- Email: [email protected]
The p53-associated protein MDM2 contains a newly characterized zinc-binding domain called the RING finger
PROTEINSEQUENCEMOTIFS The p53-associated protein MDM2 contains a newly characterized zinc-bindingdomain called the RING finger The p53 protein has been shown to behave as a tumour suppressor in human and mouse systems 1,2, binding to specific
DNA elements adjacent to promoters aad activating the transcription of a variety of genes 3,4.Th~.sp5~mediated growth regulation is lost in many human malignancies, either by a mutation of the gene encoding p53 itself, or by oncoproteins, such as viral oncogene products, binding to and thereby inactivating p53 (Ref. 5). Recently, another oncoprotein, MDM2, has been
(a)
MDH2 IAP DROC3H CELR05 ICP0 RING1
(b)
(434-491) (224-275) (697-754) (778-833) (4-59) (15-71)
MAY1994
TIBS 1 9 -
shown to interact directly with p53 (Ref. 6). The human homologue of mdm2 is overexpressed in a significant number of sarcomas, implying an involvement in human neoplasias 7,s. The binding of MDM2 to p53 inactivates p53 and removes growth regulation by this protein, in a manner similar to that seen in virally induced cancers 6,8. In addition to an amino-terminal p53-binding domain, MDM2 also contains a putative nuclear localization signal, an acidic region that suggests MDM2 has trans-activating ability, and two carboxy-terminal 'zinc-finger' motifs between residues 305 and 322, and between residues 438 and 478, the latter having been reported as comprising two tandem zinc fingers of the C2H2 and C4 classes, respectivelys-I°. However, c!oser inspection of the zincfinger region of MDM2. and subsequent database searches, show that this region
(438-478) has significant homology to a newly characterized zinc-binding domain called the RING finger, initially identified in the human ringl gene n-13 (Fig. la). A sequence alignment of the MDM2 RING finger with those of the existing family members was performed, and the sequences of the four most similar RING domains (IAP, DROC3H, ICPO AND CELRO5) and MDM2 are shown in Fig. la. The alignment shows that MDM2 fits well into the ascribed framework for RINGfinger proteins except for a substitution of Cys3 for Thr. This may represent a conservative substitution, with the hydroxyl of the threonine residue replacing the sulphydryl group of cysteine in the co-ordination of Zn2÷,thus forming a variant of the RING-finger domain (Fig. la). The RING-finger domain has been shown to bind two moles of Zn2~ per mole of RING finger, with the Zn2÷ binding to the conserved cysteines and
AIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKK RNKPCPVCRQPIQMIVLTYFP DSKLCKICYVEEC~VCFVP CGHVVACAKCALS VDKCPMCRKIVTSVLKVYFS SSAECTICYENPIDSVLYM CGHMCHCYDCAIEQWRGVGGGQCPLCRAVIRDVIRTYTT E T L T C P S C K T R P K D CIML K C Y H L F C E T C I K T M Y D T R Q R K C P K C N S N F G A N D F H R I F VAERCPICLEDPSNYSMAL PCLHAF CYVCITRWI RQNPTCPLCKVPVESVVHTIES * $*$ $$ * * $ $ *$ *$ * $ $ * $ $$ $$ SS SELHCPICLDHLKNTHTTK ECLHRF CSDCIVTALR SGNKECPTCRKKLVSKRSLRPD
HDM2 CELR05
1 340
MCNTNMSVPTDGAVTTSQIPASEQ ETLVRPKPLLLKLLKSVGAQKDTY QENSKLRLETQTFFSVDSIVNBEEYKNLKKYYSLAIKEYERVSKDLEDI
MDH2 CELRO5
48 390
TMKEVLPYLGQYIMTKRLYDEKOQHI VYCSNDLLGDLFGVPSFSVKEH TTERDAPRSAKEARAMLMSEEHQKTLKEIQCQSDIHNSFYKVSHDBEVLR
MDM2 CELR05
97 439
RKIYTMIYRNLVVVNQQRSSDSGTSVSENRCHLEGGSDQKDLVQBLQEEK CEFETVKEEYNKTVKQSBWDEMKATLNTLRSMNRSLKSEKIRLRBKDKQS
MDM2 CELRO5
147 489
PSSSHLVSRPSTBSRRRAISETEENSDELSGERQRKRHKSDSZSLSFDBB QKDINTLKSELTSLKEAQDKCLLVPLEDVBNAPPEDVNK Z R Q E Y RB
MDM2 CELR05
197 535
LALCVIREICCBRSSSSESTGTPSNPDLDAGVSBHSGDWLDQDSVS DQF LCKEVKRLGAMBKQEKQKQVEKEVNRQIADKLSRLETLRKTSEMI~NDEE
MDM2 CELRO5
246 585
SVEFRVBSLDSEDYSLSBEGQELSDB DDBVYQVTVYQAGESDTDSPEE CISDELRAIGTAVEEEQBRNAQLYIBKREQEDRNLKMMNDRMIQNQTPNR
MDM2 CELR05
294 635
DPBISLADYWKCTSCNEMNPPLPSHCNRCWALRE WLP DKGKDKGBISE LRB K L S C L E S K A Q T D A Q I A K M H E F E K K A N E E L V T K L S B S V Q P K S A B L T R
MDM2 CELRO5
344 685
KAKL BNSTQAEEGF VPDCKKTZVNDSRRSCVEENDDKITQASQSQRSE LTNLMBQHRKNIQEVGMSRDENQZKADRCEGQMKQIQELYAAKAREIRDF
MDM2 CELR05
393 734
DYSQPSTSSSI IYSSQBDVKEFBREETQDKEESVB SSLPLNAIEPCV KFKRQRAEEELETLRIKYBRVKRNBSVPAQSGDQVLEEANRQMKETLTCP
MDM2 CELRO5
44i 784
ICQGRPKNGCIVHGKTGHLMACFTCAKKL KKRNKPCPVCRQ;IQMIVLT S C K T R P K D CZML K C Y H L F C E T C I K T M Y D T R Q R K C P K C N S N F G A N D F H
HDM2 CELR05
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Rgure 1 (a) Amino acid sequence alignment of the MDM2 zinc-finger region with the most similar RING-fingerdomains from four proteins, lAP, DROC3H,CELR05 and ICPO. The RING1sequence is shown for reference. The absolutely conserved Zn2* ligands are shown in bold and labelled *, with the conserved hydrophobic residues labelled $. The sequences were aligned using the automated alignment package AMPStg, using a PAM matrix of 250 and a gap penalty of 12.0. Scores, calculated according to the algorithm of Needleman and Wunsch2o and based on pairwise comparisons of each sequence after 100 randomizations, were used to assess the significance of the alignment. The scores for the alignment range from 6.3 to 8.7, with identities of 26-34%. Scores above four are deemed statistically significant19, (b) Amino acid sequence alignment of MDM2 and CELR05. The alignment was based on a pairwise comparison as implemented in the AMPS19 package using a PAM matrix of 250 and gap penalty of 12.0. The score (defined above) for the pairwise alignment is 4.6. Absolutely conserved residues are in bold with conservative substitutions indicated by o.
198
© 1994,ElsevierScienceLtd 0968-0004/94/$07.00
TIBS 1 9 -
PROTEINSEQUENCE MOTIFS
MAY 1994
histidine, stabilizing the motif into a single folded domain ]2,14.As well as the conservation of the Zn ~-~ligands, there are additional conserved residues between MDM2 and the rest of the RING-finger family, including a number of hydrophobic residues implicated in stabilizing the RiNG-finger fold (Fig. la; Ref. 14). Interestingly, two of the proteins shown in the alignment are implicated in the control of cell fate, namely IAPTMand DROC3Hlo. ICPO is involved in gene regulation ~4whereas the functions of CELRO5 (a Caenorhabditis elegans protein) 17and RING1 (Ref. 12) are not known. MDM2 and CELR05 show significant sequence homologies outside the RING finger, and both can be aligned (17% identity over 491 residues; Fig. Ib). To further assess the significance of the alignment, the zinc finger region of MDM2 (434-491) was used to search the OWL database (version 22.1) with the program PROSRCH18using PAM matrices of I00 and 250. Statistically significant matches (excluding itself) were observed only between MDM2 and IAP, DROC3H, CELR05 and ICPO. Furthermore, no matches were observed in the top I00 alignments between MDM2 and any C2H2 or C4 zincfinger-containing protein, supporting the
proposal that MDM2 contains a RING finger rather than two tandemly arranged zinc fingers as previously postulated 8-]°. The extensive RING-fingerfamily contains a number of known oncoproteins including, for example, MELI8, BMI-I, RFP, PML and TI8 (reviewed in Ref. 13). A large number of proteins in this family are also implicated in the control of cell growth, differentiation and development 13. The presence of the RING.fingerdomain in MDM2 may infer a common functionality with other members of the RING-finger family. This function may involve protein-protein or proteinnucleic acid interaction, which would be distinct from the proposed inactivation of p53, but necessary for the other important biological activities of MDM2.
The modular structure of NifU proteins
frame (ORO YKL253 of chromosome XI of yeasts; a hypothetical 22 kDa protein from Haernophilus influenzaeg; and a short ORF (ORF2) in Azotobacter vinelandii l° (Fig. It)). Thus, A. vinelandii has two Nifll-like proteins, one of which contains only the carboxy-terminal domain (Fig. I a, b). An internal domain of NifU proteins from nltrogen-fixing bacteria, not present in the so-called NifU proteins from two Rhodobacter species or the NifU-like protein from yeast, shows significant sequence similarity (Fig. Ic) to two internal repeats of nitrite reductases from Klebsiella pneumoniae (NasB) II, Escherichia coli (NirB) 12, Emericella nidulans (NiiA)13and Neurospora crassa (Nit-6) 14, as well as to a single carboxyterminal domain of nitrate reductase of K. pneumoniae (NasA) n, the carboxyl terminus of Nile from Bradyrhizobium japonicum Is and a short ORF (gp64) 16 from E. coll. Interestingly, the domain is present in Nile of B. japonicum at its extreme carboxy-terminal end and is absent from the other Nile homologues. In E. nidulans, the domain is encoded by the fifth exon of the niiA (nitrite reductase) gene 13. This domain contains two pairs of conserved cysteines that might participate in the formation of the 2Fe-2S cluster in NifU and the other proteins identified (Fig. la). This cluster might serve as an electron carrier in the nitrate and nitrite reductases.
The nifcluster contains genes responsible for nitrogen fixation in several prokaryotes l-~. With the exception of the essential genes coding for and regulating the expression of the nltrogenase subunlts, the functions of other nifgenes appear to be associated with cofactor biosynthesis, amino acid interconversion and regulation of ammonia storage. However, not all the biochemical activities of the nifgene products have been identified yet. Some of these proteins might represent specialized versions of general metabolic enzymes; thus their presence cannot oe precluded from non-nitrogen fixing organisms. A recent example is the identification of the NifS family as pyridoxal-phosphatedependent aminotransferases 4. Here we report the identification by sequence analysis of two distinct domains in the NifU protein family (Fig. la). Although the biochemical function of NifU proteins remains unknown, it has been shown that NifU exists as a homodimer, containing one redox 2Fe--2S cluster per subunit s. The carboxy-terminal domain, known to be the only common region of NifU between nitrogen-fixing bacteria and several rhodobacterial species e07,is shown here to be present in: the open reading © 1994,Elsevier Science Ltd 0968-0004/94/$07.00
References 1 Eliyahu,D. et al. (1989) Prec. Natl Acad. Sci. USA 86, 8763-8767 2 Rnlay,C. A., Hinds, P. W. and Levine, A. J. (1989) Cell 57, 1083-1093 3 Kem, S. E. et al. (1991) Science 252,1708-1711 4 Zambetti, G. P. et al. (1992) Genes Dev. 6, 1143-1152 5 Levine,A. J. (1990) Virology 177,419-426 6 Momand,J. et aL (1992) Cell 69, 1237-1245 7 Fakharzadeh,S. S., Trusko, S. P. and George, D. L. (1991) EMBO J. 10, 1565-1569 80liner, J. D. et al. (1992) Nature 358, 80-83
9 Chen,J., Marechal.V. and Levine, A. £(1993) MoL Cell. Biol. 13, 4107-4114 10 Oliner, J. D. et aL (1993) Nature 362, 857-860 11 Freemont, P. S., Hanson, I. M. and Trowsdale, J. (1991) Cell 64, 483-484 12 Lovering, R. et al. (1993) Proc. Natl Acad. Sci. USA 90, 2112-2116 13 Freemont, P. S. (1993) Anal NYAcad. Sci. 684, 174-192 14 Everett, R. D. et al. (1993) J. Mol. BioL 234, 1038-1047 15 Crook, N. E., Clem, R. J. and Miller, K. L. (1993) J. ViroL 67, 2168-2174 16 Price, B. D. et al. (1993) EMBO J. 12, 2411-2418 17 Sulston, J. et al. (1992) Nature 356, 37-41 18 Collins, J. F., Coulson,A. F. W. and Lyall, A. (1988) CABIOS 4, 67-71 19 Barton, G. J. and Sternberg, M. J. F_(1990) J. Mol. Biol. 212, 389-402 20 Needleman,S. B. and Wunsch, C. D. (1970) J. MoL Biol. 48, 443-453
MICHAEL N. BODDYAND PAUL S. FREEMONT Protein Structure Laboratory,Imperial Cancer Research Fund,44 Lincoln's Inn Relds, London, UK WC2A3PX.
KATHERINE L. B. BORDEN Laboratory of Molecular Structure, National Institute for Medical Research,The Ridgeway, Mill Hill, London, UK NW7 1AA.
Modular proteins, frequently found in higher eukaryotes, are rare in prokaryotes, probably because of a lack of appropriate genetic shuffling mechanisms (see Refs 17, 18 and references therein). The mobility of the NifU-like domains in both prokaryotes and lower eukaryotes therefore comes as a surprise. It will be a challenge to identify the genetic mechanism(s) that enabled the various species to acquire the two domains, in addition, knowledge of the modular structure of NifU-like proteins might contribute towards elucidating the functions of their domains.
References 1 Beynon,J. et al. (1987) J. Bacteriol. 169, 4024-4029 2 Arnold,W. et al. (1988)J. Mol. Biol. 203, 715-738 3 Jacobson, M. R. et al. (1989) J. Bacteriol. 171, 1017-1027 40uzounis, C. and Sander,C. (1993) FEBS Lett. 322, 159-164 5 Dean, D. R. et al. (1993) J. BacterioL 175, 6737-6744 6 Meijer, W. G. and Tabita, F. R. (1992) J. BacterioL 174, 3855-3866 7 Masepohl, B. et al. (1993) Mol. Gen. Genet. 238, 369-382 8 Purnelle, B., Skala, J., Van Dyck, L. and Goffeau, A. (1992) Yeast8, 977-986 9 Tomb, J. F., EI-Hajj, H. and Smith, H. O. (1991) Gene 104,1-10 10 Joerger, R. D., Jacobson, M. R. and Bishop, P. E. (1989) J. BacterioL 171, 3258-3267 11 Lin, J. T., Goldman, B. S. and Stewart, V. (1993) J. Bacteriol. 175, 2370-2378
199
DNA elements adjacent to promoters aad activating the transcription of a variety of genes 3,4.Th~.sp5~mediated growth regulation is lost in many human malignancies, either by a mutation of the gene encoding p53 itself, or by oncoproteins, such as viral oncogene products, binding to and thereby inactivating p53 (Ref. 5). Recently, another oncoprotein, MDM2, has been
(a)
MDH2 IAP DROC3H CELR05 ICP0 RING1
(b)
(434-491) (224-275) (697-754) (778-833) (4-59) (15-71)
MAY1994
TIBS 1 9 -
shown to interact directly with p53 (Ref. 6). The human homologue of mdm2 is overexpressed in a significant number of sarcomas, implying an involvement in human neoplasias 7,s. The binding of MDM2 to p53 inactivates p53 and removes growth regulation by this protein, in a manner similar to that seen in virally induced cancers 6,8. In addition to an amino-terminal p53-binding domain, MDM2 also contains a putative nuclear localization signal, an acidic region that suggests MDM2 has trans-activating ability, and two carboxy-terminal 'zinc-finger' motifs between residues 305 and 322, and between residues 438 and 478, the latter having been reported as comprising two tandem zinc fingers of the C2H2 and C4 classes, respectivelys-I°. However, c!oser inspection of the zincfinger region of MDM2. and subsequent database searches, show that this region
(438-478) has significant homology to a newly characterized zinc-binding domain called the RING finger, initially identified in the human ringl gene n-13 (Fig. la). A sequence alignment of the MDM2 RING finger with those of the existing family members was performed, and the sequences of the four most similar RING domains (IAP, DROC3H, ICPO AND CELRO5) and MDM2 are shown in Fig. la. The alignment shows that MDM2 fits well into the ascribed framework for RINGfinger proteins except for a substitution of Cys3 for Thr. This may represent a conservative substitution, with the hydroxyl of the threonine residue replacing the sulphydryl group of cysteine in the co-ordination of Zn2÷,thus forming a variant of the RING-finger domain (Fig. la). The RING-finger domain has been shown to bind two moles of Zn2~ per mole of RING finger, with the Zn2÷ binding to the conserved cysteines and
AIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKK RNKPCPVCRQPIQMIVLTYFP DSKLCKICYVEEC~VCFVP CGHVVACAKCALS VDKCPMCRKIVTSVLKVYFS SSAECTICYENPIDSVLYM CGHMCHCYDCAIEQWRGVGGGQCPLCRAVIRDVIRTYTT E T L T C P S C K T R P K D CIML K C Y H L F C E T C I K T M Y D T R Q R K C P K C N S N F G A N D F H R I F VAERCPICLEDPSNYSMAL PCLHAF CYVCITRWI RQNPTCPLCKVPVESVVHTIES * $*$ $$ * * $ $ *$ *$ * $ $ * $ $$ $$ SS SELHCPICLDHLKNTHTTK ECLHRF CSDCIVTALR SGNKECPTCRKKLVSKRSLRPD
HDM2 CELR05
1 340
MCNTNMSVPTDGAVTTSQIPASEQ ETLVRPKPLLLKLLKSVGAQKDTY QENSKLRLETQTFFSVDSIVNBEEYKNLKKYYSLAIKEYERVSKDLEDI
MDH2 CELRO5
48 390
TMKEVLPYLGQYIMTKRLYDEKOQHI VYCSNDLLGDLFGVPSFSVKEH TTERDAPRSAKEARAMLMSEEHQKTLKEIQCQSDIHNSFYKVSHDBEVLR
MDM2 CELR05
97 439
RKIYTMIYRNLVVVNQQRSSDSGTSVSENRCHLEGGSDQKDLVQBLQEEK CEFETVKEEYNKTVKQSBWDEMKATLNTLRSMNRSLKSEKIRLRBKDKQS
MDM2 CELRO5
147 489
PSSSHLVSRPSTBSRRRAISETEENSDELSGERQRKRHKSDSZSLSFDBB QKDINTLKSELTSLKEAQDKCLLVPLEDVBNAPPEDVNK Z R Q E Y RB
MDM2 CELR05
197 535
LALCVIREICCBRSSSSESTGTPSNPDLDAGVSBHSGDWLDQDSVS DQF LCKEVKRLGAMBKQEKQKQVEKEVNRQIADKLSRLETLRKTSEMI~NDEE
MDM2 CELRO5
246 585
SVEFRVBSLDSEDYSLSBEGQELSDB DDBVYQVTVYQAGESDTDSPEE CISDELRAIGTAVEEEQBRNAQLYIBKREQEDRNLKMMNDRMIQNQTPNR
MDM2 CELR05
294 635
DPBISLADYWKCTSCNEMNPPLPSHCNRCWALRE WLP DKGKDKGBISE LRB K L S C L E S K A Q T D A Q I A K M H E F E K K A N E E L V T K L S B S V Q P K S A B L T R
MDM2 CELRO5
344 685
KAKL BNSTQAEEGF VPDCKKTZVNDSRRSCVEENDDKITQASQSQRSE LTNLMBQHRKNIQEVGMSRDENQZKADRCEGQMKQIQELYAAKAREIRDF
MDM2 CELR05
393 734
DYSQPSTSSSI IYSSQBDVKEFBREETQDKEESVB SSLPLNAIEPCV KFKRQRAEEELETLRIKYBRVKRNBSVPAQSGDQVLEEANRQMKETLTCP
MDM2 CELRO5
44i 784
ICQGRPKNGCIVHGKTGHLMACFTCAKKL KKRNKPCPVCRQ;IQMIVLT S C K T R P K D CZML K C Y H L F C E T C I K T M Y D T R Q R K C P K C N S N F G A N D F H
HDM2 CELR05
489 831
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Rgure 1 (a) Amino acid sequence alignment of the MDM2 zinc-finger region with the most similar RING-fingerdomains from four proteins, lAP, DROC3H,CELR05 and ICPO. The RING1sequence is shown for reference. The absolutely conserved Zn2* ligands are shown in bold and labelled *, with the conserved hydrophobic residues labelled $. The sequences were aligned using the automated alignment package AMPStg, using a PAM matrix of 250 and a gap penalty of 12.0. Scores, calculated according to the algorithm of Needleman and Wunsch2o and based on pairwise comparisons of each sequence after 100 randomizations, were used to assess the significance of the alignment. The scores for the alignment range from 6.3 to 8.7, with identities of 26-34%. Scores above four are deemed statistically significant19, (b) Amino acid sequence alignment of MDM2 and CELR05. The alignment was based on a pairwise comparison as implemented in the AMPS19 package using a PAM matrix of 250 and gap penalty of 12.0. The score (defined above) for the pairwise alignment is 4.6. Absolutely conserved residues are in bold with conservative substitutions indicated by o.
198
© 1994,ElsevierScienceLtd 0968-0004/94/$07.00
TIBS 1 9 -
PROTEINSEQUENCE MOTIFS
MAY 1994
histidine, stabilizing the motif into a single folded domain ]2,14.As well as the conservation of the Zn ~-~ligands, there are additional conserved residues between MDM2 and the rest of the RING-finger family, including a number of hydrophobic residues implicated in stabilizing the RiNG-finger fold (Fig. la; Ref. 14). Interestingly, two of the proteins shown in the alignment are implicated in the control of cell fate, namely IAPTMand DROC3Hlo. ICPO is involved in gene regulation ~4whereas the functions of CELRO5 (a Caenorhabditis elegans protein) 17and RING1 (Ref. 12) are not known. MDM2 and CELR05 show significant sequence homologies outside the RING finger, and both can be aligned (17% identity over 491 residues; Fig. Ib). To further assess the significance of the alignment, the zinc finger region of MDM2 (434-491) was used to search the OWL database (version 22.1) with the program PROSRCH18using PAM matrices of I00 and 250. Statistically significant matches (excluding itself) were observed only between MDM2 and IAP, DROC3H, CELR05 and ICPO. Furthermore, no matches were observed in the top I00 alignments between MDM2 and any C2H2 or C4 zincfinger-containing protein, supporting the
proposal that MDM2 contains a RING finger rather than two tandemly arranged zinc fingers as previously postulated 8-]°. The extensive RING-fingerfamily contains a number of known oncoproteins including, for example, MELI8, BMI-I, RFP, PML and TI8 (reviewed in Ref. 13). A large number of proteins in this family are also implicated in the control of cell growth, differentiation and development 13. The presence of the RING.fingerdomain in MDM2 may infer a common functionality with other members of the RING-finger family. This function may involve protein-protein or proteinnucleic acid interaction, which would be distinct from the proposed inactivation of p53, but necessary for the other important biological activities of MDM2.
The modular structure of NifU proteins
frame (ORO YKL253 of chromosome XI of yeasts; a hypothetical 22 kDa protein from Haernophilus influenzaeg; and a short ORF (ORF2) in Azotobacter vinelandii l° (Fig. It)). Thus, A. vinelandii has two Nifll-like proteins, one of which contains only the carboxy-terminal domain (Fig. I a, b). An internal domain of NifU proteins from nltrogen-fixing bacteria, not present in the so-called NifU proteins from two Rhodobacter species or the NifU-like protein from yeast, shows significant sequence similarity (Fig. Ic) to two internal repeats of nitrite reductases from Klebsiella pneumoniae (NasB) II, Escherichia coli (NirB) 12, Emericella nidulans (NiiA)13and Neurospora crassa (Nit-6) 14, as well as to a single carboxyterminal domain of nitrate reductase of K. pneumoniae (NasA) n, the carboxyl terminus of Nile from Bradyrhizobium japonicum Is and a short ORF (gp64) 16 from E. coll. Interestingly, the domain is present in Nile of B. japonicum at its extreme carboxy-terminal end and is absent from the other Nile homologues. In E. nidulans, the domain is encoded by the fifth exon of the niiA (nitrite reductase) gene 13. This domain contains two pairs of conserved cysteines that might participate in the formation of the 2Fe-2S cluster in NifU and the other proteins identified (Fig. la). This cluster might serve as an electron carrier in the nitrate and nitrite reductases.
The nifcluster contains genes responsible for nitrogen fixation in several prokaryotes l-~. With the exception of the essential genes coding for and regulating the expression of the nltrogenase subunlts, the functions of other nifgenes appear to be associated with cofactor biosynthesis, amino acid interconversion and regulation of ammonia storage. However, not all the biochemical activities of the nifgene products have been identified yet. Some of these proteins might represent specialized versions of general metabolic enzymes; thus their presence cannot oe precluded from non-nitrogen fixing organisms. A recent example is the identification of the NifS family as pyridoxal-phosphatedependent aminotransferases 4. Here we report the identification by sequence analysis of two distinct domains in the NifU protein family (Fig. la). Although the biochemical function of NifU proteins remains unknown, it has been shown that NifU exists as a homodimer, containing one redox 2Fe--2S cluster per subunit s. The carboxy-terminal domain, known to be the only common region of NifU between nitrogen-fixing bacteria and several rhodobacterial species e07,is shown here to be present in: the open reading © 1994,Elsevier Science Ltd 0968-0004/94/$07.00
References 1 Eliyahu,D. et al. (1989) Prec. Natl Acad. Sci. USA 86, 8763-8767 2 Rnlay,C. A., Hinds, P. W. and Levine, A. J. (1989) Cell 57, 1083-1093 3 Kem, S. E. et al. (1991) Science 252,1708-1711 4 Zambetti, G. P. et al. (1992) Genes Dev. 6, 1143-1152 5 Levine,A. J. (1990) Virology 177,419-426 6 Momand,J. et aL (1992) Cell 69, 1237-1245 7 Fakharzadeh,S. S., Trusko, S. P. and George, D. L. (1991) EMBO J. 10, 1565-1569 80liner, J. D. et al. (1992) Nature 358, 80-83
9 Chen,J., Marechal.V. and Levine, A. £(1993) MoL Cell. Biol. 13, 4107-4114 10 Oliner, J. D. et aL (1993) Nature 362, 857-860 11 Freemont, P. S., Hanson, I. M. and Trowsdale, J. (1991) Cell 64, 483-484 12 Lovering, R. et al. (1993) Proc. Natl Acad. Sci. USA 90, 2112-2116 13 Freemont, P. S. (1993) Anal NYAcad. Sci. 684, 174-192 14 Everett, R. D. et al. (1993) J. Mol. BioL 234, 1038-1047 15 Crook, N. E., Clem, R. J. and Miller, K. L. (1993) J. ViroL 67, 2168-2174 16 Price, B. D. et al. (1993) EMBO J. 12, 2411-2418 17 Sulston, J. et al. (1992) Nature 356, 37-41 18 Collins, J. F., Coulson,A. F. W. and Lyall, A. (1988) CABIOS 4, 67-71 19 Barton, G. J. and Sternberg, M. J. F_(1990) J. Mol. Biol. 212, 389-402 20 Needleman,S. B. and Wunsch, C. D. (1970) J. MoL Biol. 48, 443-453
MICHAEL N. BODDYAND PAUL S. FREEMONT Protein Structure Laboratory,Imperial Cancer Research Fund,44 Lincoln's Inn Relds, London, UK WC2A3PX.
KATHERINE L. B. BORDEN Laboratory of Molecular Structure, National Institute for Medical Research,The Ridgeway, Mill Hill, London, UK NW7 1AA.
Modular proteins, frequently found in higher eukaryotes, are rare in prokaryotes, probably because of a lack of appropriate genetic shuffling mechanisms (see Refs 17, 18 and references therein). The mobility of the NifU-like domains in both prokaryotes and lower eukaryotes therefore comes as a surprise. It will be a challenge to identify the genetic mechanism(s) that enabled the various species to acquire the two domains, in addition, knowledge of the modular structure of NifU-like proteins might contribute towards elucidating the functions of their domains.
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