The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a Cu(I)(4)(micro-S-Cys)(6) cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant K(D) = (K(1)K(2)K(3)K(4))(1/4) = 10(-)(19) for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (beta(2) = 10(19.8)). Equivalent experiments for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar K(D) values. The estimates of K(D) were supported by independent estimates of the equilibrium constants K(ex) for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer "free" Cu(I) concentrations in a narrow range around 10(-)(19) M. The results provide quantitative support for the proposals that, in yeast, (a) "free" copper concentrations are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc --> Atx1 --> Ccc2n metabolic pathway. In addition, both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.

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Highlights d Structures of FUS ZnF bound to GGU and FUS RRM bound to an RNA stem loop were solved d The RGG motif destabilizes the RNA structure, suggesting a role in remodeling RNA d The RGG motif increases RNA binding without forming an ordered structure d RNA binding by the ZnF and RRM domains contribute to FUSmediated splicing functions

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4-and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a b-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'. The structure reveals that each zinc knuckle recognizes an AG dinucleotide separated by a single nucleotide spacer. This defines a new 5'-NGNNG-3' consensus motif that explains how Lin28 selectively recognizes pre-let-7 family members. Binding assays in cell lysates and functional assays in cultured cells demonstrate that the interactions observed in the solution structure also occur between the full-length protein and members of the pre-let-7 family. The consensus sequence explains several seemingly disparate previously published observations on the binding properties of Lin28.

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GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1͞FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.transcription ͉ factor ͉ gene expression T he erythroid transcription factor GATA-1 has become a paradigm for understanding the control of gene expression by sequence-specific DNA-binding proteins. Numerous studies (reviewed in refs. 1 and 2) have revealed that the function of GATA-1 depends on its ability to bind both DNA and other proteins such as the DNA-binding proteins PU.1, Fli-1 Sp1, and erythroid Krüppel-like factor, as well as coregulators such as LIM-only 2 protein, cyclic AMP response element-binding protein-binding protein, and the zinc-finger protein friend of GATA (FOG). It is thought that different partnerships involving GATA-1 occur at different DNA elements, in different cell types, and at distinct stages of development, and that these regulatory networks contribute to the ability of GATA-1 to direct the complex patterns of gene expression required for the differentiation of blood cells.GATA-family proteins contain two highly conserved trebleclef zinc fingers (ZnFs) domains (3): the N-terminal (NF) and the C-terminal (CF) fingers. These domains mediate both DNA and protein interactions, but despite high sequence similarity to each other (Ͼ50% identity), have quite distinct functions. The CF appears to be the main determinant of DNA-binding and recognizes sequences of the form T͞AGATAA͞G, whereas the NF can participate in binding to tandem T͞AGATAA͞G sites (ref. 4 and references therein) and can independently bind GATC motifs (5). The protein-binding specificities of the two ZnFs also differ; most notably, the NF alone is able to recruit FOG, an Ϸ1,000-aa protein (6) that contains five classic CysCys-His-His (C 2 H 2 ) and four related Cys-Cys-His-Cys (C 2 HC) ZnFs (Fig. 1A).FOG is essential for the normal development of both erythrocytes and megakaryocytes (7) and the interaction of FOG with GATA-1 is indispensable for these events (8). Of the nine ZnFs in FOG (Fig. 1A), four are not found in the tandem arrays that are generally associated with DNA-binding activity. Intriguingly, each of these four isolated domains can independently bind NF, consistent with the emerging view that single fingers can mediate protein-protein interactions (9, 10).Here, we describe the solution structure of...

The alternative splicing of mRNA is a critical process in higher eukaryotes that generates substantial proteomic diversity. Many of the proteins that are essential to this process contain arginine/serine-rich (RS) domains. ZRANB2 is a widely-expressed and highly-conserved RS-domain protein that can regulate alternative splicing but lacks canonical RNA-binding domains. Instead, it contains 2 RanBP2-type zinc finger (ZnF) domains. We demonstrate that these ZnFs recognize ssRNA with high affinity and specificity. Each ZnF binds to a single AGGUAA motif and the 2 domains combine to recognize AGGUAA (Nx)AGGUAA double sites, suggesting that ZRANB2 regulates alternative splicing via a direct interaction with pre-mRNA at sites that resemble the consensus 5 splice site. We show using X-ray crystallography that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine ''ladder.'' A number of other human proteins that function in RNA processing also contain RanBP2 ZnFs in which the RNA-binding residues of ZRANB2 are conserved. The ZnFs of ZRANB2 therefore define another class of RNA-binding domain, advancing our understanding of RNA recognition and emphasizing the versatility of ZnF domains in molecular recognition.protein structure ͉ RanBP2 zinc fingers ͉ RNA-binding proteins ͉ splicing A lmost all human genes are thought to be alternatively spliced, and it has been estimated that at least 15% of human diseases are associated with changes in RNA processing (1). The selection of splice sites is influenced heavily by the binding of accessory splicing factors to regulatory sequences in the pre-mRNA. SR proteins are splicing factors that contain a C-terminal Arg/Ser-rich (RS) domain and either 1 or 2 N-terminal RNA recognition motifs (RRMs) (2). They play crucial roles in constitutive and alternative splicing, promoting recognition of splice sites by binding to exonic splicing enhancers (ESEs). RRM domains bind ssRNA with high affinity in a sequence-specific manner, whereas RS domains appear to facilitate both protein-protein and protein-RNA interactions (3, 4). Other RS domain-containing proteins that lack a canonical RRM, termed ''SR-like'' proteins (see, for example, ref. 5) are also known to play roles in splicing.ZRANB2 (Zis, ZNF265) is an SR-like nuclear protein that is expressed in most tissues and is conserved between nematodes and mammals. It interacts with the spliceosomal proteins U1-70K and U2AF 35 and can alter the distribution of splice variants of GluR-B, SMN2, and Tra2␤ in minigene reporter assays (6, 7). As such, ZRANB2 appears to regulate splice site choice. However, in place of the canonical RNA-binding RRM domains, ZRANB2 displays 2 N-terminal RanBP2-type zinc fingers (ZnFs).RanBP2-type ZnF domains are defined by the consensus sequence W-X-C-X 2-4 -C-X 3 -N-X 6 -C-X 2 -C. They occur multiple times in at least 21 human proteins, and the fold comprises 2 distorted ␤-hairpins sandwiching a central tryptophan residue and ...