Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1601-1606, February 1996 Biochemistry In vivo and in vitro characterization of the Bi and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML (B-box domain/nuclear bodies) KATHERINE L. B. BORDEN*, JOHN M. LALLY*, STEPHEN R. MARTINt, NICOLA J. O'REILLYT, ELLEN SOLOMON§, AND PAUL S. FREEMONT*¶ *Protein Structure Laboratory, fPeptide Synthesis Laboratory, and §Somatic Cell Genetics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom; and tDivision of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom Communicated by Paul Nurse, Imperial Cancer Research Fund, London, U.K, November 22, 1995 (received for review August 31, 1995) ABSTRACT Acute promyelocytic leukemia (APL) has (see refs. 2 and 14). The first region, the RING motif, defines been ascribed to a chromosomal translocation event which a family of proteins expressed in organisms ranging from plants results in a fusion protein comprising the PML protein and to viruses, several of which have been implicated in oncogen- retinoic acid receptor a. PML is normally a component of a esis (for review, see ref. 15). Recently the three-dimensional nuclear multiprotein complex which is disrupted in the APL structure of the PML RING finger has been determined and disease state. Here, two newly defined cysteine/histidine-rich the structural information used to probe the importance of protein motifs called the B-box (Bi and B2) from PML have various structural features in vivo (16). Within the RING finger been characterized in terms of their effect on PML nuclear family, a second cysteine-rich motif has been identified called body formation, their dimerization, and their biophysical the B-box (14, 15, 17). Most B-box family members possess a properties. We have shown that both peptides bind Zn2+, RING finger and either one or two B-box motifs followed which induces changes in the peptides' structures. We dem- closely by a predicted a-helical coiled-coil dimerization do- onstrate that mutants in both Bi and B2 do not form PML main forming a tripartite motif (5, 14). The spacing between nuclear bodies in vivo and have a phenotype that is different the three elements of the motif is highly conserved among from that observed in the APL disease state. Interestingly, family members, suggesting that the positions of each domain these mutations do not affect the ability of wild-type PML to relative to the others is of functional importance (5, 14). dimerize with mutant proteins in vitro, suggesting that the Bi The B-box family comprises a number of transcription and B2 domains are involved in an additional interaction factors, ribonucleoproteins, and protooncogene products, central to PML nuclear body formation. This report in which includes PML, the ret finger protein (RFP) and conjunction with our previous work demonstrates that the TIF1(T18) (see ref. 15 and references therein). These proteins PML RING-B1/B2 motif plays a fundamental role in forma- are oncogenic in humans and mice when found as transloca- tion of a large multiprotein complex, a function that may be tions that include the tripartite domain recombined with other common to those unrelated proteins which contain the motif. genes. Recently, TIF1 has been shown to interact with several nuclear receptors in vivo and is proposed to mediate the ligand-dependent transcriptional activation function of nu- The human PML gene is associated with APL (acute promy- clear receptors (18). PML and TIF1 possess two B-box-like elocytic leukemia), which arises due to a block in normal domains, which appear to form a subset of the B-box family as differentiation of promyelocytes (for review, see ref. 1). In highlighted by the presence of an extra conserved cysteine APL cells, PML is fused with the retinoic acid receptor a residue (see Fig. 1). EFP, an estrogen-responsive gene product, (RARa) following a reciprocal chromosomal translocation is another family member that has two B-boxes and is thought t(15;17)(q22q21) (2-5). The PML protein has been shown to to represent an estrogen-responsive transcription factor me- be part of a nuclear multiprotein complex distinct from small diating phenotypic expression due to estrogen action (19). The nuclear ribonucleoproteins and nucleoli (6-10). These PML B-box domain has been found in the breast cancer locus nuclear bodies become disrupted in leukemic cells; however, lA1.3B (20), which has one B-box and a coiled-coil domain and treatment with retinoic acid reverses these changes (8-10). It appears tightly linked to the BRCA-1 gene, which interestingly has been shown that the PML-RARa fusion product can bind possesses a RING finger but no B-box or coiled-coil domains to both normal PML (8, 10) as well as RXR, another retinoic (21, 22). Bi and B2 B-boxes are also present in a putative acid receptor family member (retinoid X receptor), forming ataxia-telangiectasia group D protein (ATDC), which possesses heterodimers (11). This leads to speculation that there is a the two B-box domains and a coiled-coil domain but no RING dominant-negative effect of PML-RARa over both the PML finger (23), suggesting that the B-box and RING fingers can protein and the RXR pathways. PML, but not PML-RARa, have independent molecular functions. has been shown to be a growth suppressor both in vitro and in The best characterized B-box domain thus far reported is vivo (12), suggesting that sequestration of normal PML by that from Xenopus nuclear factor XNF7. Our previous work PML-RARa would affect the growth suppressor activity of has shown that, although there are seven potential metal PML and sequestration of RXR, the induction of differenti- ligands, the XNF7 B-box peptide binds only one Zn2+ per ation (12). It is suggested that both events may be necessary for molecule and the Zn2+ is bound tetrahedrally (24). Therefore, leukemogenesis (12, 13). of the seven potential ligands conserved in XNF7 B-box (see The PML coding sequence contains three novel cysteine- Fig. 1), only four are used in Zn2+ ion ligation within the B-box rich metal binding regions and a predicted a-helical coiled-coil monomer. We have now determined the ligation scheme and The publication costs of this article were defrayed in part by page charge Abbreviations: APL, acute promyelocytic leukemia; RARa, retinoic payment. This article must therefore be hereby marked "advertisement" in acid receptor a; RXR, retinoid X receptor; RFP, ret finger protein. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1601 Downloaded by guest on October 2, 2021 1602 Biochemistry: Borden et al. Proc. Natl. Acad. Sci. USA 93 (1996) three-dimensional structure of the XNF7 B-box domain in the A presence of Zn2+ (25). The B-box structure comprises two RING finger B1 B2 Coiled-coil (3-strands, two helical turns, and three extended loop regions and represents a novel zinc finger fold with an unusual ligation scheme. The conserved residues used in ligation are labeled in Fig. 1. Interestingly, two of the three remaining conserved residues, Cys-17 and Cys-25 as well as Asp-20, which is a Eag deletion cysteine in PML Bl and B2, are on a flexible loop in the XNF7 B B-box structure (Fig. 1B). Mutations of Cys-17, Cys-25, and PML Bi (124-166) DAQAVC TRC KESADFWCFECEQLLCAKCFEA HQWFLKHEARPL Asp-20 did not affect the ability of the XNF7 B-box monomer PML B2 (184-232) TNNIFCSNPNH RTPTLTSIYCRGCSKPLCCSCALLDSSH SELKCDISAE to fold or bind Zn2+ (25). This suggests that these conserved XNF7 (219-260) RPLEKg SEP DERLKLYCKDDGTLSCVIqRDSLK g ASHNFLPI residues might form a potential interface for association with 1 10 20 30 40 other proteins via metal-mediated intermolecular interaction. Tat protein, for FIG. 1. (A) Tripartite domain for PML. Bi and B2 refer to B-boxes In the human immunodeficiency virus (HIV) and coiled-coil refers to the predicted a-helical coiled-coil domain. example, conserved metal ligands are used to form intermo- Boundaries of the Eag deletion are also shown as well as the position lecular dimers using divalent cations (26). of the double point mutations (v). (B) Sequence alignment of PML Bi A number of studies have recently been initiated aimed at and B2 B-boxes and the XNF7 B-box for which the three-dimensional determining the molecular function of the B-box domain. structure is known. Conserved cysteine and histidine residues are Deletions of the XNF7 B-box domain results in a loss of labeled (*) and are shown in boldface with those residues known to be binding to mitotic chromosomes (27), while deletions and Zn2+ ligands from the XNF7 B-box monomer structure (underlined). point mutations in the B-box of PwA33 results in loss of Extra conserved cysteine in PML Bi and B2 is in italics and is association with the lampbrush loops of chromosomes in the equivalent to Asp-20 in XNF7 B-box. Flexible loop region in XNF7 B-box (residues 17-25) is double underlined. Numbering refers to the oocyte nucleus in Pleurodeles (28, 42). These data at least XNF7 B-box peptide numbering and bracketed numbers refer to the suggest that the B-box domain of XNF7 and PwA33 is impor- whole protein sequence. Cys-21 and Cys-24 from PML B2 are equiv- tant for the association of these proteins with subcellular alent to Cys-17 and Cys-20, respectively, in PML Bi and to Cys-20 and structures during Xenopus and Pleurodeles development, al- Asp-20 in XNF7 B-box.