Structure and Ubiquitin Interactions of the Conserved Zinc Finger Domain of Npl4

Abstract

Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel β-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the “Ile-44” surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin. Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel β-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the “Ile-44” surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin. Protein ubiquitylation plays an important role in a large number of biological processes, including intracellular proteolysis, DNA repair, transcription, translation, signal transduction, cell cycle progression, organelle assembly, protein trafficking, and virus budding (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6906) Google Scholar, 2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, 3Hicke L. Nat. Rev. Mol. Cell. Biol. 2001; 2: 195-201Crossref PubMed Scopus (994) Google Scholar). Cells therefore dedicate an extensive array of machinery to the enzymology of ubiquitin transfer. Upon ubiquitylation, proteins must be directed to the correct intracellular locale, and pathways that utilize ubiquitin as a targeting signal therefore have effector proteins that specifically recognize ubiquitylated proteins. A number of such ubiquitin binding proteins are now known, and their study has led to the identification and characterization of several conserved ubiquitin binding motifs, including the ubiquitin-interacting motif (UIM), 1The abbreviations used are: UIM, ubiquitin-interacting motif; Ub, ubiquitin; NZF, Npl4 zinc finger; BME, β-mercaptoethanol; GST, glutathione S-transferase; EXAFS, extended x-ray absorption fine structure; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single-quantum coherence; E2, ubiquitin conjugating enzyme; E3, ubiquitin-protein isopeptide ligase. the ubiquitin-associated domain, and the ubiquitin E2 variant/UBC-like domain (2Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, 4Buchberger A. Trends Cell Biol. 2002; 12: 216-221Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 5Madura K. Cell Cycle. 2002; 1: 235-244Crossref PubMed Scopus (43) Google Scholar, 6Hofmann K. Falquet L. Trends Biochem. Sci. 2001; 26: 347-350Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 7Hofmann K. Bucher P. Trends Biochem. Sci. 1996; 21: 172-173Abstract Full Text PDF PubMed Scopus (352) Google Scholar). Each of these motifs forms an independent folding domain that can bind ubiquitin in vitro, although the molecular details of ubiquitin recognition are not yet understood in any case. Given the generality of ubiquitin (Ub) as a targeting signal, it is likely that additional ubiquitin recognition motifs remain to be identified and characterized. One example is the recently discovered Npl4 zinc finger/RanBP2 zinc finger domain (NZF) (see Fig. 1A), which was first identified in Npl4, a protein that binds ubiquitin in vitro and mediates several different ubiquitin-dependent functions of the AAA-ATPase p97/Cdc48p. A hallmark of NZF domains is the presence of four regularly spaced cysteine residues, which suggests that the motif may be a metal binding module (8Delaney S.J. Hayward D.C. Barleben F. Fischbach K.F. Miklos G.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7214-7218Crossref PubMed Scopus (77) Google Scholar, 9Nakielny S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (182) Google Scholar, 10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar, 11Babst M. Katzmann D. Snyder W. Wendland B. Emr S. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). NZF motifs appear to be quite prevalent, and our protein data base searches revealed 243 unique peptide sequences with four cysteines and an asparagine with the same spacing as those of Npl4 NZF (Fig. 1A). This ensemble of sequences also exhibited significant conservation at 12 of 24 of the remaining residues, which presumably reflects the conserved structure and function(s) of this common domain. In addition to Npl4 itself, several other NZF-containing proteins are known to be involved in ubiquitin-dependent processes. One example is Vps36p, a protein required for vacuolar protein sorting of ubiquitylated proteins in yeast (11Babst M. Katzmann D. Snyder W. Wendland B. Emr S. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). Isolated NZF domains from both Npl4 and Vps36p bind mono- and poly-Ub chains in vitro, consistent with the idea that these NZF domains serve to recognize ubiquitylated protein substrates in vivo (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). However, not all NZF-like domains bind ubiquitin, suggesting that those that do may represent a specialized subset of a much larger domain superfamily that shares a common three-dimensional fold (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). The prevalence of the NZF domain and its apparent role in ubiquitin recognition in several important biological pathways led us to study the molecular basis for NZF/Ub interactions. Toward this end, we have analyzed the sequence conservation across putative NZF domains, characterized the metal and ubiquitin binding properties of the Npl4 and Vps36p NZF domains, determined the solution structure of the NZF domain from Npl4, mapped the interaction surfaces in the Npl4 NZF/Ub complex, and tested the importance of a conserved, dipeptide motif (TF) at NZF positions 13 and 14 for ubiquitin binding. The SwissProt, PIR, PRF, and KEGG GENE data bases were searched with the pattern X6CX2CX3NX6CX2CX5, which yielded 243 unique matches in 166 different proteins (duplicates were removed manually). The apparent correlation between Asn-16 and Trp-7 (72%; see Fig. 1A) was tested by a search with the pattern X4WXCX2CX10CX2CX (5Madura K. Cell Cycle. 2002; 1: 235-244Crossref PubMed Scopus (43) Google Scholar), which produced 161 matches (in 114 proteins), 84% of which had asparagine at the variable position corresponding to Asn-16 in Npl4 NZF. Searches with the more stringent RanBP2-like pattern (12Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; PubMed Scopus Google the with both the and positions revealed conservation at the variable positions but was it the known NZF domain of Vps36p (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). Searches of the data base yielded that the motif has not characterized DNA NZF domains from Npl4 and yeast Vps36p were into proteins with between the and NZF domains A. 2002; PubMed Scopus Google Scholar). were into the Npl4 by Protein in the was with at the were by and at to protein in Npl4 NZF protein were at from of were in of A to the and for at to The NZF protein was by an The protein was and with of A and with glutathione in BME, were and for of BME, by absorption at for with of of NZF, and to by Npl4 NZF was from and protein by in BME, This yielded of Npl4 NZF. and NZF were and as that was in with or as the and to absorption and extended x-ray absorption fine structure zinc was removed by of Npl4 NZF in a zinc two at the of Npl4 NZF are in our but are not in any of the Npl4 NZF was characterized by and The of zinc was analyzed by the protein using and the zinc was analyzed by absorption and ubiquitin were and as G. M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). Npl4 NZF was of or by to a of and in a cell in at a protein of were at at the using an for were using a array were at using a were at in the in the and in the from in a data represent the of were with of a zinc absorption the first to were at both and data were analyzed using the of the which to the in the and the were to using data were to to data of characterized were using and functions in for a This in a of and an of which were used for data of the NZF were at and were to the for structure were Npl4 NZF in BME, in were and in to cysteine and were at a with a and and were using a of PubMed Scopus Google Scholar, S. EMBO J. 2002; 21: PubMed Scopus Google Scholar), that of the and were and analyzed (Fig. were using TOCSY, and with using a of and the for of were using a of A. S. F. Wang G. PubMed Scopus Google Scholar), TOCSY, and were for and for the first three and S. J. B. PubMed Scopus Google Scholar, J. PubMed Scopus Google and three-dimensional J. 3: Scopus Google Scholar, J. Scopus Google were used to were from a three-dimensional S. F. A. J. PubMed Scopus Google Scholar). were identified using a with the to for the one A. J. PubMed Scopus Google B. 2002; PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar, PubMed Scopus Google were from the in at and for the free and forms of NZF were characterized in M. D. J. 2001; PubMed Scopus Google to a of were using NZF were using in P. K. J. Mol. Biol. PubMed Scopus Google and in P. J. M. Warren G.L. Biol. 1998; PubMed Scopus Google through a with NOE four and 13 J. J. B. PubMed Scopus Google Scholar). of used NOE which the fold of the domain and revealed the the coordinated for and to zinc as as and NOE and structure were using the P. K. J. Mol. Biol. 2002; PubMed Scopus Google Scholar), which produced the same structure as was determined were analyzed using J. 1996; PubMed Scopus Google Scholar), M. K. J. Mol. 1996; PubMed Scopus Google Scholar), and were with for NZF is the ensemble of using the is the same ensemble of at to and of in is the ensemble of using the is the same ensemble of at to and of in and as determined by the were used to not for into were the of of or from have of from are in of of or from have of from are in of or from have of from are in using to the and were using the is the ensemble of using the P. K. J. Mol. Biol. PubMed Scopus Google Scholar). is the same ensemble of at to and of in P. J. M. Warren G.L. Biol. 1998; PubMed Scopus Google and as determined by the were used to not for into were the from have of from are in using J. 1996; PubMed Scopus Google and were using the M. K. J. Mol. 1996; PubMed Scopus Google in a were at in the ubiquitin binding the Npl4 NZF domain, ubiquitin was into Npl4 NZF at of the Npl4 NZF binding ubiquitin, Npl4 NZF was into ubiquitin at of S. J. B. PubMed Scopus Google were at and were using the Emr M. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar, S. A. J. S.J. Nat. Biol. 1996; 3: PubMed Scopus Google Scholar). for ubiquitin were from the at binding for and NZF proteins and NZF surfaces were as Wang M. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). were at in BME, The NZF a Npl4 and Vps36p NZF used in our studies the conserved NZF and the sequence and for the Npl4 NZF are in Fig. Npl4 NZF was in and the protein was to (see The presence of a zinc ion was using absorption of of Npl4 and (Fig. and The to the revealed that the zinc four and an of The was consistent with a the zinc therefore that Npl4 NZF a ion using the four conserved of Npl4 and for Npl4 NZF were using heteronuclear (see Fig. and for a metal the four cysteine exhibited that were between free cysteine and cysteine The structure of Npl4 NZF was using NOE which were to both the protein structure and the zinc for and zinc The ensemble of was of (Fig. and with from the structure of and of Npl4 NZF four β-strands that form two and The a and the three were in A. J. PubMed Scopus Google Scholar). In the of two antiparallel that The four are by a compact that and and two metal binding and that form and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Mol. Biol. 2001; PubMed Scopus Google Scholar, Scholar). The rubredoxin was in the of and has at the zinc in a number of proteins Scholar, A. K. G. 1996; PubMed Scopus Google Scholar, M. J. Mol. Biol. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar). are that two β-strands and two metal binding positions and The the at positions and to with the first cysteine and the at position to with the cysteine This metal binding is by a at position which a in the details of this are in proteins the rubredoxin for example in M. S. J. J. Biol. PubMed Scopus Google Scholar). The in Npl4 NZF are different from rubredoxin both NZF at position and This the and the pattern (see Fig. and in the the of Asn-16 a to the first cysteine position the in the two Npl4 are to one and to other rubredoxin (Fig. in all of the that are conserved across NZF domains The NZF structure is by a which the zinc and of the conserved Trp-7 and The Trp-7 is to the of and the a of with from both and One of the Trp-7 is from by the of and the other is by the Asn-16 The conserved Asn-16 also to and its and across to the and (Fig. for conserved NZF in NZF domain (see Fig. The NZF is in it not to the of metal binding at position of the rubredoxin ubiquitin binding position of from NZF at position of the rubredoxin of in NZF domain (see Fig. The NZF is in it not to the sequence in a in the Npl4 NZF motif can bind both mono- and as analyzed by (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). were used to the interaction surfaces of both proteins the Npl4 (Fig. The Npl4 was in as is for with in the (see in therefore the of for the free and A of of ubiquitin were the of of Npl4 NZF (Fig. of the revealed that of ubiquitin see Fig. The ubiquitin the three of the the of which presumably to the NZF binding A of the binding is an surface by and in Fig. This to the surface of ubiquitin, which has by to in and virus budding C.M. L. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, B. A. J. 2002; PubMed Scopus Google Scholar). also NZF which is chains linked the proteins for Our studies do not NZF binding is or by at this In the a of of NZF were the of of ubiquitin (Fig. of NZF ubiquitin binding were the rubredoxin the zinc and the zinc and as as and its ubiquitin, the binding surface NZF is but also it that the between NZF and is likely to other that as as interactions that provide and by the NZF the interaction between Npl4 NZF and free ubiquitin, we binding in which ubiquitin was to bind to (Fig. to the Npl4 NZF surface with and the interaction was as ubiquitin not bind to a surface (Fig. A of the Npl4 NZF/Ub binding data to a yielded a of that this binding may the that Npl4 NZF ubiquitylated proteins free and may therefore additional binding through with the of ubiquitin, additional in the Npl4 protein complex, that the of the Npl4 NZF/Ub interaction is to those for other characterized ubiquitin recognition motifs. One example is the UIM, a motif with ubiquitin binding from several that bind to ubiquitin with in the and A. D. Biochem. Res. 2002; PubMed Scopus Google used to that the motif from ubiquitin with a of and Nat. Cell Biol. 2002; PubMed Scopus Google to that ubiquitin to a the with a of binding were also to the interaction of ubiquitin with the NZF domain of yeast Vps36p (Fig. the and of the Vps36p NZF/Ub interaction were to those of the Npl4 NZF/Ub The that Vps36p NZF binds ubiquitin is not from a biological Vps36p is required for the sorting of ubiquitylated proteins into the yeast (11Babst M. Katzmann D. Snyder W. Wendland B. Emr S. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). However, the is of from a Vps36p NZF has an asparagine in of the conserved Trp-7 that forms the of Npl4 NZF. Our that the Vps36p NZF can form a ubiquitin binding the of this Our that ubiquitin binds to Npl4 NZF by the 13TF14 Npl4 NZF motifs appear to into several different that are characterized by dipeptide sequences at positions 13 and that NZF motifs that bind ubiquitin contain those that bind other have other of at positions 13 and this the Npl4 NZF 13TF14 were and in to and which are the in NZF motifs from and in Fig. all four single the for ubiquitin binding at and the two not bind to ubiquitin therefore that the 13TF14 dipeptide is an of the ubiquitin binding of Npl4 NZF. Our studies that the Npl4 NZF domain forms a zinc binding module and provide a for conserved in this domain NZF now the ubiquitin-associated domain J. Nat. Biol. 1998; PubMed Scopus Google Scholar, J. J. Mol. Biol. 2002; PubMed Scopus Google Scholar), ubiquitin E2 variant/UBC-like domain S. EMBO J. 2002; 21: PubMed Scopus Google Scholar, S. L. W. Nat. Biol. 2001; PubMed Scopus Google Scholar, Hofmann C.M. 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In all of these a single ion is coordinated by four by the that two in Fig. zinc binding from Npl4 NZF and are and with of The of the zinc binding domains of and Npl4 are also although Npl4 NZF is more compact the other two and to represent a of the the zinc of Npl4 NZF is also to the binding of the rubredoxin protein to (Fig. these are between Npl4 NZF and the other the that all may interactions. the appear to the that the NZF fold a for metal binding. and of the NZF have identified 243 sequences from 166 different proteins that the and spacing of the four cysteines and single asparagine of Npl4 NZF (see and Fig. 1A). had the presence of a sequence motif in the nuclear J. G. Cell. Full Text PDF PubMed Scopus Google Scholar, J. D. G. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, N. N. N. K. K. U. M. PubMed Scopus Google Scholar, S. A. 2001; 12: PubMed Scopus Google Scholar). Searches the RanBP2/Nup358 zinc finger motif have required the presence of to the Npl4 NZF cysteines as as Trp-7 and Asn-16 but the spacing between the first two cysteines to between and (12Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; PubMed Scopus Google Scholar). search a number of proteins with that do not our stringent spacing but NZF proteins Vps36p the Trp-7 it that more known proteins contain NZF motifs, with the the search it be to with that the NZF motif that may be a of protein that the NZF and zinc motifs. The prevalence of the NZF motif it important to the that different NZF the Npl4 and Vps36p NZF motifs both bind specifically to ubiquitin, as do the NZF motifs from two other and (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). However, the NZF domains from two other and do not bind ubiquitin in vitro (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). all four known NZF motifs with ubiquitin binding the 13TF14 the two known NZF motifs that do not bind ubiquitin have different at these positions in and in that the basis of be at this position in the rubredoxin a Scholar). the presence of at position 13 suggests a role in binding. with this our studies that the 13TF14 dipeptide forms the binding for Npl4 NZF the the of any NZF The chains of and form an surface the Npl4 NZF motif, and our that this is the of ubiquitin binding. that and also may be used by NZF domains biological is to recognize ubiquitin. with these our that both and are for ubiquitin and studies of the Npl4 that the chains the ubiquitin L. M. D. and W. this idea we tested for between the of at positions 13 and 14 of the 243 NZF domains identified by our search we that a is at position a large at position 14 In any is at position a large of the This suggests that the NZF motif forms a that the 13TF14 dipeptide to recognize the surface of ubiquitin. Our sequence also revealed that the dipeptide at NZF positions 13 and 14 that this dipeptide may also form a recognition for NZF domain. several including RanBP2/Nup358 and contain of NZF domains that bind the form of the S. Shaikh S. Burke B. Dreyfuss G. EMBO J. 1999; 18: 1982-1995Crossref PubMed Scopus (182) Google Scholar, G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus (77) Google Scholar). are composed of of NZF domains, suggesting that may be the protein binding for this class of NZF domain. The zinc also bind H.H. D. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus (77) Google Scholar), which is yet protein that can be by the NZF domain. NZF motif was first in the Npl4, which is a of the with (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). is one of at two that the AAA-ATPase to protein The is required for at three including of ubiquitylated proteins from the into the and the ubiquitin-dependent of and in the and by Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, B. Y. Nat. Rev. Mol. Cell. Biol. 2002; 3: PubMed Scopus Google Scholar). is also required for an as yet that to the of a nuclear M. H.H. D. Warren G. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). the role of in and to be can bind ubiquitin by means of the Npl4 NZF domain and can the (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). One is therefore that functions by ubiquitylated protein substrates in the and which the of to its substrates of the or to from other proteins. the other known which functions in the of the D. P. Warren G. PubMed Scopus Google and of the nuclear M. H.H. D. Warren G. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar), also the to ubiquitin (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar). In that ubiquitin recognition is by a ubiquitin-associated domain in and the ubiquitylated to be Vps36p example in which an NZF motif to ubiquitylated proteins into a biological have that the Vps36p NZF can bind ubiquitin and this and have that Vps36p is a of the complex, which is required for sorting of ubiquitylated proteins into (11Babst M. Katzmann D. Snyder W. Wendland B. Emr S. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). is therefore to that Vps36p its NZF motif to recognize ubiquitylated protein are through the In to Npl4, is the that Vps36p also as an for a that functions in this M. Wendland B. Emr EMBO J. 1998; PubMed Scopus Google Scholar, S. B. A. S. K. J. Mol. Biol. 2001; PubMed Scopus Google Scholar, N. P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Our studies also that other characterized proteins that contain NZF motifs of the class also ubiquitin binding this is the that a number of these proteins contain additional domains that are linked to of ubiquitin proteins with finger motifs that bind (10Meyer H.H. Wang Y. Warren G. EMBO J. 2002; 21: 5645-5652Crossref PubMed Scopus (296) Google Scholar, G. K. 1999; PubMed Scopus Google Scholar), proteins that bind ubiquitin M. M. EMBO J. 1999; 18: PubMed Scopus Google Scholar), proteins with their domains and proteins that contain other known ubiquitin binding domains are also a number of proteins with NZF motifs of the class known to ubiquitin, and we that in this are likely to be In the NZF motif is a zinc binding module that can be into proteins and used to bind ubiquitin, and other proteins. Our studies reveal the NZF the NZF/Ub and provide for of this versatile and of Protein for of DNA and for protein for absorption the of for protein the for for the ubiquitin and and and for of the