Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation.

Hub proteins have central roles in regulating cellular processes. By targeting a single cellular hub, a viral oncogene may gain control over an entire module in the cellular interaction network that is potentially comprised of hundreds of proteins. The adenovirus E1A oncoprotein is a viral hub that interacts with many cellular hub proteins by short linear motifs/molecular recognition features (MoRFs). These interactions transform the architecture of the cellular protein interaction network and virtually reprogram the cell. To identify additional MoRFs within E1A, we screened portions of E1A for their ability to activate yeast pseudohyphal growth or differentiation. This identified a novel functional region within E1A conserved region 2 comprised of the sequence EVIDLT. This MoRF is necessary and sufficient to bind the N-terminal region of the SUMO conjugase UBC9, which also interacts with SUMO noncovalently and is involved in polySUMOylation. Our results suggest that E1A interferes with polySUMOylation, but not with monoSUMOylation. These data provide the first insight into the consequences of the interaction of E1A with UBC9, which was initially described in 1996. We further demonstrate that polySUMOylation regulates pseudohyphal growth and promyelocytic leukemia body reorganization by E1A. In conclusion, the interaction of the E1A oncogene with UBC9 mimics the normal binding between SUMO and UBC9 and represents a novel mechanism to modulate polySUMOylation.

Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail.

BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.

A logical sequence search for S100B target proteins.

The EF-hand calcium-binding protein S100B has been shown to interact in vitro in a calcium-sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (alpha and beta), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled-coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of alpha and beta tubulin found at the alpha beta tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.

The solution structure of the C-terminal domain of the Mu B transposition protein.

Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available. Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate. We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B(223-312)) in 1.5 M NaCl using NMR spectroscopic methods. The structure of Mu B(223-312) comprises four helices (backbone r.m.s.d. 0.46 A) arranged in a loosely packed bundle and resembles that of the N-terminal region of the replication helicase, DnaB. This structural motif is likely to be involved in the inter-domainal regulation of ATPase activity for both Mu A and DnaB. The approach described here for structural determination in high salt may be generally applicable for proteins that do not crystallize and that are plagued by solubility problems at low ionic strength.

Sequence specific analysis of the heterogeneous glycan chain from peanut peroxidase by 1H-NMR spectroscopy.

The cationic peanut peroxidase is a complex enzyme consisting of a heme group, two calcium ions and three complex carbohydrate chains at positions Asn60, 144 and 185. Details of the heme and calcium ligation, necessary for oxidation, have recently been revealed from the three-dimensional structure of the peroxidase. However, the three glycans that may be important for the stability of the enzyme as well as its activity were not resolved. In order to determine the configuration of one of these glycans, PNGase A was used to cleave the glycan from the enzyme at Asn-144. This glycan was studied by two dimensional 1H-NMR spectroscopy to identify the sugar linkages. The results indicated a glycan structure comprising a Man alpha1-6(Xyl beta1-2)Man beta1-4GlcNAc beta1-4(Fuc alpha1-3)GlcNAc beta core but with an additional Man alpha1-3 appendage linked to Man3. The glycan also appeared to be heterogeneous as was noted from a single terminating galactose being linked to approximately 20-25% glycan.

Identification of the ubiquitin interfacial residues in a ubiquitin-E2 covalent complex.

One of the key intermediates formed during the protein ubiquitination cycle is a covalent complex between ubiquitin (Ub) and the conjugation enzyme, UBC1. In order to probe the interface between these two proteins we have formed the covalent complex in situ (in the NMR tube) using Ub, the catalytic domain of UBC1, UBC1 delta450, an activation enzyme, E1, and Mg2+-ATP. The size of the Ub-UBC1 delta450 complex (25 kDa) and its relatively short lifetime (approximately 4 h) makes assignment of the backbone resonances in the covalent species difficult. In order to monitor the formation and identify the interface in the complex we have used fast 1H-15N HSQC spectra to monitor the decay of 1H-15N correlations as a function of time until the complex formed reached about 90%. The residual peak intensities were used to probe the surface of interaction between Ub and UBC1 delta450 and provided a clear surface of interaction on Ub.

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.

Specificity and Zn2+ enhancement of the S100B binding epitope TRTK-12.

The calcium-binding protein S100B (an S100 dimer composed of two S100beta monomers) is proposed to act as a calcium-sensory protein through interactions with a variety of proteins. While the nature of the exact targets for S100B has yet to be defined, random bacteriophage peptide mapping experiments have elucidated a calcium-sensitive "epitope" (TRTK-12) for S100B recognition. In this work, interactions of TRTK-12 with S100B have been shown to be calcium-sensitive. In addition, the interactions are enhanced by zinc binding to S100B, resulting in an approximate 5-fold decrease in the TRTK-12/S100B dissociation constant. Moreover, Zn2+ binding alone has little effect. TRTK-12 showed little evidence for binding to another S100 protein, S100A11 or to a peptide derived from the N terminus of S100B, indicating both a level of specificity for TRTK-12 recognition by S100B and that the N-terminal region of S100B is probably not involved in protein-protein interactions. NMR spectroscopy revealed residues most responsive to TRTK-12 binding that could be mapped to the surface of the three-dimensional structure of calcium-saturated S100B, revealing a common region indicative of a binding site.

Solution and solid state conformation of the human EGF receptor transmembrane region.

The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase family of signalling cell surface molecules. Signalling by this protein is mediated through binding of epidermal growth factor to its extracellular region ultimately leading to phosphorylation of several residues on the intracellular portion of the receptor. The only means of communication between the intracellular and extracellular domains is via the transmembrane region of the protein. In this work we describe the first structural studies of a 34-residue synthetic peptide (hEGFRp), representative of the human EGFR transmembrane region, using two-dimensional and 2H wideline NMR and CD spectroscopies. In water the peptide demonstrated a lack of regular secondary structure and existed as oligomers. Addition of the lipomimetic solvent, trifluoroethanol (TFE), led to the production of monomeric structured species. Analysis of NMR spectra of the hEGFRp indicated that an alpha-helix was present between residues M626 and R647. This observation was reinforced by solid state 2H NMR studies in lipid bilayers which showed typical 'Pake' spectra indicating axially symmetric motion. The helical region in hEGFRp commences four residues later than predicted via hydrophobicity profiles, and extends to include several charged arginine residues which would lie on the cytosolic side of the membrane. These observations provide the first evidence that the transmembrane alpha-helical region in EGFR may not only traverse the membrane but may continue to the cytosolic region near T654, an important phosphorylation site.

A novel calcium-sensitive switch revealed by the structure of human S100B in the calcium-bound form.

BACKGROUND: S100B is a homodimeric member of the EF-hand calcium-binding protein superfamily. The protein has been implicated in cellular processes such as cell differentiation and growth, plays a role in cytoskeletal structure and function, and may have a role in neuropathological diseases, such as Alzheimers. The effects of S100B are mediated via its interaction with target proteins. While several studies have suggested that this interaction is propagated through a calcium-induced conformational change, leading to the exposure of a hydrophobic region of S100B, the molecular details behind this structural alteration remain unclear. RESULTS: The solution structure of calcium-saturated human S100B (Ca(2+)-S100B) has been determined by heteronuclear NMR spectroscopy. Ca(2+)-S100B forms a well defined globular structure comprising four EF-hand calcium-binding sites and an extensive hydrophobic dimer interface. A comparison of Ca(2+)-S100B with apo S100B and Ca(2+)-calbindin D9k indicates that while calcium-binding to S100B results in little change in the site I EF-hand, it induces a backbone reorientation of the N terminus of the site II EF-hand. This reorientation leads to a dramatic change in the position of helix III relative to the other helices. CONCLUSIONS: The calcium-induced reorientation of calcium-binding site II results in the increased exposure of several hydrophobic residues in helix IV and the linker region. While following the general mechanism of calcium modulatory proteins, whereby a hydrophobic target site is exposed, the 'calcium switch' observed in S100B appears to be unique from that of other EF-hand proteins and may provide insights into target specificity among calcium modulatory proteins.

A change-in-hand mechanism for S100 signalling.

S100 proteins are a group of small dimeric calcium-binding proteins making up a large subclass of the EF-hand family of calcium-binding proteins. Members of this family of proteins have been proposed to act as intracellular calcium modulatory proteins in a fashion analogous to that of the EF-hand sensor proteins troponin-C and calmodulin. Recently, NMR spectroscopy has provided the three-dimensional structures of the S100 family members S100A6 and S100B in both the apo- and calcium-bound forms. These structures have allowed for the identification of a novel calcium-induced conformational change termed the change-in-hand mechanism. Helix III of the C-terminal calcium-binding loop changes its helix-helix interactions (or handness) with the remainder of the molecule primarily owing to the reorientation of the backbone in an effort to coordinate the calcium ion. This reorientation of helix III exposes several residues in the C-terminus and linker regions of S100B resulting in the formation of a hydrophobic patch surrounded be a number of acidic residues. This site is the proposed region for protein-protein recognition.

Assignment and secondary structure of calcium-bound human S100B.

The NMR assignments of backbone 1H, 13C, and 15N resonances for calcium-bound human S100B were completed via heteronuclear multidimensional NMR spectroscopic techniques. NOE correlations, amide exchange, 3JHNH alpha coupling constants, and CSI analysis were used to identify the secondary structure for Ca-S100B. The protein is comprised of four helices (helix I, Glu2-Arg20; helix II, Glu31-Asn38; helix III, Gln50-Thr59; helix IV, Phe70-Phe87), three loops (loop I, Glu21-His25; loop II, Glu39-Glu49; loop III, Leu60-Gly66), and two beta-strands (strand I, Lys26-Lys28; strand II, Glu67-Asp69) which form a short antiparallel beta-sheet. Helix IV is extended by approximately one turn when compared to the secondary structures of apo-rat [Drohat et al. (1996) Biochemistry, 35, 11577-11588] and bovine S100B [Kilby et al. (1996) Structure, 4, 1041-1052]. In addition, several residues outside the calcium-binding loops in S100B undergo significant backbone chemical shift changes upon binding calcium which are not observed in the related protein calbindin D9k. Together these observations support previous site-directed mutagenesis, absorption spectroscopy, and cysteine chemical reactivity experiments, suggesting that the C-terminus in Ca-S100B is important for interactions with other proteins.

Identification and structural influence of a differentially modified N-terminal methionine in human S100b.

The calcium-binding protein S100b is a homodimer comprised of two identical 91-residue beta-subunits. Recombinant S100b is a heterogeneous protein, although the basis of this heterogeneity has not been established. We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in S100b arises from a mixture of formyl-S100b and desformyl-S100b when expressed in Escherichia coli. Reversed-phase HPLC purification of these two forms of S100b has allowed the differences in N-terminal composition to be used as a probe for tertiary contacts in the protein. The presence or absence of the N-terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40-43), indicating that these two regions are close in space.

Transmembrane region of the epidermal growth factor receptor: behavior and interactions via 2H NMR.

The first wide-line 2H NMR investigation of a receptor tyrosine kinase is reported. Selectively deuterated peptides from the membrane-associated portion of the human epidermal growth factor (EGF) receptor were synthesized for examination in lipid bilayers mimicking certain natural membrane features. The peptide sequence included the 23-amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Dispersion of the peptide with lipid in the lipomimetic solvent, trifluoroethanol (TFE), was found to be a very useful initial step for sample preparation. TFE readily dissolved all components and was then easily removed in vacuo to yield thin films which could be subsequently hydrated to produce bilayers incorporating homogeneously dispersed peptide. Samples extensively studied consisted of 6 mol % peptide in multilamellar liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine and similar liposomes containing cholesterol. 2H NMR spectra of the resulting unsonicated model membranes indicated the existence of peptide monomers undergoing rapid axially symmetric diffusion. It was possible to examine structural and behavioral effects of events often suggested as pivotal in signaling mechanisms and to consider by wide-line NMR for the first time the effect of cholesterol on hydrophobic peptides. When it was incorporated into bilayers by an alternative method involving dialysis of aqueous solutions prepared using a cationic detergent, spectra suggested that the peptide existed primarily as irreversibly aggregated oligomers which were relatively immobile on a time scale of 10(-3)-10(-4) s. For liposomes prepared by hydration of thin films, deuterated methyl groups on the peptide at locations corresponding to Ala623, Met644, and Val650 of the human EGF receptor were individually distinguishable. In highly fluid matrices, spectra suggested the presence of peptide monomers, diffusing symmetrically about axes perpendicular to the membrane. Studied as a function of temperature, 2H NMR spectra of such samples permitted independent consideration of membrane/peptide relationships at separate locations in the receptor tyrosine kinase. None of the locations probed demonstrated significant conformational sensitivity to temperature over a wide range. Effects seen at Ala623 and Met644, at opposite ends of the putative membrane-spanning domain, suggested slight increases in motional order with decreasing temperature. Addition of 33% cholesterol to the membrane caused little apparent conformational change at Val650 or Met644. However, in the presence of the sterol, Met644 and Ala623 exhibited nonaxially symmetric motion at low temperatures, perhaps as a result of peptide oligomerization. Moreover, the presence of cholesterol led to considerable change in spatial arrangement or order at Ala623. There was little evidence to support transmission of conformational changes along the peptide segment probed.

Structural influence of cation binding to recombinant human brain S100b: evidence for calcium-induced exposure of a hydrophobic surface.

The dimeric calcium-binding protein S100b is proposed to undergo a calcium-induced structural change allowing it to interact, via a hydrophobic surface, with other proteins. Previously it has been suggested that calcium binding to S100b leads to the exposure of at least one phenylalanine residue (Mani et al., 1982, 1983). This effect appears to be "reversed" at higher ionic strength, leading to a possible reburying of phenylalanine residues (Mani et al., 1982, 1983). To study these effects, we monitored calcium binding to recombinant human S100b by NMR spectroscopy under different salt (KCI) conditions. 15N-Labeled glycine residues in S100b showed calcium-induced chemical shift changes similar to those reported for the related monomeric protein calbindin D9k, suggesting similar conformational changes are occurring in the calcium-binding loops of these two proteins. Calcium binding to S100b also resulted in a shifting and broadening of several 1H resonances from the Ca-S100b form only including those from the side chains of residues F14, F70, and F73 but not those of residue Y17. This broadening was enhanced with increased ionic strength (KCI). However, small additions ( < 15% v/v) of the hydrophobic solvent trifluoroethanol relieved this phenomenon, leading to narrower line widths. These observations are consistent with the calcium-induced exposure of at least one of these hydrophobic residues, resulting in self-association of the S100b dimer. Trifluoroethanol serves to dissociate these complexes back to the dimeric calcium species. We propose that this cluster of hydrophobic residues which include F14, F73, and F88 may be important for interactions with a target protein.

NMR solution structure of a synthetic troponin C heterodimeric domain.

The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides (residues 93-126, SCIII, and 129-162, SCIV) in the presence of calcium only. It is therefore of great interest to know how closely the structure of this heterodimeric domain is to the intact protein domain. Analysis of such a structure has important implications in protein engineering and in understanding the stability of calcium-binding proteins in terms of biological function. The solution structure of this heterodimeric protein was determined by 1H NMR spectroscopy using 802 NOE derived distance restraints and 23 phi and 22 chi angle restraints. Distance geometry-simulated annealing calculations yielded a family of 42 converged structures (rmsd 0.86 +/- 0.17 A) showing an arrangement of four alpha-helices similar in fold to the C-terminal of troponin C. The dimer interface has several important interactions between helix pairs E/H and F/G responsible for the association of the two peptides. However, neither the peptide complex nor the solution NMR structure of TnC pack as tightly as that observed in the TnC X-ray structure. The interhelical distance between the F/G helix is about 1.4 A greater in solution than in the crystal. A comparison of the exposed surface area of the hydrophobic residues in the SCIII/SCIV heterodimer revealed that residues 1104, Y112, and 1121 are more exposed than in the previously determined solution structure of the SCIII homodimer. These residues are important for the interaction with the inhibitory region of TnI and provide evidence for their involvement in the regulation of muscle contraction.

Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate.

SUMMARY: Escherichia coli HU, a nonsequence-specific histone- and HMG-like DNA-binding protein, was chemically converted into a series of HU-nucleases with an iron-EDTA-based cleavage moiety positioned at 16 rationally selected sites. Specific DNA cleavage patterns from each of these HU-nucleases allowed us to determine the precise localization, stoichiometry, and orientation of HU binding in the Mu transpososome, a multiprotein structure that mediates the chemical reactions in DNA transposition. Correlation of the DNA cleavage data with the position of the cleavage moiety in the HU three-dimensional structure indicates the presence of a dramatic DNA bend, for which the bend center, direction, and magnitude were assessed. The data, which directly localize selected HU amino acids with respect to DNA in the transpososome, were used as constraints for computer-based molecular modeling to derive the first snapshot of an HU-DNA interaction.

Structural influence of calcium on the heme cavity of cationic peanut peroxidase as determined by 1H-NMR spectroscopy.

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (LiP) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of LiP have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 1H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 1H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 microM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.

Human S100b protein: formation of a tetramer from synthetic calcium-binding site peptides.

Human brain S100b protein is a unique calcium-binding protein comprised of two identical 91-amino acid polypeptide chains that each contain two proposed helix-loop-helix (EF-hand) calcium-binding sites. In order to probe the assembly of the four calcium-binding sites in S100b, a peptide comprised of the N-terminal 46 residues of S100b protein was synthesized and studied by CD and 1H NMR spectroscopies as a function of concentration and temperature. At relatively high peptide concentrations and in the absence of calcium, the peptide exhibited a significant proportion of alpha-helix (45%). Decreasing the peptide concentration led to a loss of alpha-helix as monitored by CD spectroscopy and coincident changes in the 1H NMR spectrum. These changes were also observed by 1H NMR spectroscopy as a function of temperature where it was observed that the Tm of the peptide was lowered approximately 14 degrees C with a 17-fold decrease in peptide concentration. Sedimentation equilibrium studies were used to determine that the peptide formed a tetramer in solution in the absence of calcium. It is proposed that this tetrameric fold also occurs in S100b and is a result of the interaction of portions of all four calcium-binding sites.