Characterization of the Conserved Interaction between GATA and FOG Family Proteins

Abstract

The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His → Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His → Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo. The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His → Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His → Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo. Zinc binding domains (often referred to as zinc fingers, or ZnFs) 1The abbreviations used are: ZnF, zinc finger; USH-F1, finger 1 from U-shaped; HSQC, heteronuclear single quantum coherence; Pnr-NF, N-terminal ZnF of Pannier; MOPS, 4-morpholinepropanesulfonic acid: NOE, nuclear Overhauser effect; FOG, friend of GATA-1; d4-TSP, sodium 3-trimethylsilyl[2,2,3,3-2H]propionate; CREB, cAMP-response element. 1The abbreviations used are: ZnF, zinc finger; USH-F1, finger 1 from U-shaped; HSQC, heteronuclear single quantum coherence; Pnr-NF, N-terminal ZnF of Pannier; MOPS, 4-morpholinepropanesulfonic acid: NOE, nuclear Overhauser effect; FOG, friend of GATA-1; d4-TSP, sodium 3-trimethylsilyl[2,2,3,3-2H]propionate; CREB, cAMP-response element. are extremely common in eukaryotes; for example, perhaps 3–4% of human genes encode proteins that contain such domains. At least 20 different classes of zinc binding domains have been identified, differing in the number of zinc ions they bind and the identity and spacing of the ligating amino acids. Since the initial discovery of the classical zinc fingers in TFIIIA (1Miller J. McLachlan A.D. Klug A. EMBO J. 1985; 4: 1609-1614Crossref PubMed Scopus (1645) Google Scholar), it has become clear that many zinc binding domains function as sequence-specific DNA (or RNA) binding motifs. However, it has also become apparent that many classes of zinc binding domains, including LIM domains and PHD domains, have roles as mediators of protein-protein interactions. GATA and classical ZnFs have also been shown to be capable of contacting other proteins, especially in the context of the regulation of gene expression (for review, see Ref. 2Mackay J.P. Crossley M. Trends Biochem. Sci. 1998; 23: 1-4Abstract Full Text PDF PubMed Scopus (378) Google Scholar). It has been demonstrated, both in Drosophila melanogasterand in mammals, that interactions between ZnFs of GATA and FOG family transcription factors are essential for normal development. In mammals, GATA-1 and FOG-1 cooperate to drive erythroid development (3Tsang A.P. Fujiwara Y. Hom D.B. Orkin S.H. Genes Dev. 1998; 12: 1176-1188Crossref PubMed Scopus (294) Google Scholar), whereas the Drosophila proteins Pannier (a GATA family protein; Ref.4Ramain P. Heitzler P. Haenlin M. Simpson P. Development. 1993; 119: 1277-1291PubMed Google Scholar) and U-shaped (a FOG family protein; Refs. 5Haenlin M. Cubadda Y. Blondeau F. Heitzler P. Lutz Y. Simpson P. Ramain P. Genes Dev. 1997; 11: 3096-3108Crossref PubMed Scopus (165) Google Scholar and 6Cubadda Y. Heitzler P. Ray R.P. Bourouis M. Ramain P. Gelbart W. Simpson P. Haenlin M. Genes Dev. 1997; 11: 3083-3095Crossref PubMed Scopus (122) Google Scholar) combine to direct the development of both the heart (7Fossett N. Zhang Q. Gajewski K. Choi C.Y. Kim Y. Schulz R.A. Proc. Natl Acad. Sci. U. S. A. 2000; 97: 7348-7353Crossref PubMed Scopus (41) Google Scholar) and sensory bristles (6Cubadda Y. Heitzler P. Ray R.P. Bourouis M. Ramain P. Gelbart W. Simpson P. Haenlin M. Genes Dev. 1997; 11: 3083-3095Crossref PubMed Scopus (122) Google Scholar). GATA proteins (Fig. 1 A; Ref.8Weiss M.J. Orkin S.H. Exp. Hematol. 1995; 23: 99-107PubMed Google Scholar) typically contain two ZnFs with a CX 2CX 17CX 2C consensus. The more C-terminal of these two domains (CF) binds DNA sequences containing GATA sites, whereas the N-terminal zinc finger (NF) may contact promoters with different motifs (9Newton A. Mackay J. Crossley M. J. Biol. Chem. 2001; 276: 35794-35801Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In addition, the zinc finger domains have been shown to mediate interactions with other proteins, including CREB-binding protein (10Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl Acad. Sci., U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (311) Google Scholar), erythroid Kruppel-like factor (11Merika M. Orkin S. Mol. Cell. Biol. 1995; 15: 2437-2447Crossref PubMed Scopus (435) Google Scholar), and members of the FOG family (12Tevosian S.G. Deconinck A.E. Cantor A.B. Rieff H.I. Fujiwara Y. Corfas G. Orkin S.H. Proc. Natl Acad. Sci. U. S. A. 1999; 9: 950-955Crossref Scopus (173) Google Scholar, 13Holmes M. Turner J. Fox A. Chisholm O. Crossley M. Chong B. J. Biol. Chem. 1999; 274: 23491-23498Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Svensson E.C. Tufts R.L. Polk C.E. Leiden J.M. Proc. Natl Acad. Sci. U. S. A. 1999; 96: 956-961Crossref PubMed Scopus (235) Google Scholar, 15Tsang A.P. Visvader J.E. Turner C.A. Fujiwara Y., Yu, C. Weiss M.J. Crossley M. Orkin S.H. Cell. 1997; 90: 109-119Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). FOG family proteins (Fig. 1 B) contain either eight or nine ZnFs that are related to the classical CCHH ZnFs (which have a conserved C-X 2–5CX12HX 2–5H sequence). Several of the fingers in each FOG protein, however, have an altered consensus sequence in which the final zinc binding histidine is replaced with a cysteine. Interestingly, it is only these variant CCHC ZnFs that mediate interactions with GATA NFs. The structures of two of these domains (fingers 1 and 9 from D. melanogaster U-shaped) have recently been determined (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and to classical CCHH fingers in The only is a of the the zinc The GATA binding of CCHC finger 1 from U-shaped; has been determined (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A.H. C. M. Kowalski K. Mackay J. Crossley M. EMBO J. 1999; PubMed Scopus Google Scholar). of the in GATA binding are the of the the final and of the to histidine the finger to a classical CCHH is to the GATA-FOG interaction in J.M. Kowalski K. C.K. Sharpe B.K. Fox A.H. Crossley M. J.P. J. Biochem. 2000; PubMed Scopus Google Scholar). The CCHC topology to play a in the but the for is The that are in GATA binding are conserved FOG family CCHC ZnFs (Fig. 1 although of the that the binding show to and weak as a A.H. C. M. Kowalski K. Mackay J. Crossley M. EMBO J. 1999; PubMed Scopus Google Scholar). It is however, such differences are or result from the and of the The interactions between the GATA and FOG proteins have become a of that with the interactions are with human gene is the and GATA-1 including and different in the GATA-1 have been K. K. G. R. C. K. J. C. 2001; PubMed Scopus Google Scholar, K. G. C. P. J. C. Mol. 11: PubMed Google Scholar, Newton M.J. Crossley M. 2001; PubMed Scopus Google Scholar, M. Orkin S.H. J.M. Weiss M.J. 2000; PubMed Scopus Google Scholar). of these with the interaction between the GATA-1 and two to that mediate GATA-FOG whereas two to a to the FOG binding The with the DNA binding of the but not the interaction with The that the interaction with FOG to different and different the binding of the GATA-1 to FOG fingers and 9 and a and M. Orkin S.H. J.M. Weiss M.J. 2000; PubMed Scopus Google Scholar). Newton M.J. Crossley M. 2001; PubMed Scopus Google Scholar) and K. K. G. R. C. K. J. C. 2001; PubMed Scopus Google Scholar), the other the interaction with FOG finger 9 but have the with FOG fingers 1 and these The K. G. C. P. J. C. Mol. 11: PubMed Google Scholar) also the interaction with FOG, although has not been with the binding of FOG It is that an of the of different FOG fingers contact GATA-1 may these different Here we the interaction between U-shaped finger 9 as a and the N-terminal ZnF of Pannier The in the interaction are a and with for the between U-shaped finger 1 a and GATA-1 results suggest a common of interaction and interacting FOG We also to the for a number of of these are weak and the is to be the the of the CCHC topology in FOG zinc fingers is The of a in but not interaction with of the structure of mutant finger the basis for a subtle change in the at the C-terminal of the USH-F1, and GATA-1 and as (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. R. Crossley M. Mackay J.P. J. 1999; PubMed Scopus Google Scholar). Pannier and the same as that used for the GATA-1 that the for of the from a D. melanogaster The mutant of U-shaped finger 9 of a the Cys → His The the expression The including and which are the of the and and as K. R. Crossley M. Mackay J.P. J. 1999; PubMed Scopus Google Scholar). The of of The identity of the of a as (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). for or structure of Pnr-NF, or of in containing of both and concentrations of and 1 for and The to and a with a at of for and for The the A. J. 1985; Scholar) with and Ref. A. K. Biochem. 9: Scopus Google Scholar), U. J. Chem. Scopus Google Scholar), and C. J. Chem. 1985; Scopus Google Scholar). A. M. R. J. Scholar, J. J.E. N. J. Scholar) and G. A. J. Chem. 1993; Scopus Google Scholar) the of and the histidine an in which the to S. Sci. 1993; PubMed Scopus Google Scholar), and the and to and either or and as K. R. Crossley M. Mackay J.P. J. 1999; PubMed Scopus Google Scholar) and C. P. P. K. J. 1995; PubMed Scopus Google Scholar). The of to sodium at whereas the to the of the D. J. Chem. Scopus Google Scholar). as and at concentrations of and at a of 1 with Pnr-NF, at of and The same and The binding of and to a can be as the is given 1 and are the concentrations of and Pnr-NF, at a given in the a and can be as the of the of the and the in between the and is the is to be in these a single is at the of the in the and is the of a in is the of the same in the and are the of the and 1 (3Tsang A.P. Fujiwara Y. Hom D.B. Orkin S.H. Genes Dev. 1998; 12: 1176-1188Crossref PubMed Scopus (294) Google Scholar) is M. Cubadda Y. Blondeau F. Heitzler P. Lutz Y. Simpson P. Ramain P. Genes Dev. 1997; 11: 3096-3108Crossref PubMed Scopus (165) Google Scholar), the expression can be used to a of from the of with the least in the of a and to the in the is the of and is the of the between the and The for a in not the between the and of the protein and can be determined a expression such as However, the of a from as a function of for a can be determined from the of the J. J.P. J. Scholar), which is given is the for the is a factor (which the of the but has or The and are given and 9 and the for and To the binding for a in the each in the each of of and and the as the of and the at which the a binding with the the of the of the differences between the and The of in a to the of the to the in to a final of and with both of and of The to and sodium to a final of 20 The to the required concentrations of each to that they and a containing sodium and at in 20 for the a the two proteins to be used in each of the protein the whereas the with the more protein and at and The the containing either binding or each at with each of of The at and the at the of the of the protein from the to for the of and least to a single binding used to for the binding and of concentrations used in of protein in a the The used to of in the in and to the of P. C. K. J. Mol. Biol. 1997; PubMed Scopus Google Scholar) and the of the In with a of the of for of from an of and in with used to a of in the of used to structures from in the of zinc The with the function used as for in P. J. M. T. Biol. 1998; PubMed Scopus Google Scholar). Zinc the of to and to and with of the zinc to the for for for and for with a of The protein and in with initial The of a of at with of and a low the for of the and and The of with of in which the in The the to and The and final of of (for with for and to and of structures and the 20 with the used to the structure of The structures and the R. M. K. J. Mol. PubMed Scopus Google Scholar) and R.A. R. J.M. J. PubMed Scopus Google Scholar). We to the of that for interaction with GATA family The GATA binding of been a with (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the same for However, the of GATA-1 to in in the HSQC, which the of in the Furthermore, it clear that a more have been required to these that the and of interaction different from for formation of the In an to the the Pannier Pannier is the of U-shaped; sequence identity with that of GATA-1 including of the in FOG binding (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A.H. Kowalski K. Mackay J.P. Crossley M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with in a similar of in the but the of of in the for the GATA-1 to be The the an change and to have a change the change and these and are the structure of in C that to be in the interaction and from not show a perhaps the and from the of Pannier similar result for (Fig. Ref. C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the of and each In addition, the of and in The that in the and are in in C. The of in binding of USH-F1, A.H. C. M. Kowalski K. Mackay J. Crossley M. EMBO J. 1999; PubMed Scopus Google Scholar) that a common of interaction between GATA and FOG CCHC ZnFs that the for and the differences that as a result of given that the of these the of to the of the a and (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that the interaction between be of moderate We to the of the interaction Several and in and of a and an a of for that to be in the of the that the of the the between the and for these It is also that the binding for these from the binding of a in (Fig. are of S. 2001; PubMed Scopus Google Scholar), and the 9 to these In the of of the change in a a of a of and a of It is clear from the that the is more for (Fig. of and 20 used for and determined from of the in the and in To an of the of the and to to the of the (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and the in an of the from (Fig. and (Fig. B) of and of the same two fingers with the GATA-1 that used for (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that the for are similar to for the interactions Furthermore, it that a a in the not is an important result given that structural have been in the It has been shown in a that of FOG fingers 1 and 9 in which the final zinc-ligating to a histidine a classical CCHH to with the GATA-1 J.M. Kowalski K. C.K. Sharpe B.K. Fox A.H. Crossley M. J.P. J. Biochem. 2000; PubMed Scopus Google Scholar). with the that fingers in FOG family proteins the CCHC to the of To we the final zinc-ligating of to histidine → His or → His in the a CCHH similar to classical and that the folded not We determined the structure of and heteronuclear The of with wild-type (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google to the between the two zinc-ligating and and to and which are in the of in wild-type The of the zinc and the identity of the structures in the of zinc the basis of these it that the of and and the of and the zinc binding and that the in The of the zinc binding the for the histidine in an for In the final structure the of the zinc binding and the used in final of structure used in the for the family of 20 differences from is the as in differences from differences differences are given as the the and differences are given as the is the as in differences are given as the the and differences are given as the in a of structures in and the 20 structures with overall used to the structure of (Fig. The structures from the from and and as shown the low of the are of or and that for of the or of the the same differences for the final 20 structures with to the are for the and for The for family of have been with the The structure of (Fig. B) a and an and is the same as the structure of (Fig. for the of the in are a with a the and the The are the are not in and the However, whereas in the of with the of both and the only with the of in is of a change in of the two zinc binding a of the to histidine which the zinc is the only change in of the of the zinc binding that are subtle in the of the zinc binding or the zinc (Fig. and the same in both and whereas a change in the of is In the a similar to the histidine of classical CCHH with the more of of the histidine to the zinc from a different with The of the histidine not the of in the and The of and are to which the of the Cys → His substitution apparent (Fig. the the final zinc binding in an in and the same a that is to in and and in also an and not In and both to of although the is not as change in from the in of the and histidine The from to the zinc binding is and in the two different to the zinc binding of in the same in which the overall of the the in however, the in are both and with O. The of the histidine substitution is apparent the of the the and a subtle change of and the same of is from (Fig. The is and the of the of and with the of classical CCHH to the of to bind the of of to a number of either in or the same that in the wild-type that the of interaction of and each containing 1 of Pnr-NF, that the interaction that in the of in the of we to zinc fingers that contact other proteins the CCHC fingers of FOG have from DNA binding classical zinc sequence of zinc finger domains that are to with DNA with the protein binding zinc fingers of Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar), FOG-1 A.P. Visvader J.E. Turner C.A. Fujiwara Y., Yu, C. Weiss M.J. Crossley M. Orkin S.H. Cell. 1997; 90: 109-119Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, A.H. C. M. Kowalski K. Mackay J. Crossley M. EMBO J. 1999; PubMed Scopus Google Scholar), A. K. EMBO J. 15: PubMed Scopus Google Scholar), and Y. Proc. Natl Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar) the from the sequence The between each of sequences and a final in which the related sequences are to each other in the whereas the more related are that the sequences of many of the protein binding domains are overall from DNA binding zinc In the that important for DNA (for example, the in that the are not conserved in the We have shown that the of the interaction between the N-terminal zinc finger of GATA family proteins and zinc fingers from FOG family proteins are conserved between different fingers and different the finger 9 from D. melanogaster U-shaped that the N-terminal ZnF is the same as that in a U-shaped finger (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the of contact in the CCHC fingers (Fig. as as the of GATA family proteins. Interestingly, two of the contact and are the the final zinc-ligating the the His → Cys substitution in of classical zinc fingers may be required to the two in such a as to with GATA NFs. two are conserved FOG family CCHC ZnFs that are capable of interacting with the and it has been C. J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar) that protein-protein a of protein as a however, are not conserved in other zinc finger domains that are to mediate protein-protein interactions as fingers from and In to be more sequence protein binding classical fingers DNA binding the of protein that are these zinc fingers with the of a DNA In it is also interesting to that the sequences of many DNA binding zinc fingers are conserved not only the in DNA but also the other may that such domains have the finger of erythroid Kruppel-like factor it mediates interactions both with DNA and with GATA-1 (11Merika M. Orkin S. Mol. Cell. Biol. 1995; 15: 2437-2447Crossref PubMed Scopus (435) Google Scholar). of and has been used to demonstrate that these interactions are with in the the domains are the of and it has become clear in that weak interactions can play very important roles in The GATA-FOG interactions are also the C-terminal zinc finger from GATA-1 is to bind FOG proteins A.P. Visvader J.E. Turner C.A. Fujiwara Y., Yu, C. Weiss M.J. Crossley M. Orkin S.H. Cell. 1997; 90: 109-119Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar), and FOG CCHC fingers with GATA proteins A.H. C. M. Kowalski K. Mackay J. Crossley M. EMBO J. 1999; PubMed Scopus Google Scholar). has that interactions are essential for normal development Mackay J.P. Orkin S.H. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and it is interesting to as to weak interactions such as these are between proteins that are at low in the nuclear It is that concentrations of transcription factors as a result of either nuclear or other factors or It is also that the very of the in the the of these interactions for example, the other it is that such as that for the to an for the of of binding and are for the of gene expression that is required development. We that of the final in to histidine a classical CCHH the interaction of with GATA-1 in a J.M. Kowalski K. C.K. Sharpe B.K. Fox A.H. Crossley M. J.P. J. Biochem. 2000; PubMed Scopus Google Scholar). To the basis for we determined the structure of the mutant of mutant a structure with a very similar to that of the wild-type It although the of the histidine substitution are subtle in of the protein the change in the of and has a the GATA-FOG of the binding of and with the of the two the binding are The two to of the binding of wild-type but are in the binding of change in to be to the of the The results the of zinc finger domains as protein-protein interaction domains and the that weak interactions can play important roles in the regulation of development. The loss of in (16Liew C.K. Kowalski K. Fox A.H. Newton A. Sharpe B.K. Crossley M. Mackay J.P. Structure 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that results from the C → substitution the that binding is only however, suggest that such interactions are and not to can see protein-protein interactions can as a result of in domains that may have a in DNA We for of the at the of and for with