Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence.

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.

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.

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.

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 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.

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.

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.

Relative stabilities of synthetic peptide homo- and heterodimeric troponin-C domains.

It has previously been shown that synthetic peptides corresponding to calcium-binding sites III (SCIII) and IV (SCIV) from troponin-C can undergo a calcium-induced dimerization to form the respective homodimers (Shaw GS, Hodges RS, Sykes BD, 1990, Science 249:280-283; Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). In addition, an equimolar mixture of SCIII and SCIV has been shown to form preferentially the SCIII/SCIV heterodimer (Shaw GS et al., 1992a, J Am Chem Soc 114:6258-6259). The stabilities of these dimers have been investigated by using 1H-NMR and circular dichroism spectroscopies to follow temperature- and guanidine hydrochloride (GuHCl)-induced denaturations. It has been found that the most stable species, the SCIII/SCIV heterodimer (delta GuH2O = -64.8 kJ/mol), is about 13 kJ/mol more stable than the least stable species, the SCIV homodimer, while the SCIII homodimer is of intermediate stability. This trend of free energies agrees well with the trend of delta G0 values derived from the products of the dissociation constants for calcium binding and peptide association determined from earlier calcium-titration studies. These observations provide evidence that calcium affinity and the association of 2-calcium binding sites are tightly linked. However, it was noted that in all cases delta G0 was considerably more negative than delta GuH2O determined from GuHCl experiments. This difference increased as the stability of the peptide complex increased, providing evidence that linear extrapolation of GuHCl data for very stable proteins may significantly underestimate the value for delta G0.

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.

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.

Effects of dopamine-receptor blockade on self-stimulation in the monkey.

In a dose-response experiment it was shown that intraperitoneal injections of 0.062 mg/kg, and 0.1 mg/kg of the dopamine-receptor blocking agent and neuroleptic spiroperidol severely attenuate self-stimulation in the orbitofrontal cortex, hypothalamus, and in the region of the locus coeruleus, in the rhesus monkey and in the squirrel monkey. In the rhesus monkey intracranial injections of 6 mug of spiroperidol bilaterally into the nucleus accumbens or the hypothalamus attenuated self-stimulation of the amygdala, and injections into the orbitofrontal cortex attenuated self-stimulation of the amygdala and lateral hypothalamus. Self-stimulation at other sites tested (including the region of the locus coeruleus) was much less affected by the injections, and injections into the region of the locus coeruleus were ineffective. These results together with other control experiments suggest that spiroperidol can attenuate self-stimulation in the monkey independently of any motor impairment or sedation produced, and that dopamine receptors in particular brain regions are involved in self-stimulation of particular brain sites.

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.

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.

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.

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.

Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides.

Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.

Determination of the solution structure of a synthetic two-site calcium-binding homodimeric protein domain by NMR spectroscopy.

The solution structure of a 34-residue synthetic calcium-binding peptide from site III of chicken troponin-C has been determined by 1H NMR spectroscopy. In solution and in the presence of calcium this peptide forms a symmetric two-site homodimeric calcium-binding domain (Shaw et al., 1990). The solution structure of this dimer was determined from the measurement of 470 NOEs from a 75-ms NOESY data set. For the dimer structure determination, the constraint list included 868 distance restraints, 44 phi angles, and 24 chi 1 and 2 chi 2 angles. Seven structures were calculated by restrained molecular dynamics using a procedure in which intramonomer distances were used first and then all distances, intra- and intermonomer, were input during further dynamics. The structures exhibited a fold very similar to the C-terminal domain of troponin-C comprised of a pair of helix-loop-helix calcium-binding sites. The rms deviation of these structures for backbone atoms between residues 97-122 and 97'-122' for the dimer was 0.82 A. The dimer structure was also calculated to be more symmetric than sites III and IV in troponin-C.