (20 mM phosphate buffer, pH 7.4, 500 mM NaCl), protein was eluted in buffer using a linear gradient of imidazole (0-500 mM). Peak fractions were concentrated and applied to a HiLoad Superdex 75 pg 26/60 column equilibrated in buffer (20 mM phosphate buffer, pH 7.4, 100 mM NaCl). Trx tag was removed by cleavage with 28 units of thrombin (Novagen) per about 13 mg of protein at 37 °C for 12 h. The solution was applied to a Ni-chelating column. Remaining tag was removed by cleavage with 130 units of enterokinase per about 5 mg of protein at 37 °C for 16 h. Then the solution was applied to a HiLoad Superdex 75pg 26/60 column. Peak fractions were dialyzed against NMR buffer (100 mM NaCl, 20 mM Tris-HCl buffer, pH 7.0, 5 mM dithiothreitol) and concentrated using a Centricon spin dialysis tube (30-kDa cutoff; Amicon, Inc.).

NMR Spectroscopy-- All NMR spectra were recorded at 35 °C on a Bruker DMX750 spectrometer equipped with a 5-mm inverse triple resonance probe head with three-axis gradient coils. 1H, 13C, and 15N sequential resonance assignments were obtained using two-dimensional double resonance and three-dimensional double and triple resonance through-bond correlation experiments (16-18): two-dimensional 1H-15N HSQC, two-dimensional 1H-13C CT-HSQC optimized for observation of either aliphatic or aromatic signals, three-dimensional 15N-separated HOHAHA-HSQC, three-dimensional HNHA, threedimensional HNHB, three-dimensional HNCO, three-dimensional CBCA(CO)HN, three-dimensional HBHA(CBCACO)NH, three-dimensional HNCACB, three-dimensional C(CO)NH, three-dimensional HCABGCO, and three-dimensional HCCH-TOCSY. Stereospecific assignments of alpha - and beta -protons and the methyl side chains of Val and Leu residues were achieved by a combination of quantitative J measurements and NOESY data. 3J couplings were measured using quantitative two-dimensional and three-dimensional J correlation spectroscopy (18). Interproton distance restraints were derived from multidimensional NOE spectra (16-18): three-dimensional 15N-separated NOESY-HSQC spectrum with a mixing time of 100 ms, three-dimensional 13C/15N-separated NOESY-HSQC spectrum with a mixing time of 100 ms, and four-dimensional 13C/13C-separated HMQC-NOESY-HMQC spectrum with a mixing time of 100 ms. Amide-proton exchange rates were determined by recording a series of two-dimensional 1H-15N HSQC spectra at different time points immediately after the H2O buffer was changed to D2O buffer. These spectra were processed using NMRPipe software (19) and were analyzed using Capp/Pipp/Stapp software (20). 1H, 13C, and 15N chemical shifts were referenced to HDO (4.68 ppm at 35 °C), indirectly to TSP (13C) (21), and to liquid ammonia (15N), respectively (22).

Structure Calculations-- NOE-derived interproton distance restraints were classified into four ranges: 1.8-2.7, 1.8-3.3, 1.8-4.3, and 1.8-5.0 Å, corresponding to strong, medium, weak, and very weak NOEs. The upper limit was corrected for constraints involving methyl protons and methylene protons that were not assigned stereospecifically (23). Hydrogen bond distance restraints were applied to N and O atoms (2.8-3.3 Å) and to HN and O atoms (1.8-2.3 Å), in regular secondary structures that had small amide exchange rates. Torsion angle restraints on phi  and psi  were derived from 3JHNHalpha coupling constants (24), short-range NOEs (alpha Hi to NHi+1 and beta Hi to NHi+1), and a data base analysis of backbone (13Calpha , 13Cbeta , 13C', 1Halpha , 15N) chemical shifts using the program TALOS (25). Side-chain chi 1 angle restraints were derived from HOHAHA connectivities and the distances between alpha H and beta 1H and between alpha H and beta 2H, which were estimated from NOEs and ROEs. Values for 3JHalpha Hbeta and 3JNHbeta coupling constants were also taken into consideration. The structures of the RING-H2 finger domain of EL5 were calculated using the hybrid distance geometry-dynamical simulated annealing method (26), as contained in X-PLOR 3.1 (27). For structure calculations, we used 755 interproton distance restraints (comprising 290 intraresidue, 185 sequential (|i - j| = 1), 73 medium range (1 < |i - j| < 5), and 207 long range (|i - j| >=  5) restraints) obtained from heteronuclear three- and four-dimensional NOE spectra. In addition to the NOE-derived distance restraints, 18 distance restraints for 9 hydrogen bonds and 140 dihedral angle restraints (46 phi , 47 psi , 38 chi 1, and 9 chi 2) were included in the calculation. A final set of 15 lowest energy structures was selected from 100 calculations. None of them had NOE and dihedral angle violations of >0.5 Å and >5°, respectively. Structural statistics calculated for the final 20 structures are summarized in Table I. Statistics did not change significantly when the 40 lowest energy structures were used for calculation. The average coordinate of the ensembles of the final 15 structures was subjected to 500 cycles of Powell restrained energy minimization to improve stereochemistry and non-bonded contacts. The structural statistics for the restrained energy minimized average structure are also summarized in Table I. Figures were generated using MOLMOL (28).

A series of two-dimensional 1H-15N HSQC spectra was recorded for the RING-H2 finger domain in 20 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl and 2 mM dithiothreitol, as a function of OsUBC5b concentration. Solutions containing 0.25 mM of either 15N-labled RING-H2 finger domain or non-labeled OsUBC5b were mixed to yield molecular ratios (OsUBC5b : RING-H2 finger domain) of 0, 0.25, 0.5, 0.75, 1, 1.25, and 2. Assignments of the HSQC signals of EL5 RING finger domain bound to E2 were made by tracing the peaks during titration and were confirmed by analyzing three-dimensional triple resonance experiments carried out on the solution containing 0.6 mM 15N/13C-labeled EL5 RING finger domain and 0.72 mM OsUBC5b.

Cloning and Purification of Recombinant MBP Fusion Proteins-- The EL5 fragments Gly96-Asn325 and Gly96-Val181 were amplified by PCR using 5'-GAATTCGGAGGAGGGGTCGACCCG-3' and 5'-GGAATTCTCAATCCGGACATGCGC-3' primers or 5'-CGAATTCGGAGGAGGGGTCGACCCG-3' and 5'-GGAATTCTCAATCCGGACATGCGC-3' primers, respectively. The PCR products were digested with EcoRI and inserted into the EcoRI site of pMAL-p2 (New England BioLabs, Beverly, MA) to express MBP-EL5(96-325) and MBP-EL5(96-181). To yield MBP for negative control experiments, pMAL-p2 was digested with EcoRI, blunted, and self-ligated. All PCR products were verified by DNA sequencing. The plasmids were introduced into the E. coli strain JM109 to produce the recombinant proteins and were purified by amylose affinity chromatography according to the manufacturer's instructions (New England BioLabs).

In Vitro Ubiquitination Experiments-- The ubiquitination reaction was performed in 150 µl of ubiquitination buffer containing 300 ng/µl bovine ubiquitin (Sigma), 50 ng of mouse E1, 10 ng OsUBC5b as E2, and 250 ng of MBP fusion protein. The reaction buffer was incubated at 35 °C for 1 h, and the reaction was stopped by the addition of SDS sample buffer. After boiling for 5 min, the samples were separated by 7.5% SDS-PAGE and subjected to immunoblotting using anti-MBP antibody (New England BioLabs).

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Determination of NMR Experimental Conditions-- EL5 consists of a transmembrane domain (domain I), a basic domain (domain II), a conserved domain (domain III), and the RING-H2 finger domain (domain IV) followed by the C terminus (Fig. 1a). For NMR experiments, two protein fragments were prepared: EL5 (129-181, domain IV), containing the RING-H2 finger domain only, and EL5 (96-181, domain III + IV), which additionally contained the conserved domain. The 1H-15N HSQC spectrum of EL5(129-181) obtained at 35 °C showed dispersed, intense signals, indicating a well-defined molecular structure (Fig. 1b). In the spectrum of EL5(96-181), the strengths of peaks differed considerably (Fig. 1c). The weak signals corresponded to those of EL5(129-181), that is, to the RING-H2 finger domain. The intense signals were concentrated in the center of the spectrum, indicating that domain III is a highly flexible random coil structure.


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Fig. 1.   a, structure of EL5. b-d, two-dimensional 1H-15N HSQC spectra of EL5-(129-181) (b), EL5-(96-181) (c), and EL5-(129-181) unfolded upon addition of EDTA (d). Sequential assignments of backbone amides are shown in b.

To investigate the role of Zn2+ in the folding of the EL5 RING-H2 domain, Zn2+ was chelated by EDTA. Removal of zinc from the purified domain by addition of EDTA led to a loss of the chemical shift dispersion characteristics of the folded proteins (Fig. 1d). The protein was reversibly refolded upon addition of excess Zn2+, as indicated by the reappearance of a spectrum that was identical to that in Fig. 1b. Therefore we decided to carry out the purification of recombinant EL5(129-181) and its NMR analysis in the presence Zn2+.

1H, 15N, and 13C Signal Assignments and Secondary Structure of the EL5 RING-H2 Finger Domain-- The backbone amide resonances in the 1H-15N HSQC spectrum were sequentially assigned to all non-proline residues based upon the analysis of correlations observed in CBCA(CO)NH and HNCACB spectra, respectively. The former type of spectrum correlates each 15N, HN signal pair to the Calpha and Cbeta signals of the preceding residue, while the latter additionally provides the 15N, HN, Calpha , and 15N, HN, Cbeta intraresidue correlations. The assignments were checked and extended to backbone Halpha and CO signals, and to 1H and 13C side-chain signals by correlations observed in the three-dimensional spectra, 15N-separated HOHAHA-HSQC, HBHA(CO)NH, HNCO, C(CO)HN, and HCCH-TOCSY. Side-chain 1H and 13C signals in aromatic regions were assigned to aromatic amino acids according to cross-peak patterns observed in 1H-13C CT-HSQC and CT-HSQC-relay spectra that were optimized for aromatic side chains. The aromatic Hdelta and Cdelta signals were then correlated to sequentially assigned backbone signals through cross-peaks observed in three-dimensional 15N-separated NOESY-HSQC and 13C/15N-separated NOESY-HSQC spectra and a four-dimensional 13C/13C-separated NOESY spectrum.

In order to obtain a high-resolution NMR structure, the resonance assignments were refined by stereospecific assignments of numerous methylene and methyl protons of Val and Leu residues. Stereospecific assignments of Gly alpha -protons were derived from 3JHNHalpha coupling constants (24), intraresidue NOEs between HN and alpha -protons, and sequential NOEs between Gly alpha -protons (i) and HN protons (i+1). beta -protons were stereospecifically assigned using J couplings obtained from HNHB (29) and HOHAHA-HSQC spectra, and by intraresidue alpha -beta distances estimated from NOE/ROE spectra, as described by Clore and Gronenborn (17). In some cases, information from local secondary structure and short-range NOEs was used to facilitate stereospecific assignments. Signals from all Val gamma -methyl groups were stereospecifically assigned from estimates of 3JNCgamma (30), 3JNHbeta (29), and 3JHNHalpha coupling constants and from NOEs between Val alpha -beta and alpha -gamma protons. Leu delta -methyl group signals were stereospecifically assigned from NOE patterns observed in three-dimensional NOESY, ROESY, and four-dimensional NOESY spectra.

On the basis of essentially complete signal assignments, interproton distance constraints were derived from short range NOE connectives obtained from three-dimensional 15N-separated NOESY-HSQC, 13C/15N-separated NOESY-HSQC, and four-dimensional 13C/13C-separated HSQC-NOESY-HSQC spectra. Dihedral constraints for phi  angles were determined from 3JHNHalpha coupling constants derived from Halpha /HN intensity ratios measured in HNHA experiments. Slowly exchanging amide protons were assigned in 1H-15N HSQC hydrogen exchange experiment and were identified as protons involved in interresidue hydrogen bonds. The beta -strand and helical domains indicated by the NOE and J coupling data were corroborated by secondary structural elements predicted by the observed displacements of Calpha and Cbeta chemical shifts from their random-coil values (31). In summary, the analysis indicated that the RING-H2 finger domain of EL5 contained two beta -strands and an alpha -helix in a beta beta alpha arrangement.

Tertiary Structure of RING-H2 Finger Domain of EL5-- The three-dimensional structure of the RING-H2 finger domain of EL5 was determined by a hybrid distance geometry/dynamic-simulated annealing approach (26) based upon 913 experimental restraints derived from NMR spectroscopy. Structural statistics for the EL5 RING-H2 finger domain are shown in Table I. The superposed backbone N, Calpha , and C' coordinates of the final 15 structures (Fig. 2a) were well aligned, except for residues 129-131 at the N terminus and residues 179-181 at the C terminus. For the residues 132-178, the root mean square deviation (r.m.s.d.) for backbone heavy atoms was 0.34 Å, compared with 0.72 Å for all heavy atoms. A ribbon diagram (Fig. 2b) representing the backbone conformation of the restrained, energy-minimized mean structure of the RING-H2 finger domain illustrates its beta beta alpha fold (beta 1, Ala147-Phe149; beta 2, Gly156-His158; alpha 1, Cys161-Leu166). There is a long flexible loop at each side of the beta beta alpha structure (N-loop, Val133-Glu146; C-loop, Gly167-Val180).


                              
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Table I
Structural statistics for the RING-H2 finger domain of EL5


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Fig. 2.   a, best-fit superposition of the backbone (N, Calpha , and C') atoms of the final 15 structures of the RING-H2 finger domain of EL5. The structures are superimposed on the energy-minimized average structure using the backbone coordinates of residues 132-178. b, ribbon diagram of the energy-minimized average structure of the RING-H2 finger domain of EL5.

The three-dimensional structures of a few RING finger domains have been determined. The molecular function of two of them, RAG1 and c-Cbl, is known. The V(D)J recombination-activating protein, RAG1, is a dimer. Its dimerization domain consists of a zinc finger and a RING finger (14). It also contains a unique binuclear zinc cluster instead of the mononuclear zinc site in the RING finger. The determination of the crystal structure of c-Cbl bound to a specific ubiquitin-conjugating enzyme (E2), UbcH7, has revealed that the RING finger domain of c-Cbl recruits the E2 (15). The RING finger domain of EL5 is structurally similar to those of RAG1 and c-Cbl (Fig. 3a). The backbones of the secondary structural elements of the proteins are almost superimposable. The backbone (N, Calpha , C', O) atomic r.m.s.d. values for the beta -strand- and alpha -helix-forming residues between the EL5 RING finger domain and RAG-1 or c-Cbl are 0.73 and 1.30 Å, respectively.


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Fig. 3.   a, comparison of the free RING finger domain of EL5 with the E2-bound form of c-Cbl and the dimerization domain of RAG1. The additional alpha -helices at the N and C termini of RAG1 are shown in yellow. b, surface potential representations of these domains. Positively charged areas are blue, and negatively charged areas are red. The hydrophobic grooves in c-Cbl and EL5 are indicated by arrows. The atomic coordinates for c-Cbl and RAG1 were downloaded from the Protein Data Bank at Brookhaven National Laboratory (accession numbers: 1FBV and 1RMD, respectively).

Although the overall three-dimensional structures of the RING finger domains of RAG1 and c-Cbl are similar, their molecular functions are different. To obtain information on the molecular function of the RING-H2 finger domain of EL5, we compared the RING finger domains of the three proteins at the atomic level. The RAG1 RING finger domain has one additional alpha -helix each at its N- and C terminus (Fig. 3a). The RAG1 dimer interface is stabilized by an extensive hydrophobic core containing two clusters of three Phe residues in these additional alpha -helices (14). In accordance with the observation that c-Cbl is a monomer, its RING finger domain lacks the dimer-stabilizing N- and C-terminal alpha -helices. As the EL5 RING-H2 finger domain resembles c-Cbl in this respect, it is not expected to form dimers. The c-Cbl RING finger domain binds E2 along a hydrophobic groove (indicated by arrows in Fig. 3b) that is formed by the alpha -helix of the beta beta alpha structure and the two zinc-chelating N- and C- terminal loops (Ref. 15, compare electron potential map in Fig. 3b). On the contrary, the RING finger domain of RAG1, which is a DNA-binding protein without ubiquitin ligase (E3) activity (32), does not possess this groove. Its N- and C-terminal loops are closer together with the remaining space occupied by the side chain of Arg57 (Fig. 3b). Thus, the groove that enables the hydrophobic interaction between c-Cbl and E2 is occupied by a basic group (Arg57) in RAG1, preventing interactions between RAG1 and E2. The residues in c-Cbl that form the hydrophobic contact with E2 (Ile383, Cys404, Ser407, Trp408, Ser411, and Pro417) (15) are mostly conserved in the RING-H2 finger domain of EL5 (Val136, Cys161, Trp165, Ser168, and Pro173) as shown in Fig. 3b. The spatial arrangements of these hydrophobic residues is quite similar in the two molecules (Fig. 3b), suggesting that EL5 should bind E2 similarly as c-Cbl does.

The EL5 RING-H2 Finger Domain Catalyzes Auto-ubiquitination in Vitro-- Takai et al. (6) showed that the fusion protein of EL5(96-325) and maltose-binding protein (MBP-EL5(96-325)) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and UbcH5a or OsUBC5a/b, a rice E2. Thus, the EL5 domains III, IV, and the C-terminal region are sufficient to catalyze ubiquitin transfer to the MBP moiety in cooperation with E2. It was also demonstrated that replacement of Cys153 by Ser abolished the E3 activity. Our structural analysis suggested that the RING-H2 finger domain of EL5 binds E2. We expressed the EL5 RING-H2 finger domain as a fusion protein with MBP (MBP-EL5(96-181) and MBP-EL5(129-181), respectively) and determined its E3 activity. When MBP-EL5(129-181) was incubated with ubiquitin, E1 and OsUBC5b in an in vitro ubiquitination assay, only one ubiquitinated derivative of the fusion protein was detected. When MBP-EL5(96-181) was used in the same experiment, several ubiquitinated derivatives of the fusion protein were observed (Fig. 4, lanes 3 and 4). The latter result was similar to that obtained with MBP-EL5(96-325) (Fig. 4, lanes 1 and 2). These findings showed that only the RING-H2 finger domain was sufficient to bind E2, but that the polyubiqutination chain reaction was disturbed in the small construct, MBP-EL5(129-181), probably because the MBP moiety sterically hindered the development of a polyubiquitin chain. Therefore we decided to carry out the in vitro ubiquitination assay using MBP-EL5(96-181). Since Zn2+ is needed for the correct folding of the EL5 RING finger domain, we tested its effect in the ubiquitination assay. No ubiquitination was detectable in the presence of EDTA (Fig. 4, lanes 5 and 6), but the activity was restored by addition of excess ZnSO4 (Fig. 4, lanes 7 and 8). Thus, Zn2+ facilitates effective EL5-mediated ubiquitination by structurally stabilizing the EL5 RING-H2 finger domain.


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Fig. 4.   EL5 RING-H2 finger domain-catalyzed auto-ubiquitination in vitro. MBP-EL5(96-325) (lanes 1 and 2), MBP-EL5(96-181) (lanes 3 and 4) and MBP (lanes 9 and 10) were incubated at 35 °C with ATP, ubiquitin, E1, and OsUBC5b (E2) for 1 h, and were then subjected to SDS-PAGE followed by immunoblotting with an anti-MBP antibody. MBP-EL5(96-181) was incubated with EDTA before starting the assay (lanes 5-8), in the absence (lanes 5 and 6) or in the presence of an excess amount of ZnSO4 (lanes 7 and 8). Ladders of bands at higher molecular weights (lanes 2, 4, and 8) indicate the occurrence of ubiquitination.

Identification of Residues in the EL5 RING-H2 Finger Domain That Interact with OsUBC5b-- To identify EL5 residues that interact with E2, we recorded amide 15N and 1H chemical shifts of the EL5 RING-H2 finger domain as a function of the concentration of OsUBC5b. Residues located close to OsUBC5b in the E3/E2 complex were anticipated to exhibit large changes in their chemical shifts, because the shift depends on the residue's magnetic environment. The HSQC signal of EL5(96-181) had indicated that the conserved domain remained unchanged (i.e. unstructured) in the presence of OsUBC5b, implying that it was not involved in the EL5/OsUBC5b interaction. Therefore we used EL5(129-181) in the NMR titration experiments.

In these experiments, the extreme broadening of the signals observed in several residues indicated intermediate exchange on the NMR time scale. From the titration curve that was plotted on the basis of the chemical shift variations observed in the RING-H2 finger domain, the stoichiometry between RING-H2 finger domain and OsUBC5b (1:1) and the dissociation constant (Kd ~1 × 10-5 M) were calculated. Seven residues (Val136, Cys137, Ala147, Arg148, Glu160, Thr171, and Leu174) displayed significant chemical shift perturbations upon complex formation with OsUBC5b (Fig. 5a). The amide signals of 5 residues (Leu138, Val162, Asp163, Met164, and Trp165) were not detectable due to extreme broadening of the signal. We ascribe this phenomenon to an exchange between the free and bound forms on the chemical shift time scale. The residues with the chemical shifts most sensitive (chemical shift perturbations, Delta delta   =  Delta delta 1HN   + 0.1 Delta delta 15N   greater than 0.15 ppm) to complex formation were mapped on the free form structure of the EL5 RING-H2 finger domain (Fig. 5b). These residues are all localized on one side of the molecule where they form the E2 binding surface. The location of this binding site is in good agreement with that of the binding site of the c-Cbl RING finger domain for E2 (15).


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Fig. 5.   a, NMR chemical shift perturbation of the EL5 RING-H2 finger domain upon binding to OsUBC5b. Changes in the NMR chemical shifts of RING-H2 finger domain (Delta delta ) as induced by complex formation with OsUBC5b, were calculated by the function  Delta delta   =  Delta delta HN   (pink) + 0.10  Delta delta 15N   (magenta). The light blue bars indicate resonances broadened beyond recognition. b, two views of the surface of the EL5 RING-H2 finger domain. Residues that showed highly sensitive backbone amide chemical shift (Delta delta  > 0.15 ppm) are colored magenta. Residues marked in red were not detectable due to extreme broadening of the signal after binding of OsUBC5b. The left view is in the same orientation as the models in Fig. 3, while the right view is from the opposite side.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intense interest in RING finger proteins has arisen because of their role in human disease and their widespread occurrence. In higher plants, numerous genes for RING finger proteins have been identified. For example, 387 RING finger proteins have been predicted from a search of the Arabidopsis genome data base (33). Although RING finger proteins play important roles in plants, there is no information on their structure and/or the relationship between the structure and function.

The EL5 RING-H2 Finger Domain Interacts with UbcH4/5a-Type Ubiquitin-conjugating Enzymes-- Our structural-based analysis suggested that the RING-H2 finger domain of EL5 could bind E2. NMR titration experiments and an in vitro ubiquitination assay showed that the RING-H2 finger domain of EL5 could bind the E2, OsUBC5b, along a groove formed by a cluster of hydrophobic residues. The primary sequence of the EL5 RING-H2 finger domain shows high similarity with other RING finger domains that interact with UbcH4/5a (Fig. 6a). In fact, the fusion protein of EL5 with MBP was polyubiquitinated by incubation with E1 and UbcH5a. OsUBC5b, the E2 used in the present study, is highly similar to UbcH4/5a (6). Thus, available evidence suggests that the EL5 RING-H2 finger domain belongs to the group of UbcH4/5a-type E2 binding domains. Consequently, EL5 is a UbcH4/5a-type E2 binding E3.


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Fig. 6.   a, alignment of amino acid sequences of RING finger domains. The Cys and His residues responsible for zinc binding are marked yellow. Residues that are conserved or conservatively exchanged with respect to the EL5 RING-H2 finger domain are marked blue and light blue, respectively. Asterisks indicate residues that are crucial for the binding of E2 by EL5, c-Cbl and CNOT4, respectively. b, EL5 RING finger domain. Residues that exhibited a high sensitive amide chemical shift in NMR titration experiments are highlighted. Residues that are well and poorly conserved between E2 binding RING finger domains (Group 1, 2, and 3) are shown in magenta and gray, respectively. Pro173, which cannot be detected in a 15N HSQC spectrum, is shown in yellow. c, c-Cbl RING finger domain. Residues that interact with UbcH7 are colored magenta (14). d, CNOT4 RING finger domain. Residues that showed a high sensitive amide chemical shift in NMR titration experiment, and those that are critical for UbcH5b interaction are colored magenta (39).

Identification of Residues in the RING Finger Domain That Are Critical for E2 Interaction-- The primary sequences of various RING finger domains are compared in Fig. 6a. Four groups can be distinguished on the basis of RING finger domain interaction with other proteins. The first group consists of ubiquitin ligases that cooperate with UbcH4/5a, but not with UbcH7/8; it includes EL5 and AO7 (34). Members of the second group, including HHARI (35), interact with UbcH7/8, but not with UbcH1 and UbcH5. c-Cbl forms a group on its own; it is E3-interacting both with UbcH7 (36) and UbcH4 (37). The fourth group includes KAP-1 (38) and RAG1 (14). Its members appear to function in the formation of macromolecular assemblages.

The result of the EL5-E2 NMR titration experiment detects an altered chemical environment for amide groups on EL5. The observed chemical shift changes reveal direct contacts as well as dynamic conformational changes at a particular position or its close proximity. Our NMR titration experiments also shed some light on the structural basis of the functional classification of the four groups of RING finger domains. Seven residues of the EL5 RING-H2 finger domain (Val136, Cys137, Ala147, Arg148, Glu160, Thr171, Leu174) displayed significant perturbations of chemical shift. Five residues (Leu138, Val162, Asp163, Met164, Trp165) were undetectable in EL5/OsUBC5b complex (Fig. 5). Among these twelve residues (highlighted in Fig. 6b), six (Val136, Cys137, Leu138, Val162, Trp165, and Leu174; magenta in Fig. 6b) are well conserved in E2 binding RING finger domains (Group 1, 2, and 3; Fig. 6a). All of these residues are hydrophobic and may be the most important ones for hydrophobic interaction with E2. In fact, the spatial position of these residues is almost identical in c-Cbl and CNOT4 (Fig. 6, b-d). Interestingly, the pair of hydrophobic and Trp residues located at the first and fourth position after the third pair of zinc chelating residues (positions 162 and 165 in EL5) is conserved in almost all RING finger domains that are known or expected to act as E3. Only in CNOT4 and HHARI, the position of the two residues are interchanged as shown Fig. 6d. This suggests that this pair of hydrophobic and Trp residues may be important for E2 binding. The residues map to identical positions on the alpha -helix of the E2 binding RING finger domains and form the center of the hydrophobic groove together with the hydrophobic residue (Val136 for EL5, Ile383 for c-Cbl and Leu16 for CNOT4) of loop 1. A detailed comparison of the free EL5 RING finger domain with the E2-bound form of c-Cbl suggested that the side-chain conformations (chi 1) of the Trp residues in both molecules are different (Fig. 3). This difference of conformations may not reflect a principle difference between the structures of EL5 and c-Cbl, but may be caused by E2 binding. In fact, the Trp at this position is required for E3 activity, because its replacement by Ala reduced E3 activity in c-Cbl (36). Moreover, E2 binding was completely abolished in a CNOT4 mutant in which Ile45 was replaced by Ala (13).

In this study, we determined the three-dimensional structure of the RING-H2 finger domain of EL5, and found that it closely resembles other RING finger domains. The surface and charge distribution of the putative E2 binding site, a groove formed by N-loop, C-loop, and alpha -helix, is essentially identical to that of the RING finger domain of c-Cbl, a UbcH7-binding protein. We demonstrated that the EL5 RING-H2 finger domain is crucial for E3 activity. Moreover, we identified key residues for EL5 interaction with a rice E2, OsUBC5b. We clarified for the first time the functional and structural basis of E2 binding by RING finger domain in plants. It is hoped that these insights will facilitate the understanding of E2 recognition by ATL family RING finger proteins, which are widespread in higher plants.

PDB and BMRB Accession Code-- The coordinates of the final structures and the structural constraints used for the calculations have been deposited in the RCSB Protein Data Bank (accession code 1IYM). The chemical shift values of the 1H, 13C, and 15N resonances have been deposited in the BioMagResBank data base (accession number 5459).

    ACKNOWLEDGEMENTS

The purified recombinant mouse E1 was kindly provided by Dr. Keiji Tanaka and Dr. Toshiaki Suzuki of the Tokyo Metropolitan Institute of Medical Science.

    FOOTNOTES

* This work was supported in part by a grant from the Rice Genome Project PR-2201, MAFF, Japan (to E. K.) and by a grant from the Bio-oriented Technology Research Advancement Institution, Japan (to T. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1IYM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The chemical shift values of the 1H, 13C, and 15N resonances have been deposited in the BioMagResBank data base (accession number 5459).

To whom correspondence should be addressed. Tel. and Fax: 81-298-38-7006; E-mail: ekatoh@nias.affrc.go.jp.

Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M210531200

    ABBREVIATIONS

The abbreviations used are: MBP, maltose-binding protein; r.m.s.d., root mean square deviation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Boller, T. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 189-214[CrossRef]
2. Shibuya, N., and Minami, E. (2001) Phys. Mol. Plant Path. 59, 223-233[CrossRef]
3. Takai, R., Hasegawa, K., Kaku, H., Shibuya, N., and Minami, E. (2001) Plant Sci. 160, 577-583[CrossRef][Medline] [Order article via Infotrieve]
4. Martinez-Garcia, M., Garciduenas-Pina, C., and Guzman, P. (1996) Mol. Gen. Genet. 252, 587-596[CrossRef][Medline] [Order article via Infotrieve]
5. Salinas-Mondragon, R. E., Garciduenas-Pina, C. G., and Guzman, P. (1999) Plant. Mol. Biol. 40, 579-590[CrossRef][Medline] [Order article via Infotrieve]
6. Takai, R., Matsuda, N., Nakano, A., Hasegawa, K., Akimoto, C., Shibuya, N., and Minami, E. (2002) Plant J. 30, 447-455[CrossRef][Medline] [Order article via Infotrieve]
7. Berg, J. M., and Shi, Y. (1996) Science 271, 1081-1085[Abstract]
8. Borden, K. L. B., and Freemont, P. S. (1996) Curr. Opin. Struct. Biol. 6, 395-401[CrossRef][Medline] [Order article via Infotrieve]
9. Saurin, A. J., Borden, K. L., Boddy, M. N., and Freemond, P. S. (1996) Trends Biochem. Sci. 21, 208-214[CrossRef][Medline] [Order article via Infotrieve]
10. Borden, K. L. B. (2000) J. Mol. Biol. 295, 1103-1112[CrossRef][Medline] [Order article via Infotrieve]
11. Pickart, C. M. (2001) Annu. Rev. Biochem. 70, 503-533[CrossRef][Medline] [Order article via Infotrieve]
12. Borden, K. L. B. (1998) Biochem. Cell Biol. 76, 351-358[CrossRef][Medline] [Order article via Infotrieve]
13. Hanzawa, H., Ruwe, M. J., Albert, T. K., Vliet, P. C., Timmers, H. T. M., and Boelens, R. (2001) J. Biol. Chem. 276, 10185-10190[Abstract/Free Full Text]
14. Bellon, S. F., Rodgers, K. K., Schatz, D. G., Coleman, J. E., and Steitz, T. A. (1997) Nat. Struct. Biol. 4, 586-591[Medline] [Order article via Infotrieve]
15. Zheng, N., Wang, P., Jeffrey, P. D., and Pavletich, N. P. (2000) Cell 102, 533-539[Medline] [Order article via Infotrieve]
16. Bax, A., and Grzesiek, S. (1993) Acc. Chem. Res. 26, 131-138
17. Clore, G. M., and Gronenborn, A. M. (1991) Science 252, 1390-1399[Medline] [Order article via Infotrieve]
18. Clore, G. M., and Gronenborn, A. M. (1998) Trends Biotech. 16, 22-34[CrossRef][Medline] [Order article via Infotrieve]
19. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biol. NMR 6, 277-293
20. Garrett, D. S., Powers, R., Gronenborn, A. M., and Clore, G. M. (1991) J. Magn. Reson. 95, 214-220
21. Bax, A., and Subramanian, S. (1986) J. Magn. Reson. 67, 565-569
22. Live, D. H., Davis, D. G., Agosta, W. C., and Cowburn, D. (1984) J. Am. Chem. Soc. 106, 1934-1941
23. Wüthrich, K., Billetter, M., and Braun, W. (1983) J. Mol. Biol. 169, 949-961[Medline] [Order article via Infotrieve]
24. Vuister, G. W., and Bax, A. (1993) J. Am. Chem. Soc. 115, 7772-7777
25. Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biol. NMR 13, 289-302
26. Nilges, M., Gronenborn, A. M., Brunger, A. T., and Clore, G. M. (1988) Protein Eng. 2, 27-38[Abstract]
27. Brünger, A. T. (1993) X-PLOR Manual Version 3.1 , Yale University, New Haven, CT
28. Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 51-55[CrossRef][Medline] [Order article via Infotrieve]
29. Archer, S. J., Ikura, M., Torchia, D. A., and Bax, A. (1991) J. Magn. Reson. 95, 636-641
30. Vuister, G. W., Wang, A. C., and Bax, A (1993) J. Am. Chem. Soc. 115, 5334-5335
31. Spera, S., and Bax, A. (1991) J. Am. Chem. Soc. 111, 8317-8318
32. Silver, D. E., Sponopulou, E., Mulligan, R. C., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. 90, 6100-6104[Abstract]
33. Kosarev, P., Mayer, K. FX., and Hardtke, C. S. (2002) Genome Biol. 3, 0016.1-0016.12
34. Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S., and Weissman, A. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11364-11369[Abstract/Free Full Text]
35. Moynihan, T. P., Ardley, H. C., Nuber, U., Rose, S. A., Jones, P. F., Markham, A. F., Scheffner, M., and Robinson, P. A. (1999) J. Biol. Chem. 274, 30963-30968[Abstract/Free Full Text]
36. Yokouchi, M., Kondo, T., Sanjay, A., Houghton, A., Yoshimura, A., Komiya, S., Zhang, H., and Baron, R. (2001) J. Biol. Chem. 276, 35185-35193[Abstract/Free Full Text]
37. Joazeiro, C. A. P., Wing, S. S., Huang, H., Leverson, J. D., Hunter, T., and Liu, Y. C. (1999) Science 286, 309-312[Abstract/Free Full Text]
38. Peng, H., Begg, G. E., Schultz, D. C., Friedman, J. R., Jensen, D. E., Speicher, D. W., and Rauscher III, F. J. (2000) J. Mol. Biol. 295, 1139-1162[CrossRef][Medline] [Order article via Infotrieve]
39. Albert, T. K., Hanzawa, H., Legtenberg, Y. I. A., Ruwe, M. J., Heuvel, F. A. J., Collart, M. A., Boelens, R., and Timmers, H. T. M. (2002) EMBO J. 21, 355-364[Abstract/Free Full Text]
40. Laskowski, R. A., Rullmann, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, 477-486[Medline] [Order article via Infotrieve]


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