Solution Structure of a CUE-Ubiquitin Complex Reveals a Conserved Mode of Ubiquitin Binding

AbstractMonoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins

Sequence-Specific DNA Recognition by Steroidogenic Factor 1: A Helix at the Carboxy-Terminus of the DNA Binding Domain is Necessary for Complex Stability

Abbreviated title: Structure-Function of SF1-DNA Interactions Key words: SF1; nuclear hormone receptor; DNA recognition; NMR; solution structure Disclosure of Potential Conflicts of Interest: K.E.M. has consulted for World Book Science Inc., has equity interests in Ligand Pharmaceuticals Inc., and received lecture fees from Serono Inc., but has no conflicts with entities directly related to the material being published. All other authors have nothing to disclose. This is an un-copyedited author manuscript copyrighted by The Endocrine Society. This may not be duplicated or reproduced, other than for personal use or within the rule of "Fair Use of Copyrighted Materials" (section 107, Title 17, U.S. Code) without permission of the copyright owner, The Endocrine Society. From the time of acceptance following peer review, the full text of this manuscript is made freely available by The Endocrine Society at http://www.endojournals.org/. The final copy edited article can be found at http://www.endojournals.org/. The Endocrine Society disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it by the National Institutes of Health or other parties. base-pair DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibingene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, NGFI-B and ERR2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

Solution NMR studies of apo-mSin3A and -mSin3B reveal that the PAH1 and PAH2 domains are structurally independent

The evolutionarily conserved mammalian Sin3 (mSin3) transcriptional corepressor interacts with a diverse array of transcription factors mainly through two PAH (paired amphipathic helix) domains located near the N terminus. Previous studies suggested the possibility of interdomain interactions involving the PAH domains. Here, we show that the domains are structurally independent and the properties of the individual domains, such as the conformational heterogeneity and the ability of mSin3A PAH2 to homodimerize, are preserved in constructs that span both PAH domains. Our results thus suggest that the N-terminal segments of the Sin3 proteins are broadly available for interactions with other proteins and that the PAH domains are organized into structurally independent modules. Our data also rule out any heterotypic association between the paralogous mSin3A and mSin3B proteins via interactions involving the mSin3A PAH2 domain

Monster: inferring non-covalent interactions in macromolecular structures from atomic coordinate data

A web application for inferring potentially stabilizing non-bonding interactions in macromolecular structures from input atomic coordinate data is described. The core software, called Monster, comprises a PERL wrapper that takes advantage of scripts developed in-house as well as established software in the public domain to validate atomic coordinate files, identify interacting residues and assign the nature of these interactions. The results are assembled and presented in an intuitive and interactive graphical format. Potential applications of Monster range from mining and validating experimentally determined structures to guiding functional analysis. Non-commercial users can perform Monster analysis free of charge at http://monster.northwestern.edu

NMR structural studies of intramolecular (Y+)_n•(R+)n(Y-)n DNA triplexes in solution: imino and amino proton and nitrogen markers of G.cntdot.AT base triple formation

We reported previously on NMR studies of (Y+)_n•(R+)_n(Y-)_n DNA triple helices containing one oligopurine strand (R)_n and two oligopyrimidine strands (Y)_n stabilized by T-AT and C^+•GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidineadenosine base pair to form a G•TA triple in an otherwise (Y+)_n•(R+)_n(Y-)_n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)_n•(R+)_n(Y-)_n triplex with the strands linked through two T_5 loops and that contains a central G•TA triple flanked by T•AT triples. The G•TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H_2O solution, pH 4.85, at 5°C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T•AT triples, as well as the guanosine imino and cytidine amino protons involved in Watsonxrick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C^+•GC triples in the NOESY spectrum of the G•TA triplex. The NMR data are consistent with the proposed pairing alignment for the G•TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair. We detect a set of NOES between adjacent triples that establishes that the G•TA triple stacks between flanking T•AT triples in the G•TA triplex. The imino protons of the G•TA triplex in H_2O, pH 4.85, broaden sequentially between 32 and 42 °C with the thymidine imino protons in the central G•TA triple most stable to increasing temperature. The intramolecular G-TA triplex is stable at pH 5.0 and 5 °C but converts fully to the Watson-Crick hairpin duplex on raising the pH to 6.5 at this temperature. The assigned guanosine and thymidine imino protons have been linked to their attached ring nitrogens in the G•TA triplex from an analysis of the proton-detected natural abundance nitrogen-proton two-dimensional correlation spectrum. The guanosine N 1 nitrogen in the G•TA triple resonates 10 ppm to high field of the loop thymidine N3 nitrogens in contrast to their attached imino protons, which exhibit similar chemical shifts. These results demonstrate the capabilities of the NMR approach in monitoring individual base triples and their pairing alignments, as well as establishing that the G•TA triple can be readily accommodated in an otherwise intramolecular (Y+)_n•(R+)_n(Y-)_n triple helix in solution

NMR structural studies of intramolecular (Y+)_n•(R+)n(Y-)n DNA triplexes in solution: imino and amino proton and nitrogen markers of G.cntdot.AT base triple formation

We reported previously on NMR studies of (Y+)_n•(R+)_n(Y-)_n DNA triple helices containing one oligopurine strand (R)_n and two oligopyrimidine strands (Y)_n stabilized by T-AT and C^+•GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidineadenosine base pair to form a G•TA triple in an otherwise (Y+)_n•(R+)_n(Y-)_n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)_n•(R+)_n(Y-)_n triplex with the strands linked through two T_5 loops and that contains a central G•TA triple flanked by T•AT triples. The G•TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H_2O solution, pH 4.85, at 5°C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T•AT triples, as well as the guanosine imino and cytidine amino protons involved in Watsonxrick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C^+•GC triples in the NOESY spectrum of the G•TA triplex. The NMR data are consistent with the proposed pairing alignment for the G•TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair. We detect a set of NOES between adjacent triples that establishes that the G•TA triple stacks between flanking T•AT triples in the G•TA triplex. The imino protons of the G•TA triplex in H_2O, pH 4.85, broaden sequentially between 32 and 42 °C with the thymidine imino protons in the central G•TA triple most stable to increasing temperature. The intramolecular G-TA triplex is stable at pH 5.0 and 5 °C but converts fully to the Watson-Crick hairpin duplex on raising the pH to 6.5 at this temperature. The assigned guanosine and thymidine imino protons have been linked to their attached ring nitrogens in the G•TA triplex from an analysis of the proton-detected natural abundance nitrogen-proton two-dimensional correlation spectrum. The guanosine N 1 nitrogen in the G•TA triple resonates 10 ppm to high field of the loop thymidine N3 nitrogens in contrast to their attached imino protons, which exhibit similar chemical shifts. These results demonstrate the capabilities of the NMR approach in monitoring individual base triples and their pairing alignments, as well as establishing that the G•TA triple can be readily accommodated in an otherwise intramolecular (Y+)_n•(R+)_n(Y-)_n triple helix in solution

NMR structural studies on a nonnatural deoxyribonucleoside which mediates recognition of GC base pairs in pyrimidine•purine•pyrimidine DNA triplexes

As a part of our ongoing efforts to define the structural aspects of unusual pairing alignments in DNA triplexes by nuclear magnetic resonance spectroscopy, we have examined the structural role of a nonnatural deoxyribonucleoside, P1, that has been shown to mediate the recognition of GC base pairs in pyrimidine-purine-pyrimidine DNA triplexes [Koh, J.S., & Dervan, P.B. (1992) J. Am. Chem Soc. 114, 1470]. A qualitative interpretation of the NMR data indicates that this analog of protonated cytosine is readily accommodated in the third strand segment of an intramolecular triplex system. Furthermore, the observed NOE patterns position the imino and amino protons of P1 opposite the N^7 and O^6 atoms of guanine, respectively, consistent with the previously proposed pairing scheme

NMR structural studies on a nonnatural deoxyribonucleoside which mediates recognition of GC base pairs in pyrimidine•purine•pyrimidine DNA triplexes

As a part of our ongoing efforts to define the structural aspects of unusual pairing alignments in DNA triplexes by nuclear magnetic resonance spectroscopy, we have examined the structural role of a nonnatural deoxyribonucleoside, P1, that has been shown to mediate the recognition of GC base pairs in pyrimidine-purine-pyrimidine DNA triplexes [Koh, J.S., & Dervan, P.B. (1992) J. Am. Chem Soc. 114, 1470]. A qualitative interpretation of the NMR data indicates that this analog of protonated cytosine is readily accommodated in the third strand segment of an intramolecular triplex system. Furthermore, the observed NOE patterns position the imino and amino protons of P1 opposite the N^7 and O^6 atoms of guanine, respectively, consistent with the previously proposed pairing scheme

Solution Nuclear Magnetic Resonance Studies of the Ligand-Binding Domain of an Orphan Nuclear Receptor Reveal a Dynamic Helix in the Ligand-Binding Pocket

The ligand-binding domains (LBDs) of the NR5A subfamily of nuclear receptors activate transcription via ligand-dependent and ligand-independent mechanisms. The <i>Drosophila</i> Ftz-F1 receptor (NR5A3) belongs to the latter category, and its ligand independence is attributed to a short helical segment (α6) within the protein that resides in the canonical ligand-binding pocket (LBP) in the crystalline state. Here, we show that the α6 helix is dynamic in solution when Ftz-F1 is bound to the LxxLL motif of its cofactor Ftz, undergoing motions on the fast (picosecond to nanosecond) as well as slow (microsecond to millisecond) time scales. Motions on the slow time scale (∼10^–3 s) appear to pervade throughout the domain, most prominently in the LBP and residues at or near the cofactor-binding site. We ascribe the fast time scale motions to a solvent-accessible conformation for the α6 helix akin to those described for its orthologs in higher organisms. We assign this conformation where the LBP is “open” to a lowly populated species, while the major conformer bears the properties of the crystal structure where the LBP is “closed”. We propose that these conformational transitions could allow binding to small molecule ligands and/or play a role in dissociation of the cofactor from the binding site. Indeed, we show that the Ftz-F1 LBD can bind phospholipids, not unlike its orthologs. Our studies provide the first detailed insights into intrinsic motions occurring on a variety of time scales in a nuclear receptor LBD and reveal that potentially functionally significant motions pervade throughout the domain in solution, despite evidence to the contrary implied by the crystal structure