Predicting the three-dimensional structure of alpha- and beta-interferons

Secondary structures of leukocyte alpha 1- and alpha 2-interferons and of fibroblast beta-interferon are calculated using the molecular theory of protein secondary structures. The common secondary structure calculated for alpha- and beta-interferons is used to predict the three-dimensional structures of fragments 1-110 and 111-166 of the chains (which are supposed to be quasi-independent domains). The predicted structure of the active domain I (1-110) is an "up-and-down" tetrahelical complex (in which the second helix is shorter than the others and can be missed in alpha 1-interferon) similar to the mirror-image of myohaemoerythrin. The predicted structure of domain II (111-166) is either a three-stranded beta-sheet screened from one side by two alpha-helices or a three-helical complex (similar to that in the N-domain of papain), the first structure being better consistent with the circular dichroism data of alpha-interferon and its C-end fragment.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma

A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.     

Solution structure of the tumor necrosis factor receptor-1 death domain.

Tumor necrosis factor receptor-1 death domain (TNFR-1 DD) is the intracellular functional domain responsible for the receptor signaling activities. The solution structure of the R347K mutant of TNFR-1 DD was solved by NMR spectroscopy. A total of 20 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 1167 distance constraints and 117 torsion angle constraints. The atomic rms distribution about the mean coordinate positions for the 20 structures for residues composing the secondary structure region is 0.40 A for the backbone atoms and 1.09 A for all atoms. The structure consists of six antiparallel alpha-helices arranged in a similar fashion to the other members of the death domain superfamily. The secondary structure and three-dimensional structure of R347K TNFR1-DD are very similar to the secondary structure and deduced topology of the R347A TNFR1-DD mutant. Mutagenesis studies identified critical residues located in alpha2 and part of alpha3 and alpha4 that are crucial for self-interaction and interaction with TRADD. Structural superposition with previously solved proteins in the death domain superfamily reveals that the major differences between the structures reside in alpha2, alpha3, and alpha4. Interestingly, these regions correspond to the binding sites of TNFR1-DD, providing a structural basis for the specificity of death domain interactions and its subsequent signaling event.     

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.     

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C.

The structure of a peptide analog of the inhibitory region of cardiac troponin-I (N-acetyl-G110-TnI(104-115) amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405-421). This structure shows a helical like peptide backbone 'bent' around P109-G110 to bring the hydrophobic residues F106, L111 and V114 closer together. The other 'side' of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.     

Conformation studies of biologically active fragments of bovine growth hormone

Conformations of bovine growth hormone active fragments were studied using far ultraviolet circular dichroism and intrinsic fluorescence emission spectroscopy. The small fragment, A-II (segment 96-133 of bovine growth hormone), undergoes a helix to random coil structural transition between pH 5 and 10 (pKa = 7.15). At pH9, the random coil state of A-II reverts back to helix conformation as ionic strength increases from 0.01 to 1. The A-II fluorophore, Tyr-110, is quenched by a neighboring carboxyl group of Glu-111, but is only slightly affected by the secondary structural transition. The large fragment, A-I (segments 1-95 and 134-191, connected via a disulfide linkage, of bovine growth hormone), is a rigidly structured molecule with a large amount of beta-sheet structure. Trp-86 of A-I was found to reside in an aromatic and hydrophobic amino acid cluster which is only destroyed by a high concentration of denaturant. Based on the primary sequence of bovine growth hormone, conformation predictions were made using the Chou-Fasman method ((1974) Biochemistry 13, 222). Bovine growth hormone helical structures are predicted to be in segments 10-34, 66-87, 111-127, and 186-191, beta-Sheet structures are predicted to be in segments 45-54, 90-94, 101-105, 136-142, 161-165, and 174-179. Tetrapeptides 37-40, 41-44, 60-63, 129-132, 146-149, and 156-159 were predicted to be beta turns. The prediction scheme confirmed several spectroscopic observations, but it did not completely explain the behavior of bovine growth hormone peptide fragments.     

A new protein folding algorithm based on hydrophobic compactness: Rigid Unconnected Secondary Structure Iterative Assembly (RUSSIA). II: Applications

The RUSSIA procedure (Rigid Unconnected Secondary Structure Iterative Assembly) produces structural models of cores of small- and medium-sized proteins. Loops are omitted from this treatment and regular secondary structures are reduced to points, the centers of their hydrophobic faces. This methodology relies on the maximum compactness of the hydrophobic residues, as described in detail in Part I. Starting data are the sequence and the predicted limits and natures of regular secondary structures (alpha or beta). Helices are treated as rigid cylinders, whereas beta-strands are collectively taken into account within beta-sheets modeled by helicoid surfaces. Strands are allowed to shift along their mean axis to allow some flexibility and the alpha-helices can be placed on either side of beta-sheets. Numerous initial conformations are produced by discrete rotations of the helices and sheets around the direction going from the center of their hydrophobic face to the global center of the protein. Selection of proposed models is based upon a criterion lying on the minimization of distances separating hydrophobic residues belonging to different regular secondary structures. The procedure is rapid and appears to be robust relative to the quality of starting data (nature and length of regular secondary structures). This dependence of the quality of the model on secondary structure prediction and in particular the beta-sheet topology, is one of the limits of the present algorithm. We present here some results for a set of 12 proteins (alpha, beta and alpha/beta classes) of lengths 40-166 amino acids. The r.m.s. deviations for core models with respect to the native proteins are in the range 1.4-3.7 A.     

Dissection of an enzyme by protein engineering. The N and C-terminal fragments of barnase form a native-like complex with restored enzymic activity.

A method is described for producing fragments of a protein suitable for studies of protein folding. The codon for a single methionine residue is introduced into the cloned gene of barnase, and the gene product cleaved with cyanogen bromide. The site of mutation was chosen to be at the surface of the protein in a region connecting segments of secondary structure in the native enzyme. The alpha + beta protein was mutated from Val36----Met, and split into two fragments, B(1-36) containing the alpha-helical regions and B(37-110), the beta-sheet. The fragments were purified by ion exchange chromatography. Neither retains catalytic activity. Fluorescence, circular dichroism, and 1H nuclear magnetic resonance data indicate that their structures are each close to that of random-coil peptides. The two fragments associate to form a tight complex (Kd = 0.2 to 0.6 microM), which displays spectroscopic properties similar to those of the uncleaved protein. The catalytic activity is restored in the complex with a value for Km similar to that for native enzyme but with kcat reduced about three- to fourfold. The second-order rate constant for association on mixing fragments in the concentration range 2.5 to 7.5 microM is 1 x 10(5) s-1 M-1.     

Two beta-rich structural domains in GABA(A) receptor alpha(1) subunit with different physical properties: Evidence for multidomain nature of the receptor

The type A gamma-aminobutyric acid (GABA(A)) receptor is a major inhibitory neurotransmitter-gated ion channel. Previously, we identified a membrane-proximal beta-rich (MPBR) domain in fragment C166-L296 of GABA(A) receptor alpha(1) subunit, forming nativelike pentamers. In the present study, another structural domain, the amino-terminal domain, was shown to exist in the fragment Q28-E165. The secondary structures of both fragments were beta-rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% beta-strand for Q28-E165 and C166-L296, respectively. The CD spectrum of the combined fragment Q28-L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domainlike nature. C166-L296 required 6.5 M of guanidine chloride for total denaturation, therefore it is extraordinarily stable, more so than Q28-E165. Moreover, effects of detergent on the molecular masses of Q28-E165 and C166-L296, as monitored using laser-scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166-L296 than in Q28-E165. Effects of pH on their molecular masses suggested that ionic forces were involved in these interactions. Together the results show that the two adjacent fragments form independent folding units, MPBR and amino-terminal domains, different in secondary structure content, denaturation profile, and polymerization status, and suggest that the former may play a more important role in receptor assembly and that the extraordinary stability may underlie its intrinsic tendency to form oligomers. More significantly, the present study has provided direct evidence for the long-postulated multidomain nature of this family of receptors.     

Diabetes mutations delineate an atypical POU domain in HNF-1alpha

Mutations in Hnf-1alpha are the most common Mendelian cause of diabetes mellitus. To elucidate the molecular function of a mutational hotspot, we cocrystallized human HNF-1alpha 83-279 with a high-affinity promoter and solved the structure of the complex. Two identical protein molecules are bound to the promoter. Each contains a homeodomain and a second domain structurally similar to POU-specific domains that was not predicted on the basis of amino acid sequence. Atypical elements in both domains create a stable interface that further distinguishes HNF-1alpha from other flexible POU-homeodomain proteins. The numerous diabetes-causing mutations in HNF-1alpha thus identified a previously unrecognized POU domain which was used as a search model to identify additional POU domain proteins in sequence databases.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the alpha/beta-barrel packing).

We present here a spectroscopic structural characterization of octarellin, a recently reported de novo protein modelled on alpha/beta-barrel proteins [K. Goraj, A. Renard and J.A. Martial (1990) Protein Engng, 3, 259-266]. Infrared and Raman spectra analyses of octarellin's secondary structure reveal the expected percentage of alpha-helices (30%) and a higher beta-sheet content (40%) than predicted from the design. When the Raman spectra obtained with octarellin and native triosephosphate isomerase (a natural alpha/beta-barrel) are compared, similar percentages of secondary structures are found. Thermal denaturation of octarellin monitored by CD confirms that its secondary structures are quite stable, whereas its native-like tertiary fold is not. Tyrosine residues, predicted to be partially hidden from solvent, are actually exposed as revealed by Raman and UV absorption spectra. We conclude that the attempted alpha/beta-barrel conformation in octarellin may be loosely packed. The criteria used to design octarellin are discussed and improvements suggested.     

Disordered C-terminal domain of tyrosyl-tRNA synthetase: secondary structure prediction.

The C-terminal domain (residues 320-419) of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus is disordered in the crystal structure and involved in the binding of the anticodon arm of tRNA(Tyr). The sequences of 11 TyrRSs of prokaryotic or mitochondrial origins were aligned and the alignment showed the existence of conserved residues in the sequences of the C-terminal domains. A consensus could be deduced from the application of five programs of secondary structure prediction to the 11 sequences of the query set. These results suggested that the sequences of the C-terminal domains determined a precise and conserved secondary structure. They predicted that the C-terminal domain would have a mixed fold (alpha/beta or alpha+beta), with the alpha-helices in the first half of the sequence and the beta-strands mainly in its second half. Several programs of fold recognition from sequence alone, by threading onto known structures, were applied but none of them identified a type of fold that would be common to the different sequences of the query set. Therefore, the fold of the C-terminal, anticodon binding domain might be novel.     

Crystal structure of a protein-toxin alpha 1-purothionin at 2.5A and a comparison with predicted models

Alpha 1-Purothionin (alpha 1-P), a wheatgerm protein and lytic toxin, has a secondary and tertiary structure similar to that of crambin as revealed by CD and NMR studies. alpha 1-P crystallizes in the tetragonal space group 1422 with unit cell dimensions: a = b = 53.59 and c = 69.79 A. X-ray diffraction data have been measured to 2.5 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using an energy-minimized alpha 1-P model structure derived from crambin (Whitlow and Teeter: Journal of Biomolecular Structure and Dynamics 2:831-848, 1985, Journal of the American Chemical Society 108:7163-7172, 1986). The energy-minimized model gives a slightly cleaner rotation solution and better refinement against the x-ray data than do the crambin or unminimized alpha 1-P structures. The final crystallographic residual with the data in the 10-2.5 A resolution range is 0.216. The refined alpha 1-P structure has a backbone rms difference of 0.74 A from crambin and 0.55 A from the energy-minimized alpha 1-P model. A low resolution NMR model of alpha 1-P calculated from metric matrix distance geometry and restrained molecular dynamics differs from crambin's backbone by 2.3 A rms deviation (Clore et al.: EMBO Journal 5:2729-2735, 1986). Backbone dihedral angles for our predicted model differ from the refined alpha 1-P structure in only one region (at a turn where there is a deletion relative to crambin). The NMR model had differences in four regions.     

Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures.

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein.     

Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (epsilon) subunit of HE provides the 3'-->5' exonucleolytic proofreading activity for this complex. Epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). In addition to alpha, epsilon also binds the small (8 kDa) theta (theta) subunit. The function of theta is unknown, although it has been hypothesized to enhance the 3'-->5' exonucleolytic proofreading activity of epsilon. Using NMR analysis and molecular modeling, we have previously reported a structural model of epsilon186, the N-terminal catalytic domain of epsilon [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and C(beta) resonances of [U-(2)H,(13)C,(15)N] methyl protonated epsilon186 in complex with unlabeled theta. A structural comparison of the epsilon186-theta complex with free epsilon186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound epsilon186 revealed a potential binding surface on epsilon for interaction with theta involving structural elements near the epsilon catalytic site. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1 and beta-strands 2 and 3 and to the region near the beginning of alpha-helix 7. Additionally, a small stretch of residues (K158-L161), which previously had not been assigned in uncomplexed epsilon186, is predicted to adopt beta-strand secondary structure in the epsilon186-theta complex and may be significant for interaction with theta. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of epsilon may be stabilized by binding theta.     

Identification of a follicle-stimulating hormone receptor-binding region in hFSH-beta-(81-95) using synthetic peptides.

Pituitary and placental glycoprotein hormones are heterodimers with alpha-subunits of identical primary structure, but dissimilar beta-subunits. Regions of structural similarity between the beta-subunits might be involved in interaction with the homologous alpha-subunits, and regions of structural dissimilarity could, therefore, be candidates for receptor interactions. A restrained matrix dot-plot analysis identified hFSH-beta-(8-32) and hFSH-beta-(55-65) as candidates for interaction with alpha-subunit. Therefore, by subtraction, hFSH-beta-(33-54) and hFSH-beta-(66-111) seemed candidates for regions of interaction with receptor. In a previous report we demonstrated that hFSH-beta-(33-53) represented a receptor-binding region of hFSH-beta. Analysis of structural parameters (flexibility, surface probability, secondary structure prediction, etc.) indicates similarities between hFSH-beta-(33-53) and hFSH-beta-(85-95), suggesting the latter might be the component of hFSH-beta-(61-111) interacting with the receptor. Testing of 11 synthetic peptides, corresponding to the primary structure of hFSH-beta, demonstrated that hFSH-beta-(31-45)-peptide amide, were unique in ability to inhibit 125I-follicle-stimulating hormone binding to receptor. hFSH-beta-(81-95)-peptide amide also stimulated estradiol biosynthesis in Sertoli cell cultures. The correlation between binding inhibition and surface probability, flexibility, and predicted secondary structure (alpha, extended, and turn) was highly significant (R2 = 0.87, p less than 0.0001). Regression significance for these parameters, taken individually, were very poor. Receptor-binding regions, therefore, appear to be characterized by a particular and complex arrangement of secondary structure motifs, surface probability, and flexibility.     

The dimerization domain of LFB1/HNF1 related transcription factors: a hidden four helix bundle?

LFB1/HNF1 alpha and LFB3/HNF1 beta bind DNA as dimers and form heterodimers together in vivo and in vitro. The dimerization domain has been located in both proteins in the 32 N-terminal residues. In previous papers we have described the conformational stability as determined by CD and the secondary structure by NMR studies of a peptide with the amino acid sequence of the dimerization domain of LFB1/HNF1 alpha. This study presents a more complete characterization of similar synthetic peptides spanning the LFB3/HNF1 beta dimerization domain and the alpha/beta heterodimer. The HNF1 peptides represent an example of structures which cannot be determined by NOE data alone because they are not sufficient to define one unique solution. An approach is presented which combines NMR data, the protein structure database and structural analyses according to known principles of protein structure. On this basis we are able to determine possible solutions and to identify a four helix bundle as the structure most consistent with the experimental evidence.