The nuclear factor CREB activates transcription of target genes in part through direct interactions with the KIX domain of the coactivator CBP in a phosphorylation-dependent manner. The solution structure of the complex formed by the phosphorylated kinase-inducible domain (pKID) of CREB with KIX reveals that pKID undergoes a coil-->helix folding transition upon binding to KIX, forming two alpha helices. The amphipathic helix alphaB of pKID interacts with a hydrophobic groove defined by helices alpha1 and alpha3 of KIX. The other pKID helix, alphaA, contacts a different face of the alpha3 helix. The phosphate group of the critical phosphoserine residue of pKID forms a hydrogen bond to the side chain of Tyr-658 of KIX. The structure provides a model for interactions between other transactivation domains and their targets.

Monoubiquitination 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.

Monoubiquitylation is a well‐characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin‐binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded α‐helices that bind ubiquitin independently, non‐cooperatively and with modest affinity. The Vps27 N‐terminal UIM engages the Leu8–Ile44–Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8–Ile44–Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin‐ binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.

Non-natural polymers with well-defined three-dimensional folds offer considerable potential for engineering novel functions that are outside the scope of biological polymers. Here we describe a family of N-substituted glycine or "peptoid" nonamers that folds into an unusual "threaded loop" structure of exceptional thermal stability and conformational homogeneity in acetonitrile. The structure is chain-length-specific and relies on bulky, chiral side chains and chain-terminating functional groups for stability. Notable elements of the structure include the engagement of the positively charged amino terminus by carbonyl groups of the backbone through hydrogen bonding interactions and shielding of polar groups from and near-complete exposure of hydrophobic groups to solvent, in a manner resembling a folded polypeptide globular domain turned inside-out. The structure is stable in a variety of organic solvents but is readily denatured in any solvent/cosolvent milieu with hydrogen bonding potential. The structure could serve as a scaffold for the elaboration of novel functions and could be used to test methodologies for predicting solvent-dependent polymer folding.

Phosphorylation of SerIQQ within the kinase inducible transactivation domain (KID) of the transcription factor CREB potentiates interaction with the KIX domain of coactivator CBP. Heteronuclear NMR spectroscopic analyses reveal that the KID domain is largely unstructured except for residues that comprise the K KA helix in the pKID-KIX complex, which populate helical conformations to a significant extent ( s 50%). The helical content in the K KB region is very small in the non-phosphorylated form (V10%) although a small increase is detected upon Ser IQQ phosphorylation. The intrinsic bias towards helical conformations probably facilitates folding of the KID domain upon binding to KIX while the principal role of the phosphate group appears to be largely in mediating the intermolecular interactions in the pKID-KIX complex.z 1998 Federation of European Biochemical Societies.

Historical Perspective. The formation of a specific complex between two strands of poly(uridylic acid) with one strand of poly(adenylic acid) in the presence of divalent cations was first described by Felsenfeld et al. (1957). Subsequently, triple-stranded complexes containing other combinations of polynucleotide strands were described including those belonging to the deoxyribose series [reviewed in Felsenfeld and Miles (1967), Wells et al. (1988), Cheng and Pettitt (1992b), and Sun and H61Sne (1993)]. These studies culminated in lowresolution, X-ray fiber-diffraction models for the (A)"-2(U)", (A)"'2(I)", and (A)"-2(T)" combinations (Arnott & Seising, 1974, and references cited therein). Further, a potential biological function for these structures was identified when they were found to inhibit RNA1 polymerase-mediated transcription in vitro (Morgan & Wells, 1968).Interest in triplexes revived following the discovery of singlestrand-specific SI endonuclease hypersensitive sites in the upstream regions of several eukaryotic genes (Larsen & Weintraub, 1982). These sites were mapped to (R)"-(Y)" sequences which, when subcloned into supercoiled plasmids, exhibited the same sensitivity toward SI nuclease [reviewed + This work was supported under NIH Grant GM 34504 to D.J.P.A Coordinates for the Y-RY triplexes containing G*TA (Accession No. 149D) and T-CG (Accession No. 177D) triples and the R-RY triplex (Accession Nos. 134D, 135D, 136D) have been deposited with the Protein Data Bank, Brookhaven National Laboratory, Upton, NY 11973, from whom copies can be obtained.

We have examined structural differences between the proto-oncogene c-Myb and the cyclic AMP-responsive factor CREB that underlie their constitutive or signal-dependent activation properties. Both proteins stimulate gene expression via activating regions that articulate with a shallow hydrophobic groove in the KIX domain of the coactivator CREB-binding protein (CBP). Three hydrophobic residues in c-Myb that are conserved in CREB function importantly in cellular gene activation and in complex formation with KIX. These hydrophobic residues are assembled on one face of an amphipathic helix in both proteins, and mutations that disrupt c-Myb or CREB helicity in this region block interaction of either factor with KIX. Binding of the helical c-Myb domain to KIX is accompanied by a substantial increase in entropy that compensates for the comparatively low enthalpy of complex formation. By contrast, binding of CREB to KIX entails a large entropy cost due to a random coil-to-helix transition in CREB that accompanies complex formation. These results indicate that the constitutive and inducible activation properties of c-Myb and CREB reflect secondary structural characteristics of their corresponding activating regions that influence the thermodynamics of formation of a complex with CBP.

The formation of a G.TA triple within an otherwise pyrimidine.purine.pyrimidine DNA triplex causes conformational realignments in and around the G.TA triple. These highlight new aspects of molecular recognition that could be useful in triplex-based approaches to inhibition of gene expression and site-specific cleavage of genomic DNA.