The Structure of the C4C4 RING Finger of Human NOT4 Reveals Features Distinct from Those of C3HC4 RING Fingers*

Hiroyuki HanzawaDagger §, Marjolein J. de Ruwe||**, Thomas K. Albert||DaggerDagger, Peter C. van der Vliet||, H. T. Marc Timmers||§§, and Rolf BoelensDagger ¶¶

From the Dagger  Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, the § Biomedical Research Laboratories, Sankyo, Limited, 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140, Japan, and the || Laboratory for Physiological Chemistry and the Centre for Biomedical Genetics, University Medical Center Utrecht, 3508 AB Utrecht, The Netherlands

Received for publication, October 11, 2000, and in revised form, November 17, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The NOT4 protein is a component of the CCR4·NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C4C4 type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a 113Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 ± 0.13 Å for the backbone atoms and 1.08 ± 0.12 Å for heavy atoms. The hNOT4 RING finger consists of an alpha -helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C3HC4 RING fingers. The relative orientation of the two zinc-chelating loops and the alpha -helix is well conserved. However, for the other regions, the secondary structural elements are distinct.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The CCR4·NOT complex was first detected in Saccharomyces cerevisiae as a global transcription regulator, affecting transcription of multiple functionally unrelated genes positively as well as negatively (1). The complex consists of CCR4 (carbon catabolite repressor 4), CAF1 (CCR4-associated factor 1, also known as POP2), the five NOT proteins (NOT1-5), and several unidentified proteins (1). The yeast NOT genes have been identified in a screen for elevated HIS3 expression (2-4). The HIS3 gene contains two core promoters, TC, a TATA-less element, and TR, a canonical TATA sequence (5, 6). Mutations in NOT genes selectively elevate transcription from TC (2-4). Besides repressing genes involved in histidine biosynthesis (HIS3 and HIS4), NOT proteins also affect transcription of genes involved in pheromone response (STE4), nuclear fusion (BIK1), and RNA polymerase II transcription (TBP) (2, 3). The CCR4 gene product regulates expression of ADH2 and other genes involved in nonfermentative growth, cell wall integrity, and ion sensitivity (7-9). CCR4 exists in a complex with other proteins (10), and two-hybrid screening with CCR4 identified CAF1 (11, 12) and DBF2 (a cell cycle-regulated kinase) (9, 13) as binding partners. Recently, it was found that CCR4 and CAF1 reside with the NOT proteins in a 1.2-MDa complex (1). Besides physical interactions between CCR4, CAF1, and NOT proteins, there is also a functional association. Mutations in the NOT, CCR4, and CAF1 genes lead to similar, but not identical, phenotypes (1, 14). Interestingly, mutations in NOT1, NOT3, NOT5, and CAF1 genes suppressed a mutation in SRB4, which is an essential component of the RNA polymerase II holoenzyme and required for the expression of most protein-coding genes. This suggests that the yeast CCR4·NOT complex has a very general role in RNA polymerase II transcription (15).

Recently, the human counterpart of the yeast CCR4·NOT complex has been identified (16). cDNAs for four subunits, hNOT2,1 hNOT3, hNOT4, and human CALIF (CAF1-like factor), were isolated and characterized. Like yeast NOT4, hNOT4 interacts with yeast NOT1 and an N-terminally truncated hNOT1 protein, and hNOT4 complements a not4-null mutation in yeast (16). Human NOT4 contains two protein motifs in its N-terminal region, a RING finger and an RNA recognition motif (16). The N-terminal part of the protein is evolutionarily conserved, in contrast to the C-terminal part (16). The RNA recognition motif has been implicated in binding of single-stranded nucleic acids (reviewed in Ref. 17). The RING finger is found in a large number of proteins in animals, plants, and viruses involved in distinct cellular functions (reviewed in Refs. 18 and 19). RING fingers are thought to mediate protein-protein interactions, and RING finger proteins are often found in large multiprotein complexes (reviewed in Refs. 18-20). Recently, an increasing amount of data showed that several RING finger-containing proteins function as E3 ubiquitin ligases, which target proteins for degradation (reviewed in Ref. 21). Examples include the proto-oncogene product c-Cbl (22), which ubiquitinates receptor protein-tyrosine kinases and the SCF complex, containing the RING finger Rbx1 protein, which targets several proteins, including G1 cyclins for degradation (23, 24). Possibly, NOT4 also functions as an E3 ligase.

The RING finger motif can be 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 using its cysteine and histidine residues (reviewed in Refs. 18 and 19). By primary sequence comparison, RING finger variants have been identified in which the zinc-coordinating ligands have been replaced with other residues (25, 26). The structure of three C3HC4 RING fingers has been solved to date (reviewed in Ref. 27). The solution structures of the immediate-early EHV-1 protein from equine herpesvirus (IEEHV) and the acute promyelocytic leukemia proto-oncogene product (PML) have been solved by NMR methods (28, 29), and the crystal structures of the immunoglobulin gene recombination enzyme RAG1 (30) and the c-Cbl RING finger bound to ubiquitin-conjugating enzyme UbcH7 (31) have been solved by x-ray diffraction. The RING finger structures of RAG1 and IEEHV are remarkably similar, but differ considerably from the PML RING finger structure. Despite this, all C3HC4 structures possess some common features, the most obvious being the coordination of the two zinc atoms in a cross-brace configuration. In this system, Cys1, Cys2, Cys5, and Cys6 coordinate the first zinc atom, and Cys3, His1, Cys7, and Cys8 share the second zinc atom. The inter-zinc distance in all three structures is 14 Å.

The consensus sequence for the RING finger of NOT4 orthologs is Cys-X2-Cys-X13-Cys-X-Cys-X4-Cys-X2-Cys-X11-16-Cys-X2-Cys. It constitutes a novel RING finger variant of a C4C4 type in which His1 is replaced with cysteine. Also, the spacing between the fourth and fifth metal-coordinating residues is different. To investigate whether this motif in NOT4 can adopt a RING finger conformation, we determined its structure by NMR methods. We found that the overall fold of the NOT4 RING finger resembles that of the C3HC4 RING fingers. However, important differences especially in the secondary structure elements are notable.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Plasmids-- pET15b-hNOT4-N78 encodes the first 78 amino acids of hNOT4 N-terminally fused to a 23-residue His6 tag. The hNOT4-N78 insert was obtained by polymerase chain reaction using pET15b-hNOT4-N227 as a template, a T7 primer (5'-TAATACGACTCACTATAGGG-3'), and a hNOT4-specific primer (5'-GCGGGATCCTATATCCTTTGCAGCTCTTCCTG-3'). The resulting polymerase chain reaction fragment was digested with XhoI and BamHI and ligated into pET15b (Novagen) digested with the same restriction enzymes. The DNA sequence of this fragment was verified by sequence analysis using an automated ABI310 sequencer (PerkinElmer Life Sciences).

Purification of hNOT4-N78-- Escherichia coli BL21(DE3) bacteria containing the pET15b-hNOT4-N78 plasmid were grown in LB medium containing 0.4% glucose at 37 °C and induced at A600 = 0.75 with 1 mM isopropyl-beta -D-thiogalactopyranoside and 10 µM ZnCl2 for 2 h at 37 °C. After harvesting, cells were washed with phosphate-buffered saline and suspended in lysis buffer (100 mM Tris-HCl (pH 7.9), 20% sucrose, 0.1 mM EDTA, 100 µM ZnCl2, 1 mM imidazole, 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM beta -mercaptoethanol, 0.2 mM sodium bisulfite, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Lysozyme was added to a final concentration of 200 µg/ml, followed by one freeze-thaw cycle to lyse the cells. After addition of KCl to 300 mM, the lysate was centrifuged for 1 h at 250,000 × g. The cleared lysate was applied to a Ni2+-nitrilotriacetic acid-agarose column (QIAGEN Inc.) equilibrated in lysis buffer containing 300 mM KCl. After washing the column with buffer A (20 mM Tris-HCl (pH 7.9), 10% glycerol, 50 mM KCl, 20 µM ZnCl2, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM beta -mercaptoethanol, 0.2 mM sodium bisulfite, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) containing 1 and 16 mM imidazole, protein was eluted in buffer A using a linear gradient from 16 to 400 mM imidazole. Peak fractions were pooled and dialyzed against buffer B (20 mM potassium phosphate (pH 7.0), 10 µM ZnCl2, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.2 mM sodium bisulfite, 0.1 µg/ml aprotinin, and 0.1 µg/ml leupeptin) containing 100 mM KCl (buffer B100) and applied to a MonoQ HR 10/10 column (Amersham Pharmacia Biotech) equilibrated in buffer B100. After washing with buffer B100, the column was developed using a linear gradient from 100 to 500 mM KCl. hNOT4-N78 eluted from the column at ~170 mM KCl and was purified to homogeneity as judged by Coomassie Blue staining of protein gels. hNOT4-N78 was concentrated using a Centricon spin dialysis tube (10-kDa cutoff; Amicon, Inc.).

To overexpress 15N-labeled, 15N/13C-labeled, and 15N/10% 13C-labeled hNOT4-N78, bacteria were grown in synthetic medium (6.0 g/liter Na2HPO4·2H2O, 3.0 g/liter KH2PO4, 0.5 g/liter NaCl, 1 mM MgSO4, 20 µM CaCl2, 36 nM FeSO4·7H2O, 20 µM ZnCl2, and 5 mg/liter thiamine) containing 0.5 g/liter 15NH4Cl as the sole nitrogen source and either 4.0 g/liter [12C]glucose or 2.5 g/liter [13C]glucose or a mixture of 2.25 g/liter [12C]glucose and 0.25 g/liter [13C]glucose as the only carbon source. Induction took place at A600 = 0.7-0.75, and proteins were purified as described above.

Protein concentrations were determined using Bio-Rad protein assays employing bovine gamma -globulin as a standard. Protein yields ranged between 10 and 15 mg/liter of bacterial culture.

NMR Measurements-- NMR experiments were carried out at 300 K and pH 7.0 on a Bruker DRX600 apparatus equipped with a triple-resonance z-gradient probe, unless indicated otherwise. For the backbone resonance assignments, three-dimensional HNCO, three-dimensional CBCA(CO)NH, three-dimensional HNHACB, three-dimensional NOESY-(15N,1H)-HSQC, and three-dimensional TOCSY-(15N,1H)-HSQC spectra were recorded; and for side chain resonance assignments, three-dimensional H(C)CH TOCSY and three-dimensional (H)CCH TOCSY spectra were recorded (reviewed in Ref. 32). For the assignments of proline residues, a CDCA(NCO)CAHA spectrum was recorded (33).

Stereospecific assignments of beta  protons and chi 1 dihedral angle restraints were obtained using three-dimensional NOESY-(13C,1H)-HSQC and TOCSY-(15N,1H)-HSQC spectra (reviewed in Ref. 32) recorded with short mixing times (50 ms and 20 ms, respectively) and a three-dimensional HNHB spectrum (34). Stereospecific assignments for the methyl protons in the prochiral center of leucine and valine were obtained by 10% 13C-labeled hNOT4-N78 (35).

3JHN-Halpha coupling constants were derived from a three-dimensional HNHA experiment (36) and converted into phi dihedral angle restraints. Hydrogen bond restraints were obtained by observing the cross-hydrogen bond 15N-13C coupling constants using a long-range HNCO spectrum (37).

113Cd-1H HSQC spectra were recorded using a broadband z-gradient probe. The delays for magnetization transfer were set to 6, 9, and 12 ms. Twenty t1 increments of 1000 scans were recorded. The 113Cd chemical shifts are reported relative to 1 M CdSO4. NOE distance restraints were obtained from two-dimensional NOE, three-dimensional NOESY-(15N,1H)-HSQC, and three-dimensional NOESY-(13C,1H)-HSQC spectra with mixing times of 50 and 150 ms. NOE cross-peaks were classified as strong (1.8-2.7 Å), medium (1.8-3.5 Å), weak (1.8-5.0 Å), and very weak (1.8-6.0 Å) using secondary structure elements for calibration. All NMR spectra were processed using the NMRpipe package (38) and analyzed using the program REGINE (39).

Structure Calculations-- Structure calculations were performed with the program X-PLOR (40, 41). To correct for multiple atom selection, we used the sum averaging option as implemented in X-PLOR. Distance restraints containing diastereotopic groups were corrected as described by Fletcher et al. (42).

The structures were calculated using the distance geometry protocol, which is followed by a simulated annealing and refinement protocol using standard parameters. Until this stage, information about the zinc coordination was not included. Then, the constraints between metal ligands (3.6 Å < Sgamma (Cys)-Sgamma (Cys) < 3.9 Å) were added to the restraints list, and an additional refinement protocol was performed. The zinc atoms were added at the average position of four metal-ligating atoms. Thereafter, the geometric restraints to the zinc atoms were added, and additional refinement was performed. The geometric restraints are as follows: bond lengths for Zn-Sgamma (Cys) are 2.3 Å and geminal bond angles of Sgamma (Cys)-Zn-Sgamma (Cys) and Zn-Sgamma (Cys)-Cbeta (Cys) are 109.5° (43).

The final set of structures was analyzed using the program PROCHECK-NMR (44). The Ramachandran plot of Fig. 4 was produced with the program PROCHECK-NMR (44). Molecular figures were prepared using the program MOLMOL (45).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Expression and Assignments-- The first 78 residues of hNOT4, which contain the C4C4 RING finger, were fused to an N-terminal His6 tag. This protein was overexpressed and 15N- and 15N/13C-isotopically labeled in bacteria and purified to homogeneity as described under "Materials and Methods." The sequence of the first 78 residues of hNOT4 is shown in Fig. 1 and is compared with the corresponding region in yeast NOT4 and with the C3HC4 RING fingers of IEEHV and RAG1.


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Fig. 1.   Comparison of the first 78 residues of hNOT4, encompassing the C4C4 RING finger, with the C4C4 RING finger of yeast NOT4 (residues 20-99) and with the C3HC4 RING fingers of IEEHV (residues 1-68), RAG1 (residues 277-350), and c-Cbl (residues 368-441). Zinc-coordinating residues are indicated with asterisks. Sequence alignment was performed using the ClustalW algorithm (46). yNOT4, yeast NOT4.

Sequence-specific assignments were obtained by the analysis of three-dimensional CBCA(CO)NH and three-dimensional HNCACB spectra and by the application of a three-dimensional CDCA(NCO)CAHA spectrum (33) for 11 proline residues. The assignments for 10 residues of the 23-residue His6-tagged region were also obtained. 1H-15N correlations for the remaining residues in the His6-tagged region were not observed, and the resonances for those residues were therefore left unassigned. Stereospecific assignments for methyl protons in the prochiral center of Val12, Leu16, Leu21, and Leu52 were obtained. In addition to this, 14 out of 64 beta -methylene protons were also obtained.

Metal-binding Sites-- To determine the number of metal ions present in the hNOT4 RING finger and the coordination system, we performed 113Cd-1H HSQC experiments using hNOT4-N78 in which the zinc ions were replaced with cadmium ions. 113Cd-substituted hNOT4-N78 was obtained by adding 113Cd- EDTA to a final concentration of 4 mM to zinc-containing hNOT4-N78, and the exchange of zinc with 113Cd was monitored using 1H-15N HSQC spectra.

Since most of the resonances were shifted by the substitution, the assignments of 1H and 15N resonances of 113Cd-hNOT4-N78 had to be confirmed by three-dimensional NOESY-(1H,15N)-HSQC and three-dimensional TOCSY-(1H,15N)-HSQC spectra. Fig. 2A shows the chemical shift differences between 113Cd-hNOT4-N78 and Zn-hNOT4-N78. Cys17, Cys33, and Cys56 displayed the largest differences in both the 1H and 15N chemical shifts, which is correlated with hydrogen bonding from amide protons to the sulfur atoms of the zinc cluster (see below). Residues other than cysteines displayed only a slight change in chemical shifts, showing that exchange took place without breaking the integrity of the whole structure. Fig. 2B shows the two-dimensional 113Cd-1H HSQC spectrum of 113Cd-hNOT4-N78 displaying the correlation between two cadmium resonances and the 1H chemical shifts belonging to the beta  protons of the coordinating cysteine residues. The observed chemical shift values of cadmium resonances, 687.5 and 714.4 ppm, are both typical for cadmium(II) coordinated by four sulfur ligands. The 113Cd-1H HSQC spectrum shows that Cys14, Cys17, Cys38, and Cys41 (site 1) share one metal ion that resonates at 687.5 ppm and that Cys33, Cys53, and Cys56 (site 2) share the other metal ion at 714.4 ppm. The correlation of the remaining Cys31 was not observed in any of the 113Cd-1H HSQC spectra with magnetization delays of 6, 9, and 12 ms. However, the last metal-coordinating ligand of site 2 was assigned to Cys31 since the resonance value for this metal is characteristic for cadmium(II) coordinated by four sulfur atoms. These results show that the NOT4 RING finger contains two zinc ions, which are ligated in a cross-brace manner, similar to the canonical RING fingers.


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Fig. 2.   A, chemical shift differences of the amide protons and nitrogens between the zinc and cadmium forms of hNOT4-N78. The values of Delta delta were calculated as follows: Delta delta  = delta (Zn form) - delta (Cd form). B, 113Cd-1H HSQC spectrum of cadmium-substituted hNOT4-N78 showing the three-bond J-coupling between 113Cd and beta  protons of the cysteine residues.

Structure of hNOT4-N78-- The three-dimensional structure was determined for the zinc-containing form of hNOT4-N78. In total, 397 distance restraints (20 intraresidue, 171 sequential, 74 medium-range, and 132 long-range) and 32 angle constraints (19 phi  and 13 chi 1) obtained from various two- and three-dimensional spectra were used for the structure calculations. Four hydrogen bond restraints, which were identified in a long-range HNCO spectrum, were also included. Finally, 200 structures were calculated, and 30 structures with a low energy were selected. Fig. 3A shows the superposition of the backbone atoms of these 30 calculated structures, and a summary of structural statistics is given in Table I and Fig. 3 (B-D). The region between residues 12 and 61 is well structured, whereas the N- and C-terminal parts are disordered due to lack of NOEs. Root mean square deviation values in the structured region versus the mean coordinates are 0.58 ± 0.13 Å for the backbone atoms and 1.08 ± 0.12 Å for all heavy atoms.


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Fig. 3.   A, superposition of the backbone atoms of 30 simulated annealing structures of hNOT4-N78. The three loop regions (residues 12-22, 27-38, and 49-61) are colored green; the region containing the alpha -helix (residues 39-48) is in red; residues 23-26 containing the helical turn are in orange; and the remaining unstructured region is in white. Zinc atoms are indicated as magenta balls. B-D, overview of the structural parameters of residues 10-65 of hNOT4-N78. B, number of distance restraints per residue; C and D, root mean square deviation (RMSD) for backbone atoms and all heavy atoms versus the residue number, respectively.

                              
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Table I
Structural statistics of hNOT4-N78

The structure of hNOT4-N78 consists of three long loops, L1 (residues 12-22), L2 (residues 27-38), and L3 (residues 49-61), and an alpha -helix (residues 39-48) between the second and third loops. Of the 8 cysteine residues that are involved in zinc coordination, the first (Cys14, Cys17), second (Cys31, Cys33), and forth (Cys53, Cys56) pairs are located in L1, L2, and L3, respectively. The remaining 2 cysteines (Cys38, Cys41) are located at the end of L2 and in the alpha -helix. Region 23-26 is recognized as a helical turn in 15 out of 30 calculated structures using secondary structure analysis in PROCHECK-NMR. All proline residues were found to have a trans-configuration on the basis of the observation of NOEs between the alpha i-1 and delta i protons. The three loops are stabilized by the coordination with the zinc ions and by hydrophobic interactions. Leu16 in L1 and Pro54 in L3 form a hydrophobic area with Ile37 in L2 and Ile45 belonging to the alpha -helix. The conformations of both L1 and L3 are remarkably similar. Val12-Pro20 can be superimposed on Gly51-Tyr60 with a root mean square deviation of 0.18 Å versus the mean coordinates. Consistent with this, hydrogen bonds were also identified for CO(Val12)-NH(Leu21) in L1 and CO(Gly51)-NH(Tyr60) in L3 on the basis of hydrogen bond J-couplings (36) in a long-range HNCO spectrum.

The Ramachandran plot for residues 12-61 of the 30 calculated structures is shown in Fig. 4A. In addition to Gly34 and Gly51, Met18 in L1 and Arg57 in L3 also have positive phi  angles in all 30 structures, although their psi  angles were not refined well. These positive phi  angles of Met18 and Arg57 were confirmed by observation of cross-correlated relaxation of HN-N and HN-H-alpha dipolar interactions of the multiple lines (47). For this, we measured the intensity ratio of the 15N-coupled amide proton resonances in 15N-labeled hNOT4-N78. Fig. 4B shows the NH multiplets of these residues together with those of Glu49 and Leu52, which have negative phi  angles. For Met18 and Arg57, the peak heights of the inner two multiplet components were lower than those of the outer two multiplets.


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Fig. 4.   A, Ramachandran plot of the 30 structures of hNOT4-N78 (residues 12-61). The shading indicates the favorable and unfavorable regions of the plot; the darker the shading, the more favorable the region. The triangles show the glycine residues, and the squares show the remaining residues. B, cross-section along the F2 dimension through selected amide proton resonances in the 15N-coupled 1H-15N HSQC spectrum.

The distance between the two zinc atoms is 14.9 ± 0.3 Å, which is slightly longer than the well conserved value of 14 Å as found in the C3HC4 RING finger structures of PML (29), IEEHV (28), RAG1 (30), and Cbl (31). This could be due to the fact that there is a 4-residue spacing between Cys33 and Cys38 in hNOT4-N78, whereas only 2 residues separate the same zinc-ligating residues in the other three C3HC4 RING fingers.

Several NH protons surrounding the metal atoms display large upfield shifts in the amide proton resonance positions upon exchanging zinc with cadmium, as shown in Fig. 2A. These protons were found within a short distance of the sulfur atoms of the cysteines involved in metal ligation in 30 structures. For example, the distances between NH and the sulfur atom are 2.90 ± 0.25 Å for NH(Cys17)-S(Cys14), 2.94 ± 0.38 Å for NH(Cys33)-S(Cys31), 2.55 ± 0.08 Å for NH(Cys56)-S(Cys53), 2.45 ± 0.06 Å for NH(Cys38)-S(Cys14), and 2.54 ± 0.31 Å for NH(Cys41)-S(Cys38). The chemical shift of the amide proton is sensitive to hydrogen bond length (37), and substitution of zinc with cadmium causes a small change in hydrogen bond length due to the increase in the metal-sulfur bond length (48, 49). It is likely that these observed large chemical shift changes upon exchanging zinc with cadmium reflect the presence of hydrogen bonds from NH protons to sulfur atoms. These hydrogen bonds could contribute to stabilize the coordination of the zinc ion.

Comparison with C3HC4 RING Finger Structures-- Fig. 5 shows a schematic drawing of the RING finger structures of hNOT4-N78, IEEHV, and RAG1. The alpha -helix in hNOT4-N78 is well conserved in IEEHV and RAG1. However, the beta -sheet that exists in both IEEHV and RAG1 is not present in hNOT4-N78. The region corresponding to the third strand of the beta -sheet in IEEHV, which is absent in RAG1, is unstructured in hNOT4-N78. The region corresponding to the first and second beta -strands adopts a loop conformation in hNOT4-N78. Although these regions have a different conformation in hNOT4-N78 and the other two RING fingers, they are located in a similar position and have a similar orientation.


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Fig. 5.   Schematic view of the RING finger structures of NOT4, IEEHV, RAG1, and c-Cbl. Please note that in RAG1, the first zinc-binding site of the RING finger is part of the binuclear zinc cluster. The conserved alpha -helix is colored red, and the remaining helices and helical turns are in yellow. The beta -sheet is colored green. Zinc ions are indicated as balls and colored magenta for conserved zinc and gray for the remaining zinc in RAG1. Residues that coordinate zinc ions are shown and colored yellow for cysteine and blue for histidine.

The root mean square deviation values of C-alpha atoms between the mean coordinates of the C4C4 RING finger and C3HC4 RING finger structures are 1.7 Å for IEEHV (45 C-alpha atoms; Protein Data Bank code 1CHC), 1.6 Å for RAG1 (45 C-alpha atoms; Protein Data Bank code 1RMD), and 1.8 Å for c-Cbl (45 C-alpha atoms; Protein Data Bank code 1FBV). Despite the differences in secondary structural elements, the overall structure of hNOT4-N78 is quite similar to the structures of IEEHV, RAG1, and c-Cbl.

In the hydrophobic region of the C3HC4 RING finger, 2 central residues are well conserved (Phe28 and Ile33 in IEEHV, Phe309 and Ile314 in RAG1, and Met400 and Leu405 in c-Cbl). In hNOT4-N78, the backbone atoms of Ile37 and Trp42 are located in a similar position in space. The orientation of the side chains, however, is slightly different. The side chain of Ile37 is still positioned in the core, similar to the conserved Phe28 of IEEHV and Phe309 of RAG1; but the side chain of Trp42 is pointing away from Ile37, although they have limited contact. Instead, the side chain of Ile45 is pointing toward the side chain of Ile37 and participating in the hydrophobic core, taking over the role of Trp42. Consistent with this, Ile45 is invariant in all known NOT4 orthologs to date (Fig. 1 and data not shown).

Recently, the crystal structure of the c-Cbl RING finger bound to the ubiquitin-conjugating enzyme UbcH7 has been reported (31). The UbcH7-binding site on the c-Cbl RING finger is provided by the shallow groove formed by the alpha -helix and two zinc-chelating loops. It is interesting to note that this region is well conserved with the hNOT4 C4C4 RING finger in structural but not chemical terms. Also, hNOT4 has a shallow groove that is formed by Leu16 in L1; Pro54 in L3; and Arg44, Ile45, and Glu49 in the alpha -helix. Although L1 and L3 come close to each other, there is a groove between the two loops and the alpha -helix. It is important to note that although the structure is conserved, the side chains are not. Ile383, Ser407, Trp408, and Ser411, which form the binding site for UbcH7 in the c-Cbl RING, are replaced by Leu16, Arg44, Ile45, and Asp48, respectively, in hNOT4.

Implication for Function-- So far, the exact role of the hNOT4 RING finger domain in the CCR4·NOT complex is unclear. Yeast complementation analysis showed that unlike full-length hNOT4, a hNOT4 protein lacking the RING finger motif does not complement its yeast counterpart.2 Surprisingly, the RING finger domain of hNOT4 is not required for interaction with hNOT1, as this is mediated by the nonconserved C-terminal part of hNOT4 (data not shown). Analogous to the RING finger of c-Cbl (22, 31), the hNOT4 RING finger may serve in a role as a E3 ligase in (poly)ubiquitination of proteins. In accordance with this proposal, we have identified in yeast two-hybrid screens components of the ubiquitin pathway as NOT4 RING interaction partners.3 The structure of the NOT4 RING finger displays the features that were observed in the Cbl-UbcH7 interaction. The described structure of the NOT4 C4C4 RING finger allows the rational design of phenotypic mutations that affect these interactions and subsequent testing of the effects on the in vivo function of NOT4. This should provide a better understanding of transcription regulation by the CCR4·NOT complex.

    FOOTNOTES

* This work was supported in part by grants from the European Communities Program "Access to Large Scale Facilities" and by the Netherlands Foundation for Chemical Research.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 1E4U) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The first two authors contributed equally to this work.

** Supported by the Dutch Cancer Foundation.

Dagger Dagger Supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research-Medical Sciences.

§§ Supported by a Pioneer grant from the Netherlands Organization for Scientific Research-Medical Sciences.

¶¶ To whom correspondence should be addressed. Tel.: 31-30-253-4035; Fax: 31-30-243-7623; E-mail: Boelens@NMR.chem.uu.nl.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M009298200

2 M. J. de Ruwe, F. A. J. van den Heuvel, M. A. Collart, and H. T. M. Timmers, unpublished observation.

3 T. K. Albert, F. A. J. van den Heuvel, Y. I. A. Legtenberg, and H. T. M. Timmers, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: hNOT, human NOT; E3, ubiquitin-protein isopeptide ligase; NOESY, nuclear Overhauser effect correlation spectroscopy; HSQC, heteronuclear single quantum correlation spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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