The structure of the C4C4 ring finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers.

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 C(4)C(4) 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 (113)Cd-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 A for the backbone atoms and 1.08 +/- 0.12 A 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 C(3)HC(4) 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.

The CCR4⅐NOT 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)(3)(4). The HIS3 gene contains two core promoters, T C , a TATA-less element, and T R , a canonical TATA sequence (5,6). Mutations in NOT genes selectively elevate transcription from T C (2)(3)(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)(8)(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 CCR4⅐NOT complex has a very general role in RNA polymerase II transcription (15).
Recently, the human counterpart of the yeast CCR4⅐NOT 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 singlestranded 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 proteinprotein 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-X 2 -Cys-X 9 -39 -Cys-X 1-3 -His-X 2-3 -(Cys/His)-X 2 -Cys-X 4 -48 -Cys-X 2 -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 C 3 HC 4 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 protooncogene 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 C 3 HC 4 structures possess some common features, the most obvious being the coordination of the two zinc atoms in a cross-brace configuration. In this system, Cys 1 , Cys 2 , Cys 5 , and Cys 6 coordinate the first zinc atom, and Cys 3 , His 1 , Cys 7 , and Cys 8 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-X 2 -Cys-X 13 -Cys-X-Cys-X 4 -Cys-X 2 -Cys-X 11-16 -Cys-X 2 -Cys. It constitutes a novel RING finger variant of a C 4 C 4 type in which His 1 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 C 3 HC 4 RING fingers. However, important differences especially in the secondary structure elements are notable.

MATERIALS AND METHODS
Plasmids-pET15b-hNOT4-N78 encodes the first 78 amino acids of hNOT4 N-terminally fused to a 23-residue His 6 tag. The hNOT4-N78 insert was obtained by polymerase chain reaction using pET15b-hNOT4-N227 as a template, a T7 primer (5Ј-TAATACGACTCACTAT-AGGG-3Ј), and a hNOT4-specific primer (5Ј-GCGGGATCCTATATC-CTTTGCAGCTCTTCCTG-3Ј). 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).
Protein concentrations were determined using Bio-Rad protein assays employing bovine ␥-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 tripleresonance 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-( 15 N, 1 H)-HSQC, and three-dimensional TOCSY-( 15 N, 1 H)-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).
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 Ͻ S ␥ (Cys)-S ␥ (Cys) Ͻ 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-S ␥ (Cys) are 2.3 Å and geminal bond angles of S ␥ (Cys)-Zn-S ␥ (Cys) and Zn-S ␥ (Cys)-C ␤ (Cys) are 109.5° (43).
The final set of structures was analyzed using the program PRO-CHECK-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
Expression and Assignments-The first 78 residues of hNOT4, which contain the C 4 C 4 RING finger, were fused to an N-terminal His 6 tag. This protein was overexpressed and 15 Nand 15 N/ 13 C-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 C 3 HC 4 RING fingers of IEEHV and RAG1.
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 His 6tagged region were also obtained. 1 H-15 N correlations for the remaining residues in the His 6 -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 Val 12 , Leu 16 , Leu 21 , and Leu 52 were obtained. In addition to this, 14 out of 64 ␤-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 113 Cd-1 H HSQC experiments using hNOT4-N78 in which the zinc ions were replaced with cadmium ions. 113 Cd-substituted hNOT4-N78 was obtained by adding 113 Cd-EDTA to a final concentration of 4 mM to zinc-containing hNOT4-N78, and the exchange of zinc with 113 Cd was monitored using 1 H-15 N HSQC spectra.
Since most of the resonances were shifted by the substitution, the assignments of 1 H and 15 N resonances of 113 Cd-hNOT4-N78 had to be confirmed by three-dimensional NOESY-( 1 H, 15 N)-HSQC and three-dimensional TOCSY-( 1 H, 15 N)-HSQC spectra. Fig. 2A shows the chemical shift differences between 113 Cd-hNOT4-N78 and Zn-hNOT4-N78. Cys 17 , Cys 33 , and Cys 56 displayed the largest differences in both the 1 H and 15 N 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 113 Cd-1 H HSQC spectrum of 113 Cd-hNOT4-N78 displaying the correlation between two cadmium resonances and the 1 H chemical shifts belonging to the ␤ 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 113 Cd-1 H HSQC spectrum shows that Cys 14 , Cys 17 , Cys 38 , and Cys 41 (site 1) share one metal ion that resonates at 687.5 ppm and that Cys 33 , Cys 53 , and Cys 56 (site 2) share the other metal ion at 714.4 ppm. The correlation of the remaining Cys 31 was not observed in any of the 113 Cd-1 H HSQC spectra with magnetization delays of 6, 9, and 12 ms. However, the last metal-coordinating ligand of site 2 was assigned to Cys 31 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.
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 and 13 1 ) obtained from various two-and threedimensional 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 Ϯ 0.13 Å for the backbone atoms and 1.08 Ϯ 0.12 Å for all heavy atoms.
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 ␣-helix (residues 39 -48) between the second and third loops. Of the 8 cysteine residues that are involved in zinc coordination, the first (Cys 14 , Cys 17 ), second (Cys 31 , Cys 33 ), and forth (Cys 53 , Cys 56 ) pairs are located in L1, L2, and L3, respectively. The remaining 2 cysteines (Cys 38 , Cys 41 ) are located at the end of L2 and in the ␣-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 ␣ iϪ1 and ␦ i protons. The three loops are stabilized by the coordination with the zinc ions and by hydrophobic interactions. Leu 16 in L1 and Pro 54 in L3 form a hydrophobic area with Ile 37 in L2 and Ile 45 belonging to the ␣-helix. The conformations of both L1 and L3 are re- FIG. 1. Comparison of the first 78 residues of hNOT4, encompassing the C 4 C 4 RING finger, with the C 4 C 4 RING finger of yeast NOT4 (residues 20 -99) and with the C 3 HC 4 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. markably similar. Val 12 -Pro 20 can be superimposed on Gly 51 -Tyr 60 with a root mean square deviation of 0.18 Å versus the mean coordinates. Consistent with this, hydrogen bonds were also identified for CO(Val 12 )-NH(Leu 21 ) in L1 and CO(Gly 51 )-NH(Tyr 60 ) 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 Gly 34 and Gly 51 , Met 18 in L1 and Arg 57 in L3 also have positive angles in all 30 structures, although their angles were not refined well. These positive angles of Met 18 and Arg 57 were confirmed by observation of cross-correlated relaxation of HN-N and HN-H-␣ dipolar interactions of the multiple lines (47). For this, we measured the intensity ratio of the 15 N-coupled amide proton resonances in 15 N-labeled hNOT4-N78. Fig. 4B shows the NH multiplets of these residues together with those of Glu 49 and Leu 52 , which have negative angles. For Met 18 and Arg 57 , the peak heights of the inner two multiplet components were lower than those of the outer two multiplets.
The distance between the two zinc atoms is 14.9 Ϯ 0.3 Å, which is slightly longer than the well conserved value of 14 Å as found in the C 3 HC 4 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 Cys 33 and Cys 38 in hNOT4-N78, whereas only 2 residues separate the same zinc-ligating residues in the other three C 3 HC 4 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.   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 C 3 HC 4 RING Finger Structures- Fig. 5 shows a schematic drawing of the RING finger structures of hNOT4-N78, IEEHV, and RAG1. The ␣-helix in hNOT4-N78 is well conserved in IEEHV and RAG1. However, the ␤-sheet that exists in both IEEHV and RAG1 is not present in hNOT4-N78. The region corresponding to the third strand of the ␤-sheet in IEEHV, which is absent in RAG1, is unstructured in hNOT4-N78. The region corresponding to the first and second ␤-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.
The root mean square deviation values of C-␣ atoms between the mean coordinates of the C 4 C 4 RING finger and C 3 HC 4 RING finger structures are 1.7 Å for IEEHV (45 C-␣ atoms; Protein Data Bank code 1CHC), 1.6 Å for RAG1 (45 C-␣ atoms; Protein Data Bank code 1RMD), and 1.8 Å for c-Cbl (45 C-␣ 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 C 3 HC 4 RING finger, 2 central residues are well conserved (Phe 28 and Ile 33 in IEEHV, Phe 309 and Ile 314 in RAG1, and Met 400 and Leu 405 in c-Cbl). In hNOT4-N78, the backbone atoms of Ile 37 and Trp 42 are located in a similar position in space. The orientation of the side chains, however, is slightly different. The side chain of Ile 37 is still positioned in the core, similar to the conserved Phe 28 of IEEHV and Phe 309 of RAG1; but the side chain of Trp 42 is pointing away from Ile 37 , although they have limited contact. Instead, the side chain of Ile 45 is pointing toward the side chain of Ile 37 and participating in the hydrophobic core, taking over the role of Trp 42 . Consistent with this, Ile 45 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 ␣-helix and two zinc-chelating loops. It is interesting to note that this region is well conserved with the hNOT4 C 4 C 4 RING finger in structural but not chemical terms. Also, hNOT4 has a shallow groove that is formed by Leu 16 in L1; Pro 54 in L3; and Arg 44 , Ile 45 , and Glu 49 in the ␣-helix. Although L1 and L3 come close to each other, there is a groove between the two loops and the ␣-helix. It is important to note that although the structure is conserved, the side chains are not. Ile 383 , Ser 407 , Trp 408 , and Ser 411 , which form the binding site for UbcH7 in the c-Cbl RING, are replaced by Leu 16 , Arg 44 , Ile 45 , and Asp 48 , respectively, in hNOT4.
Implication for Function-So far, the exact role of the hNOT4 RING finger domain in the CCR4⅐NOT 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 C 4 C 4 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 CCR4⅐NOT complex. FIG. 5. Schematic view of the RING finger structures of NOT4, IEEHV, RAG1, and c-Cbl. Please note that in RAG1, the first zincbinding site of the RING finger is part of the binuclear zinc cluster. The conserved ␣-helix is colored red, and the remaining helices and helical turns are in yellow. The ␤-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.