Most bacteriophages possess long tails, which serve as the conduit for genome delivery. We report the solution structure of the N-terminal domain of gpV, the protein comprising the major portion of the noncontractile phage tail tube. This structure is very similar to a previously solved tail tube protein from a contractile-tailed phage, providing the first direct evidence of an evolutionary connection between these 2 distinct types of phage tails. A remarkable structural similarity is also seen to Hcp1, a component of the bacterial type VI secretion system. The hexameric structure of Hcp1 and its ability to form long tubes are strikingly reminiscent of gpV when it is polymerized into a tail tube. These data coupled with other similarities between phage and type VI secretion proteins support an evolutionary relationship between these systems. Using Hcp1 as a model, we propose a polymerization mechanism for gpV involving several disorder-toorder transitions.NMR structure ͉ disordered regions ͉ macromolecular assembly

Conformational changes, including local protein folding, play important roles in protein-DNA interactions. Here, studies of the transcription factor Ets-1 provided evidence that local protein unfolding also can accompany DNA binding. Circular dichroism and partial proteolysis showed that the secondary structure of the Ets-1 DNA-binding domain is unchanged in the presence of DNA. In contrast, DNA allosterically induced the unfolding of an alpha helix that lies within a flanking region involved in the negative regulation of DNA binding. These findings suggest a structural basis for the intramolecular inhibition of DNA binding and a mechanism for the cooperative partnerships that are common features of many eukaryotic transcription factors.

The use of a short, three-residue Cu(2+)-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu(2+) can be converted into distance restraints and used for structure refinement by using a new routine, PMAG, that has been written for the structure calculation program CNS. The utility of the approach is demonstrated with an application to ATCUN-tagged ubiquitin. Excellent agreement between measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an ␣-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1⌬N301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1⌬N301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1⌬N301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1⌬N300 dimer, confirming that the latter represents a domain-swapped species.15 N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ϳ15-and ϳ75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.Gene expression can be controlled by modulating the DNA binding affinity of sequence specific transcription factors. Similar to several other transcription factors, the DNA binding of Ets-1 is modulated by an autoinhibitory module that provides a route to biological regulation (1). The Ets-1 inhibitory module is composed of sequences flanking the winged helix-turn-helix (HTH) 1 DNA binding ETS domain (2, 3). When these sequences are deleted, as in an alternatively spliced isoform of Ets-1, or when their structural elements are disrupted by mutations, as in the case of the oncogenic v-Ets, the affinity of Ets-1 for its target DNA sites is enhanced by 10-to 20-fold (4 -6). In a cellular context, this module is essential for response to different regulatory signals. DNA binding of Ets-1 is enhanced 10-to 20-fold through a partnership with the transcription factor RUNX1 (CBF␣2/AML1) (7). Conversely, in activated T-cells, phosphorylation of a serine-rich region (residues 244 -300) inhibits the DNA binding of Ets-1 by another ϳ50-fold (8). Importantly, these two effects require an intact inhibitory module.Mechanistic insight into autoinhibition has come from the observation that the Ets-1 inhibitory module changes conformation upon binding to DNA. Initial secondary structural studies performed in our laboratories demonstrated that this module is composed of four coupled ␣-helices, locat...

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA-or proteinbinding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly f lexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.Transcription factor families are defined by highly conserved DNA-binding domains that display similar DNA recognition properties. The means by which individual family members control different genes therefore must be determined by regulatory mechanisms that enhance the specificity of DNA binding. In the ets gene family, which includes at least 18 members in the human genome, partnerships with additional transcription factors, as well as posttranslational modifications, help dictate specificity for distinct targets (1). These regulatory mechanisms converge on a highly conserved Ϸ80-aa region termed the Pointed (PNT) domain (2).The PNT domain occurs in approximately one-third of the ets proteins, including Ets-1, Ets-2, GABP␣, and TEL from vertebrates, and PNT-P2 and Yan from Drosophila (Fig. 1). This domain is proposed to mediate protein-protein interactions and to be regulated by ras-dependent signaling because of the presence of an adjacent mitogen-activated protein (MAP) kinase phosphorylation site (1). In particular, the PNT domain is implicated in the self-association of chimeric oncoproteins, identified in human leukemias, that result from chromosomal translocations of the gene encoding the ets protein TEL with segments of genes encoding several tyrosine kinases or the acute myeloid leukemia (AML)-1B transcription factor (3-9).To date sequence conservation has defined the PNT domain, yet it has not been established that this region is a structural module that acts in a biological context. To create a framework for understanding the role of the PNT domain in the regulation of a variety of ets proteins and in the oncogenic potential of TEL fusion proteins, we have characterized structurally a fragment of Ets-1 that includes this domain and the adjacent MAP kinase phosphorylation site. MATERIALS AND METHODSProtein Samples. DNA sequences encoding Ets-1 , Ets-1 (29 -138) , and Ets-1 were PCR-amplified from the full-length murine ets-1 cDNA and...

The tertiary fold of the epsilon subunit of the Escherichia coli F1F0 ATPsynthase (ECF1F0) has been determined by two- and three-dimensional heteronuclear (13C, 15N) NMR spectroscopy. The epsilon subunit exhibits a distinct two domain structure, with the N-terminal 84 residues of the protein forming a 10-stranded beta-structure, and with the C-terminal 48 amino acids arranged as two alpha-helices running antiparallel to one another (two helix hairpin). The beta-domain folds as a beta-sandwich with a hydrophobic interior between the two layers of the sandwich. The C-terminal two-helix hairpin folds back to the N-terminal domain and interacts with one side of the beta-domain. The arrangement of the epsilon subunit in the intact F1F0 ATP synthase involves interaction of the two helix hairpin with the F1 part, and binding of the open side of the beta-sandwich to the c subunits of the membrane-embedded F0 part.

The solution structure of the 33 kDa complex between the dimeric DNA-binding core domain of the transcription factor MEF2A (residues 1±85) and a 20mer DNA oligonucleotide comprising the consensus sequence CTA(A/T) 4 TAG has been solved by NMR. The protein comprises two domains: a MADS-box (residues 1±58) and a MEF2S domain (residues 59± 73). Recognition and speci®city are achieved by interactions between the MADS-box and both the major and minor grooves of the DNA. A number of critical differences in protein±DNA contacts observed in the MEF2A±DNA complex and the DNA complexes of the related MADS-box transcription factors SRF and MCM1 provide a molecular explanation for modulation of sequence speci®city and extent of DNA bending (~15 versus~70°). The structure of the MEF2S domain is entirely different from that of the equivalent SAM domain in SRF and MCM1, accounting for the absence of cross-reactivity with other proteins that interact with these transcription factors.

Ets‐1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets‐1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C‐terminus. The ETS domain folds into a helix‐turn‐helix motif on a four‐stranded anti‐parallel beta‐sheet scaffold. This structure places Ets‐1 in the winged helix‐turn‐helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets‐1 with DNA. The C‐terminal sequence of Ets‐1, which is mutated in the v‐Ets oncoprotein, forms an alpha‐helix that packs anti‐parallel to the N‐terminal helix of the ETS domain. In this position, the C‐terminal helix is poised to interact directly with an N‐terminal inhibitory region in Ets‐1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets‐1 DNA binding.