Solution structure of the antiapoptotic protein bcl-2.

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-x(L) chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-x(L). These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 alpha-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-x(L). Comparison of the Bcl-2 structures to that of Bcl-x(L) shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

Identification of novel inhibitors of urokinase via NMR-based screening.

Using an NMR-based screen, a novel class of urokinase inhibitors were identified that contain a 2-aminobenzimidazole moiety. The inhibitory potency of this family of inhibitors is similar to that of inhibitors containing a guanidine or amidine group. However, unlike previously described guanidino- or amidino-based inhibitors which have pK(a) values greater than 9.0, urokinase inhibitors containing a 2-aminobenzimidazole have pK(a) values of 7.5. Thus, 2-aminobenzimidazoles may have improved pharmacokinetic properties which could increase the bioavailability of inhibitors which contain this moiety. A crystal structure of one of the lead inhibitors, 2-amino-5-hydroxybenzimidazole, complexed with urokinase reveals the electrostatic and hydrophobic interactions that stabilize complex formation and suggests nearby subsites that may be accessed to increase the potency of this new series of urokinase inhibitors.

Rationale for Bcl-xL/Bad peptide complex formation from structure, mutagenesis, and biophysical studies.

The three-dimensional structure of the anti-apoptotic protein Bcl-xL complexed to a 25-residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl-xL bound to a 16-residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl-xL. However, based upon site-directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25-mer for Bcl-xL compared to the Bad 16-mer. Rather, the increased helix propensity of the Bad 25-mer is primarily responsible for its greater affinity for Bcl-xL. Based on this observation, a pair of 16-residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl-xL. Both peptides showed an increase in helix propensity compared to the wild-type and exhibited an enhanced affinity for Bcl-xL.

Retention of immunosuppressant activity in an ascomycin analogue lacking a hydrogen-bonding interaction with FKBP12.

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Delta(23,24)-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyascomycin was found to be equipotent to the parent compound both in its immunosuppressive potency and in its interaction with the immunophilin, FKBP12. Conversely, Delta(23,24)-dehydroascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency. NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to understand this phenomenon at the molecular level. The NMR structures of the complexes formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex formation.

Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis.

Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic alpha helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.

Stoichiometry and cluster specificity of copper binding to metallothionein: homogeneous metal clusters.

Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd7-MT 2, apoMT, and Cd4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd4-alpha-domain completely displaced bound cadmium. The Cu4-alpha-domain was converted into a Cu6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. 111Cd-NMR analysis of the reaction of 111Cd7-MT with Cu(I) showed that nearly co-operative one-for-one replacement of 111Cd occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with 111Cd, NMR spectra of each protein showed that 111Cd was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu6, Zn4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu6,Cd4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver 111Cd7-MT 2 with Cu10-MT 2 resulted in interprotein metal exchange in which 111Cd-moved from the beta- to the alpha-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.

X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.

THE Bcl-2 family of proteins regulate programmed cell death by an unknown mechanism. Here we describe the crystal and solution structures of a Bcl-2 family member, Bcl-xL (ref. 2). The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins.

Solution structure of the ets domain of Fli-1 when bound to DNA.

Members of the ets family of transcription factors share a conserved DNA-binding domain, the ets domain. By using multidimensional NMR, we have determined the structure of the ets domain of human Fli-1 in the DNA-bound form. It consists of three alpha-helices and a four-stranded beta-sheet, similar to structures of the class of helix-turn-helix DNA binding proteins first found in the catabolite activator protein of Escherichia coli. NMR and mutagenesis experiments suggest that in comparison to structurally related proteins, the ets domain uses a new variation of the helix-turn-helix motif for binding to DNA.

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins.

Reaction of Cd7-metallothionein with cis-dichlorodiammine platinum (II).

The reaction of Cd7-metallothionein with cis-dichlorodiammine platinum (II) has been examined in titration and kinetic experiments. Titrations followed by 111Cd NMR spectroscopy and analysis of cadmium and platinum in the products indicated that platinum displaced cadmium in approximate 1:1 fashion, producing Pt7-metallothionein. According to NMR analysis, the overall reaction involved cooperative exchange of platinum for 111Cd in each domain of the protein. There was a modest preference for the reaction of the drug with the Cd3-containing beta-domain of the protein. The product had an apparent molecular weight that was nearly the same as that of Cd7-metallothionein as determined by Sephadex G-75 chromatography. Kinetic analysis also supported a cooperative displacement mechanism. The reaction was biphasic; neither step could be identified solely with the reaction of one cluster as indicated by the biphasic reaction of the Cd4-containing alpha-domain with the platinum complex.

Solution structure of a low molecular weight protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation. Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins. In this paper, we present the first solution structure of a protein tyrosine phosphatase. This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart. The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix. The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix. Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site. The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein.

Solution structure of the amino-terminal fragment of urokinase-type plasminogen activator.

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator is a two domain protein which consists of a growth factor and a kringle domain. The 1H, 13C, and 15N chemical shifts of this protein have been assigned using heteronuclear two- and three-dimensional NMR experiments on selective and uniformly 15N- and 15N/13C-labeled protein isolated from mammalian cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data which resulted in a total of 1299 NOE restraints. The NOE restraints were used along with 27 phi angle restraints and 21 hydrogen-bonding restraints to produce 15 low energy structures. The individual domains in the structures are highly converged, but the two domains are structurally independent. The root mean square deviations (rmsd) between residues 11-46 in the growth factor domain and the mean atomic coordinates were 0.99 +/- 0.2 for backbone heavy atoms and 1.65 +/- 0.2 for all non-hydrogen atoms. For residues 55-130 in the kringle domain, the rmsd was 0.84 +/- 0.2 for backbone heavy atoms and 1.42 +/- 0.2 for all non-hydrogen atoms. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor and other homologous proteins were observed in the region which has been implicated in binding the urokinase receptor which may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

NMR determination of the structures of peroxycobalt(III) bleomycin and cobalt(III) bleomycin, products of the aerobic oxidation of cobalt(II) bleomycin by dioxygen.

The oxidation of Co(II) bleomycin A2 by dioxygen leads to two products, HO2-Co(III) bleomycin A2 (form I) and Co(III) bleomycin A2 (form II). 1H NMR chemical shift assignments for protons of both forms have been made by two-dimensional NMR spectral techniques. The chemical shifts of protons throughout forms I and II differ from each other and from apobleomycin A2. NOESY spectra reveal a number of intermediate and long-range 1H-1H couplings within the metal-binding domain, between the metal-binding domain and the peptide linker, which connects it and the DNA-binding region of the molecule, and, in form I, between the DNA- and metal-binding domains. Molecular dynamics calculations were carried out based on the NOESY results and an adjustable square pyramidyl ligand geometry around Co(III) composed of nitrogen atoms of the primary and secondary amine groups, pyridine (N5), and amide and imidazole (N1) of the hydroxyhistidine residue. In form I, the bithiazole group folds back across the square pyramid forming a compact structure. Although this conformational feature was not observed in form II, the peptide linker between the metal- and DNA-binding domains in both species shows extensive folding based on a large number of intramolecular interactions.

1H NMR-based determination of the three-dimensional structure of the human plasma fibronectin fragment containing inter-chain disulfide bonds.

Human plasma fibronectin is a plasma glycoprotein that plays an important role in many biological processes. It consists of two identical 230-250-kDa subunits that are joined by two disulfide bonds near their carboxyl termini. Each subunit contains various binding domains composed of three types of homologous repeats. Recent work has determined the three-dimensional structures of various repeat fragments, but little is known about the three-dimensional structure of the carboxyl-terminal region. A recent NMR study of a plasmin-digested carboxyl-terminal inter-chain disulfide-linked heptapeptide dimer has proposed that the two subunits are arranged in an antiparallel fashion (An et al. (1992) Biochemistry 31, 9927-9933). We have now determined the three-dimensional structure for a substantial portion of a trypsin-digested interchain disulfide-linked 52-residue (6 kDa) fragment of the carboxyl-terminal of human plasma fibronectin (which includes the above-mentioned heptapeptide dimer) using two-dimensional NMR methods and a new strategy for NMR-based protein structure determination. The NMR data requires that the two chains in the dimer be linked in a symmetric, antiparallel arrangement. The resulting monomer conformation consists of two twisted or coiled segments, Thr3-Asn6 and Ile9-Phe12, connected by the Cys7-Pro8 residues in extended conformations, with the two monomer chains cross-linked at residues Cys7 and Cys11. The conformation of the heptapeptide dimer region differs substantially from the conformation proposed by An et al.

1H, 13C, and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin.

The 1H, 13C, and 15N resonances of FKBP when bound to the immunosuppressant, ascomycin, were assigned using a computer-aided analysis of heteronuclear double and triple resonance three-dimensional nmr spectra of [U-15N]FKBP/ascomycin and [U-15N,13C]FKBP/ascomycin. In addition, from a preliminary analysis of two heteronuclear four-dimensional data sets, 3JHN,H alpha coupling constants, amide exchange data, and the differences between the C alpha and C beta chemical shifts of FKBP to random coil values, the secondary structure of FKBP when bound to ascomycin was determined. The secondary structure of FKBP when bound to ascomycin in solution closely resembled the x-ray structure of the FKBP/FK506 complex but differed in some aspects from the structure of uncomplexed FKBP in solution.

Three-dimensional structure of the FK506 binding protein/ascomycin complex in solution by heteronuclear three- and four-dimensional NMR.

A high-resolution three-dimensional solution structure of the FKBP/ascomycin complex has been determined using heteronuclear multidimensional nuclear magnetic resonance spectroscopy (NMR) and a distance geometry/simulated annealing protocol. A total of 43 structures of the complex, including 3 tightly bound water molecules, were obtained using 1958 experimental restraints consisting of 1724 nuclear Overhauser effect (NOE) derived distances, 66 chi 1 and 46 phi angular restraints, and 122 hydrogen bond restraints. The root mean square (rms) deviations between the 43 FKBP/ascomycin solution structures and the mean atomic coordinates were 0.43 +/- 0.08 A for the backbone heavy atoms and 0.80 +/- 0.08 A for all non-hydrogen atoms. Angular order parameters for the family of 43 conformations were calculated to determine dihedral convergence. Order parameters for phi, psi, and chi 1 angles exhibited mean values of 0.98, 0.97, and 0.95, respectively, while the mean of the chi 2 order parameter was 0.63. Comparisons were made between the FKBP/ascomycin complex and two NMR-derived solution structures of unbound FKBP and the X-ray crystal structure of an FKBP/FK506 complex. Differences were observed between the FKBP/ascomycin complex and uncomplexed FKBP for residues 33-45 and 78-92. In contrast, the NMR-derived solution structure of the FKBP/ascomycin complex and the X-ray crystal structure of the FKBP/FK506 complex were very similar. Differences between the two complexes were mainly observed in the conformations of some highly solvent exposed side chains.

NMR studies of an FK-506 analog, [U-13C]ascomycin, bound to FK-506-binding protein.

Multidimensional, heteronuclear NMR methods were used to determine the complete 1H and 13C resonance assignments for [U-13C]ascomycin bound to recombinant FKBP, including stereospecific assignment of all 22 methylene protons. The conformation of ascomycin was then determined from an analysis of NOEs observed in a 13C-edited 3D HMQC-NOESY spectrum of the [U-13C]ascomycin/FKBP. This structure is found to be quite different from the solution structure of the two forms of uncomplexed FK-506. However, it is very similar to the X-ray crystal structure of FK-506 bound to FKBP, rms deviation = 0.56 A. The methods used for resonance assignment and structure calculation are presented in detail. Furthermore, FKBP/ascomycin NOEs are reported which help define the structure of the ascomycin binding pocket. This structural information obtained in solution was compared to the recently described X-ray crystal structure of the FKBP/FK-506 complex.

Sequential resonance assignments of oxidized high-potential iron-sulfur protein from Chromatium vinosum.

2D NMR spectra of the high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum have been used to obtain partial resonance assignments for the oxidized paramagnetic redox state of the protein. Sequence-specific assignments were made using NOESY and COSY spectra in H2O and D2O of the following backbone segments: Asn-5-Arg-33, Glu-39-Asp-45, Gly-55-Cys-63, Gly-68-Ala-78, and Leu-82-Gly-85. NOESY spectra with a spectral width wide enough to include the hyperfine-shifted resonances revealed numerous NOE contacts between these signals and those in the main envelope of the proton spectrum. With the aid of the X-ray crystal structure [Carter, C.W., Kraut, J., Freer, S. T., Xuong, N. H., Alden, R. A., & Bartsch, R. G. (1974) J. Biol. Chem. 249, 4212], these NOEs permitted seven of the nine hyperfine-shifted signals to be assigned to three of the cysteine residues liganded to the metal cluster (Cys-43, Cys-46, and Cys-77). The other two hyperfine-shifted signals produced no detectable NOEs to other resonances in the spectrum and were tentatively assigned to the remaining cysteinyl ligand (Cys-63). These assignments, in conjunction with recent theoretical models of the electronic structure of the Fe4S4 cluster [Noodleman, L. (1988) Inorg. Chem. 27, 3677; Bertini, I., Briganti, F., Luchinat, C., Scozzafava, A., & Sola, M. (1991) J. Am. Chem. Soc. 113, 1237], indicate that the iron atoms coordinated to Cys-63 and Cys-77 are those of the mixed-valence Fe(3+)-Fe2+ pair whereas Cys-43 and Cys-46 are ligands to the Fe(3+)-Fe3+ metal pair.

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.

Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: sequential assignment of resonances.

We report the sequential assignment of resonances to specific residues in the proton nuclear magnetic resonance spectrum of the variant-3 neurotoxin from the scorpion Centruroides sculpturatus Ewing (range southwestern U.S.A.). A combination of two-dimensional NMR experiments such as 2D-COSY, 2D-NOESY, and single- and double-RELAY coherence transfer spectroscopy has been employed on samples of the protein dissolved in D2O and in H2O for assignment purposes. These studies provide a basis for the determination of the solution-phase conformation of this protein and for undertaking detailed structure-function studies of these neurotoxins that modulate the flow of sodium current by binding to the sodium channels of excitable membranes.