Identification of the regions involved in DNA binding by the mouse PEBP2alpha protein.

The polyomavirus enhancer binding protein 2alpha (PEBP2alpha) is a DNA binding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell receptor gene enhancers. Binding of PEBP2alpha and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain known as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identification of residues of the protein that are involved in DNA binding has been obscured by the low solubility of the runt domain. Constructs of the mouse PEBP2alphaA1 gene were generated with N- and C-terminal extensions beyond the runt homology region. The construct containing residues Asp90 to Lys225 of the sequence (PEBP2alpha90-225) yielded soluble protein. The residues that participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2alpha90-225. Analysis of the changes in the NMR spectra of the two forms of the protein by chemical shift deviation mapping allowed the unambiguous determination of the regions that are responsible for specific DNA recognition by PEBP2alpha. Five regions in PEBP2alpha90-225 that are localized at one end of the beta-barrel were found to interact with DNA, similar to the DNA binding interactions of other Ig fold proteins.

Solution structure of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation.

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.

Structural analyses of CREB-CBP transcriptional activator-coactivator complexes by NMR spectroscopy: implications for mapping the boundaries of structural domains.

A number of signal-dependent and development-specific transcription factors recruit CREB binding protein (CBP) for their transactivation function. The KIX domain of CBP is a common docking site for many of these transcription factors. We recently determined the solution structure of the KIX domain complexed to one of its targets, the Ser133-phosphorylated kinase inducible transactivation domain (pKID) of the cyclic AMP response element binding protein. The NMR studies have now been extended to a slightly longer KIX construct that, unlike the original KIX construct, is readily amenable to structural analysis in both the free and pKID-bound forms. This addition of six residues (KRRSRL) to the C terminus of the original construct elongates the C-terminal alpha3 helix of KIX by about eight residues. On the basis of the NMR structure of the original KIX construct, residues in the extended helix are predicted to be solvent exposed and thus are not expected to contribute to the hydrophobic core of the domain. Their role appears to be in the stabilization of the alpha3 helix through favorable electrostatic interactions with the helix dipole, which in turn confers stability on the core of the KIX domain. These results have important implications for the identification of novel protein domain boundaries. Chemical shift perturbation mapping firmly establishes a similar mode of pKID binding to the longer KIX construct and rules out any additional intermolecular interactions between residues in the C-terminal extension and pKID.

Conformational preferences in the Ser133-phosphorylated and non-phosphorylated forms of the kinase inducible transactivation domain of CREB.

Phosphorylation of Ser133 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 alphaA helix in the pKID-KIX complex, which populate helical conformations to a significant extent (>50%). The helical content in the alphaB region is very small in the non-phosphorylated form (approximately 10%) although a small increase is detected upon Ser133 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.

Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator:coactivator interactions.

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.

Solution structure of 3-oxo-delta5-steroid isomerase.

The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

Structure of the cysteine-rich intestinal protein, CRIP.

LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha-helix appears to be packed more tightly against the preceding antiparallel beta-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.

Allosteric modulation by tertiary structure in mammalian hemoglobins. Introduction of the functional characteristics of bovine hemoglobin into human hemoglobin by five amino acid substitutions.

Bovine erythrocytes do not contain 2,3-diphosphoglycerate, the principal allosteric effector of human hemoglobin. Bovine hemoglobin has a lower oxygen affinity than human hemoglobin and is regulated by physiological concentrations of chloride (Fronticelli, C., Bucci, E., and Razynska, A. (1988) J. Mol. Biol. 202, 343-348). It has been proposed that the chloride regulation in bovine hemoglobin is introduced by particular amino acid residues located in the amino-terminal region of the A helix and in the E helix of the beta subunits (Fronticelli, C. (1990) Biophys. Chem. 37, 141-146). In accordance with this proposal we have constructed two mutant human hemoglobins, beta(V1M+H2deleted+T4I+P5A) and beta(V1M+H2deleted+T4I+P5A+A76K). These are the residues present at the proposed locations in bovine hemoglobin except for isoleucine at position 4. Oxygen binding studies demonstrate that these mutations have introduced into human hemoglobin the low oxygen affinity and chloride sensitivity of bovine hemoglobin and reveal the presence of a previously unrecognized allosteric mechanism of oxygen affinity regulation where all the interactions responsible for the lowered affinity and chloride binding appear to be confined to individual beta subunits.

Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP.

The three dimensional solution structure of the carboxy terminal LIM domain of the avian Cysteine Rich Protein (CRP) has been determined by nuclear magnetic resonance spectroscopy. The domain contains two zinc atoms bound independently in CCHC (C = Cys, H = His) and CCCC modules. Both modules contain two orthogonally-arranged antiparallel beta-sheets, and the CCCC module contains an alpha-helix at its C terminus. The modules pack due to hydrophobic interactions forming a novel global fold. The structure of the C-terminal CCCC module is essentially identical to that observed for the DNA-interactive CCCC modules of the GATA-1 and steroid hormone receptor DNA binding domains, raising the possibility that the LIM motif may have a DNA binding function.