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Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the 15 N isotopically labeled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN . By performing circular dichroism and 15 N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

The repressor of primer (Rop) protein has become a steady source of surprises concerning the relationship between the sequences and the structures of several of its mutants and variants. Here we add another piece to the puzzle of Rop by showing that an engineered deletion mutant of the protein (corresponding to a deletion of residues 30-34 of the wild-type protein and designed to restore the heptad periodicity at the turn region) results in a complete reorganization of the bundle which is converted from a homodimer to a homotetramer. In contrast (and as previously shown), a two-residue insertion, which also restores the heptad periodicity, is essentially identical with wild-type Rop. The new deletion mutant structure is a canonical, left-handed, all-antiparallel bundle with a completely different hydrophobic core and distinct surface properties. The structure agrees and qualitatively explains the results from functional, thermodynamic, and kinetic studies which indicated that this deletion mutant is a biologically inactive hyperstable homotetramer. Additional insight into the stability and dynamics of the mutant structure has been obtained from extensive molecular dynamics simulations in explicit water and with full treatment of electrostatics.

Detailed knowledge of the influence of various parameters on macromolecular solubility is essential for crystallization. The concept of so-called 'ionic strength reducers' provides insight into the changes in solubility induced by organic solvents and hydrophilic polymers in aqueous electrolytic solutions. A simple and efficient procedure is presented which exploits the properties of ionic strength reducers in the purification and crystallization of proteins. Using two designed variants of the Rop protein as model systems, superior crystals have been obtained compared with conventional techniques. This procedure is particularly useful in cases where excessive nucleation leads to the growth of a large number of tiny crystals that are useless for crystallographic analysis.

Background: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nearly identical three-dimensional structures. Here we provide a counter-example to this statement by showing that a single amino acid substitution can change the topology of a homodimeric 4-alpha-helical bundle protein., Results: We have determined the high-resolution crystal structure of a 4-alpha-helical protein with a single alanine to proline mutation in the turn region, and show that this single amino acid substitution leads to a complete reorganisation of the whole molecule. The protein is converted from the canonical left-handed all-antiparallel form, to a right-handed mixed parallel and antiparallel bundle, which to the best of our knowledge and belief represents a novel topological motif for this class of proteins., Conclusions: The results suggest a possible new mechanism for the creation and evolution of topological motifs, show the importance of loop regions in determining the allowable folding pathways, and illustrate the malleability of protein structures.

The hydrophobic core packing in four-alpha-helical bundles appears to be crucial for stabilizing the protein structure. To examine the structural basis of hydrophobic stabilization, the crystal structures of the Leu-->Val (L41V) and Leu-->Ala (L41A) substitutions of the core residue Leu41 of the ROP protein have been determined. Both substitutions are destabilizing and lead to formation of cavities. The main responses to mutations are the collapse of the central part of the alpha-helix containing the site of mutation, shifts of internal water molecules, and in L41A, the trapping of a water molecule in a cavity engineered by the mutation. For both mutants, these effects limit the increase in cavity size to less than 10 A3, while an increase of 37 A3 and 100 A3 is expected for L41V and L41A, respectively, in the absence of any cavity size reducing effects. The mobility of internal side-chains is increased and in L41A, it reaches values typical for exposed residues. A parameter (Deltanh) is introduced as a measure of the number of van der Waals contacts lost. For ROP, barnase and T4 lysozyme mutants, there is a good correlation between Deltanh and the free energy of unfolding DeltaDeltaG relative to wild-type protein. The Deltanh value turns out to be more suitable for analysing structural and energetic responses to mutation than other parameter, such as cavity volumes and packing densities. Possible evolutionary implications of the DeltaDeltaG versus Deltanh relationship are discussed., (Copyright 1999 Academic Press.)

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values., (Copyright 1998 Academic Press Limited.)

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41-->Val and Leu-41-->Ala) in the dimeric 4-alpha-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in alpha-helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. delta H zero values at the respective transition temperatures, T 1/2 (71.0, 65.3, and 52.9 degrees C at 0.5 mg/ml) decrease from 580 +/- 20 to 461 +/- 20 kJ/(mol of dimer) and 335 +/- 20 kJ/(mol of dimer) for wild-type ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L41V, and L41A, respectively. The conformational stabilities at 25 degrees C expressed by the standard Gibbs energies of denaturation, delta GzeroD, are 71.7, 61.1, and 46.1 kJ/(mol of dimer). The corresponding transition enthalpies have been obtained from extrapolation using the cDp(T) and cNp(T) functions. Their values at 25 degrees C are 176.3, 101.9, and 141.7 kJ/(mol of dimer) for wild-type ROP, L41V, and L41A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH2 group, the average delta delta GzeroD value is -5.0 +/- 1 kJ/(mol of CH2 group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu-->Val and Leu-->Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state.

Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices. Rop is dimeric, each monomer being formed by two helices connected by a sharp bend. In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information. The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure. In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor. In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts. We have shown that this property depends on the ability of the Rop moiety to fold and dimerize. The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold. In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32. Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation. This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.

Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP.

One of the two major classes of regulatory strategies that control plasmid copy number involves recognition via base pairing between two plasmid-encoded complementary RNAs. The detailed analysis of this control circuitry has revealed some features of regulatory mechanisms based on RNA-RNA interaction that distinguish them from those based on protein-nucleic acid interaction. These features provide a framework with which to understand other regulatory mechanisms based on RNA-RNA interaction, and will aid in the design of efficient artificial antisense RNA systems.

The complete resonance assignment of the ColE1 rop (rom) protein at pH 2.3 was obtained by two-dimensional (2D) proton nuclear magnetic resonance spectroscopy (1H NMR) at 500 and 600 MHz using through-bond and through-space connectivities. Sequential assignments and elements of regular secondary structure were deduced by analysis of nuclear Overhauser enhancement spectroscopy (NOESY) experiments and 3JHN alpha coupling constants. One 7.2-kDa monomer of the homodimer consists of two antiparallel helices connected by a hairpin loop at residue 31. The C-terminal peptide consisting of amino acids 59-63 shows no stable conformation. The dimer forms a four-helix bundle with opposite polarization of neighboring elements in agreement with the X-ray structure.

Repressor of primer (Rop) is a small dimeric protein that participates in the mechanism that controls the copy number of plasmid of the ColE1 family by increasing the affinity between two complementary RNAs. The Rop dimer is a bundle of four tightly packed alpha-helices that are held together by hydrophobic interactions. We have systematically altered, by site directed mutagenesis, most of the solvent exposed amino acids of the Rop bundle and we have identified the alterations that cause a decrease of the activity of the regulatory molecule. We conclude that Rop folding is rather insensitive to amino acid substitutions and to other mutations as drastic as deletions and insertions. Looking along the 2-fold symmetry axis the amino acid side chains whose alterations affect the function of Rop are all located on one side of the molecule. Furthermore they are clustered at the extremities of the alpha-helix bundle, the only exception being the aromatic ring of Phe-14.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.