Cloning and nucleotide sequencing of the antitumor antibiotic C-1027 apoprotein gene.

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met1---Ala33 leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mcmA) while the flanking regions had little homology with the corresponding ones of mcmA, except some homology at the -10th and -35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.     

Cloning and Nucleotide Sequencing of the Antitumor Antibiotic C-1027 Apoprotein Gene

The apoprotein gene for a chromoprotein antitumor antibiotic, C-1027, was cloned from the producer strain, Streptomyces globisporus C-1027, and sequenced. The process verified that; (1) the sequence included the entire structural gene directing a precursor of the apoprotein (pre-apoprotein having Met¹—Ala³³ leader peptide ahead of the apoprotein) and flanking regions, (2) the amino acid sequence of the apoprotein deduced from the base sequence perfectly matched the one based on protein analysis, ¹⁾ (3) 3rd letters of the codons were 88% G or C, while the 1st plus the 2nd letters were 63% G or C, (4) the structural gene had 57% homology with that of macromomycin apoprotein (mernA) while the flanking regions had little homology with the corresponding ones of mernA, except some homology at the – 10th and – 35th promoter regions, and (5) the gene was transcribed as a monocistronic mRNA in an early growth phase, independent of chromophore production.     

Comparison of cytochromes b5 from insects and vertebrates

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.     

Structural properties of apocytochrome b5: presence of a stable native core

Upon removal of the heme group, the water-soluble fragment of cytochrome b5 adopts a conformation less stable and compact than that of the holoprotein [Huntley, T. E., & Strittmatter, P. (1972) J. Biol. Chem. 247, 4641-4647]. This conformation, imposed by the amino acid sequence alone, has not been described in detail. One- and two-dimensional proton nuclear magnetic resonance spectroscopy techniques were applied to the apoprotein of the soluble fragment of rat liver cytochrome b5 in an effort to characterize the structure of the apoprotein. Nuclear Overhauser spectroscopy revealed a number of short interresidue distances and demonstrated that, in spite of the increased flexibility, at least one cluster of side chains exists on a time scale long enough for study. Several residues participating in the cluster, in particular the only Trp (Trp 22), were identified. Similarities with the spectrum of the reduced holoprotein were observed that led to the inspection of the cytochrome b5 crystal structure for assigning resonances. It appeared that the environment of this residue maintains its integrity in the apoprotein. Since in the holoprotein Trp 22 belongs to a hydrophobic core formed in part by beta-strands, it is proposed that some of this beta-structure is stable in the absence of the heme-protein interactions. Implications for structure and folding are discussed.     

Divergence in nonspecific hydrophobic packing interactions in the apo state, and its possible role in functional specialization of mitochondrial and microsomal cytochrome b5

The outer mitochondrial membrane isoform of mammalian cytochrome b(5) (OM b(5)) is distinguished from the microsomal isoform (Mc b(5)) by its considerably greater stability. In contrast, OM and Mc apocytochrome b(5) (apo-b(5)) exhibit similar thermodynamic stability. Contributing substantially to the greater stability of OM b(5) relative to that of Mc b(5) is the presence of Leu at position 71. Replacing Leu-71 in OM b(5) with the corresponding Mc b(5) residue (Ser) not only diminishes holoprotein stability but also markedly compromises apoprotein stability. The studies reported herein were undertaken to clarify the role played by Leu-71 in stabilizing OM b(5)s relative to Mc b(5)s, and were motivated by the possibility that stability is related to other differences in OM and Mc b(5) properties that are important for their specialized subcellular roles. The results of these studies show that Leu-71 plays an essential role in maintaining the structural integrity of the heme-independent folding core of OM apo-b(5) (core 2), despite its location in the disordered empty heme-binding pocket (core 1). The conformational integrity of core 2 in Mc apo-b(5)s is not similarly dependent on the presence of a hydrophobic residue at position 71, providing new evidence for evolution of compensating structural features not present in OM b(5)s. We propose that Leu-71 achieves its effect on OM apo-b(5) core 2 structure by participating in a nonspecific hydrophobic collapse of disordered core 1, templated by more conformationally restricted side chains of residues in the beta-sheet that separates the two cores. We hypothesize that this has the added effect of maintaining core 1 of OM apo-b(5)s in a state more compact than that which occurs in Mc apo-b(5)s, possibly contributing to stronger heme binding by limiting the number of non-native conformations that the empty heme-binding pocket can populate.     

Unfolding and refolding of hen egg-white riboflavin binding protein

The unfolding and refolding of riboflavin-binding protein (RfBP) from hen egg-white induced by addition of guanidinium chloride (GdnHCl), and its subsequent removal by dialysis have been studied by c.d. and fluorescence for both the native and reduced protein. The reduction of its nine disulphide bonds causes a reduction in the secondary structure (alpha-helix plus beta-sheet) from 63% to 33% of the amino acid residues. Unfolding of the native protein occurred in two phases; the first involving a substantial loss of tertiary structure, followed by a second phase involving loss of secondary structure at higher GdnHCl concentrations. By contrast this biphasic behaviour was not discernible in the reduced protein. The loss of ability to bind riboflavin occurred after the first phase of unfolding. Comparison of unfolding of the holoprotein and apoprotein suggested that riboflavin has only a small stabilizing effect on the unfolding process. After removal of GdnHCl, the holoprotein, apoprotein and reduced protein assumed their original conformation. The significance of the results in relation to various models for protein folding is discussed.     

A relationship between heme binding and protein stability in cytochrome b5

Conformational changes and long-range effects are often observed in proteins when they associate with their ligands. In many cases, these structural perturbations are essential to function, and they are the result of complex networks of interactions. Here we used cytochrome b(5), a protein that undergoes extensive structural rearrangement upon heme binding, to seek a relationship between affinity for the cofactor and extent of refolding induced by its binding. Three variants of the water-soluble domain of the rat microsomal protein were chosen to affect the stability of the apoprotein or the holoprotein. Sequence alterations were introduced in the heme binding loop (type I mutations, D60R and (55)TENFED --> (55)TEPFEED, or PE), which is largely unstructured in the apoprotein state, and in the folded core of the apoprotein (type II mutation, P81A). Thermal and chemical denaturation experiments and heme transfer experiments were performed on these proteins. Type I mutations left the thermodynamic stability of the apoprotein unchanged. The first mutation (D60R) stabilized the holoprotein in a probable manifestation of enhanced helical propensity or improved electrostatic interactions. The second mutation (PE) decreased heme affinity and holoprotein stability in concert. For this protein, heme transfer experiments could be used to estimate the rate constant of heme loss from each of the heme orientational isomers. In contrast, the type II mutation resulted in a marked destabilization of the apoprotein but an intermediate effect on the holoprotein stability and heme affinity. These data supported that heme affinity could be modulated by the apoprotein stability and by specific residues remote from the heme binding site.     

Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases

E. coli Par10 is a peptidyl-prolyl cis/trans isomerase (PPIase) from Escherichia coli catalyzing the isomerization of Xaa-Pro bonds in oligopeptides with a broad substrate specificity. The structure of E. coli Par10 has been determined by multidimensional solution-state NMR spectroscopy based on 1207 conformational constraints (1067 NOE-derived distances, 42 vicinal coupling-constant restraints, 30 hydrogen-bond restraints, and 68 phi/psi restraints derived from the Chemical Shift Index). Simulated-annealing calculations with the program ARIA and subsequent refinement with XPLOR yielded a set of 18 convergent structures with an average backbone RMSD from mean atomic coordinates of 0.50 A within the well-defined secondary structure elements. E. coli Par10 is the smallest known PPIase so far, with a high catalytic efficiency comparable to that of FKBPs and cyclophilins. The secondary structure of E. coli Par10 consists of four helical regions and a four-stranded antiparallel beta-sheet. The N terminus forms a beta-strand, followed by a large stretch comprising three alpha-helices. A loop region containing a short beta-strand separates these helices from a fourth alpha-helix. The C terminus consists of two more beta-strands completing the four-stranded anti-parallel beta-sheet with strand order 2143. Interestingly, the third beta-strand includes a Gly-Pro cis peptide bond. The curved beta-strand forms a hydrophobic binding pocket together with alpha-helix 4, which also contains a number of highly conserved residues. The three-dimensional structure of Par10 closely resembles that of the human proteins hPin1 and hPar14 and the plant protein Pin1At, belonging to the same family of highly homologous proteins.     

Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562

The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.     

Solution structure of Vibrio cholerae protein VC0424: a variation of the ferredoxin-like fold

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an alpha-beta sandwich architecture consisting of two layers: a four-stranded antiparallel beta-sheet and three side-by-side alpha-helices. The secondary structure elements have the order alphabetaalphabetabetaalphabeta along the sequence. This fold is the same as the ferredoxin-like fold, except with an additional long N-terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the beta-sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.     

Three-dimensional structure of DesVI from Streptomyces venezuelae: a sugar N,N-dimethyltransferase required for dTDP-desosamine biosynthesis.

D-Desosamine, or 3-(dimethylamino)-3,4,6-trideoxyglucose, is an unusual sugar found on the macrolide antibiotic erythromycin, and it has been shown to play a critical role in the biological activity of the drug. Desosamine is added to the parent aglycone via the action of a glycosyltransferase that utilizes dTDP-desosamine as its substrate. Six enzymes are required for the biosynthesis of dTDP-desosamine in Streptomyces venezuelae, with the last step catalyzed by DesVI, an N, N-dimethyltransferase. Here we describe the X-ray crystal structure determined to 2.0 A resolution of DesVI complexed with S-adenosylmethionine (SAM) and the substrate analogue UDP-benzene. Each subunit of the DesVI dimer contains a seven-stranded mixed beta-sheet flanked on either side by alpha-helices. In addition to this major tertiary structural element, there is a four-stranded antiparallel beta-sheet that provides the platform necessary for subunit-subunit assembly. On the basis of the UDP-benzene binding mode, the DesVI substrate, dTDP-3-(methylamino)-3,4,6-trideoxyglucose, has been modeled into the active site. This model places the C-6' methyl group of the sugar into a hydrophobic patch that is well-conserved among putative nucleotide-linked sugar dimethyltransferases. It is formed by Trp 140, Met 178, and Ile 200. The sugar C-2' hydroxyl sits near Tyr 14, and its C-3' amino group is properly positioned for direct in-line attack of the cofactor's reactive methyl group. While methyltransferases that catalyze single alkylations at carbons, oxygens, sulfurs, and nitrogens have been well characterized, little is known regarding enzymes capable of N,N-dimethylation reactions. As such, the ternary structure of DesVI reported here serves as a structural paradigm for a new family of dimethyltransferases that function on nucleotide-linked sugars.     

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.     

Crystal structure of ribosomal protein L27 from Thermus thermophilus HB8

Ribosomal protein L27 is located near the peptidyltransferase center at the interface of ribosomal subunits, and is important for ribosomal assembly and function. We report the crystal structure of ribosomal protein L27 from Thermus thermophilus HB8, which was determined by the multiwavelength anomalous dispersion method and refined to an R-factor of 19.7% (R(free) = 23.6%) at 2.8 A resolution. The overall fold is an all beta-sheet hybrid. It consists of two sets of four-stranded beta-sheets formed around a well-defined hydrophobic core, with a highly positive charge on the protein surface. The structure of ribosomal protein L27 from T. thermophilus HB8 in the RNA-free form is investigated, and its functional roles in the ribosomal subunit are discussed.     

The native state of apomyoglobin described by proton NMR spectroscopy: interaction with the paramagnetic probe HyTEMPO and the fluorescent dye ANS.

Proton NMR experiments were carried out on apomyoglobin from sperm whale and horse skeletal muscle. Two small molecules, the paramagnetic relaxation agent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS), were used to alter and simplify the spectrum. Both were shown to bind in the heme pocket by docking onto the hydrophobic residues lining the distal side. Only 1 extensive region of the apoprotein structure, composed of hydrophobic residues, is not affected by HyTEMPO. It includes the 2 tryptophans (located in the A helix), other nonpolar residues of the A helix and side chains from the E, G, and GH helices. The spectral perturbations induced by ANS allowed assignment of the distal histidine (His-64) in horse apomyoglobin. This residue was previously reported to titrate with a pKa below 5 and tentatively labeled as His-82 on the basis of this value (Cocco MJ, Kao YH, Phillips AT, Lecomte JTJ, 1992, Biochemistry 31:6481-6491). The packing of the side chains and the low pKa of His-64 reinforce the idea that the distal side of the binding site is folded in a manner closely related to that in the holoprotein. ANS was found to sharpen the protein signals and the improvement of the spectral resolution facilitated the assignment of backbone amide resonances. Secondary structure, as manifested in characteristic inter-amide proton NOEs, was detected in the A, B, C, E, G, and H helices. The combined information on the hydrophobic cores and the secondary structure composes an improved representation of the native state of apomyoglobin.     

Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin.

The crystal structure of the complex formed between eglin c, an elastase inhibitor from the medical leech, and subtilisin Carlsberg has been determined at 1.2 A resolution by a combination of Patterson search methods and isomorphous replacement techniques. The structure has been refined to a crystallographic R-value of 0.18 (8-1.2 A). Eglin consists of a four-stranded beta-sheet with an alpha-helical segment and the protease-binding loop fixed on opposite sides. This loop, which contains the reactive site Leu45I--Asp46I, is mainly held in its conformation by unique electrostatic/hydrogen bond interactions of Thr44I and Asp46I with the side chains of Arg53I and Arg51I which protrude from the hydrophobic core of the molecule. The conformation around the reactive site is similar to that found in other proteinase inhibitors. The nine residues of the binding loop Gly40I--Arg48I are involved in direct contacts with subtilisin. In this interaction, eglin segment Pro42I--Thr44I forms a three-stranded anti-parallel beta-sheet with subtilisin segments Gly100--Gly102 and Ser125--Gly127. The reactive site peptide bond of eglin is intact, and Ser221 OG of the enzyme is 2.81 A apart from the carbonyl carbon.     

Solid-state NMR spectroscopic study of chromophore-protein interactions in the Pr ground state of plant phytochrome A

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.     

Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.

The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. Domain II contains a four-stranded beta-sheet and an unusual 3/10 helix. Since beta-sheet residues play a role in a number of protein-RNA interactions, one or both of the beta-sheet regions in RegA protein may be involved in RNA binding. To test this possibility, mutagenesis of residues on both beta-sheets was performed, and the effects on the RNA binding affinities of RegA protein were measured. Additional sites for mutagenesis were selected from molecular modeling of RegA protein. The RNA binding affinities of three purified mutant RegA proteins were evaluated by fluorescence quenching equilibrium binding assays. The activities of the remainder of the mutant proteins were evaluated by quantitative RNA gel mobility shift assays using lysed cell supernatants. The results of this mutagenesis study ruled out the participation of beta-sheet residues. Instead, the RNA binding site was found to be a surface pocket formed by residues on two loops and an alpha-helix. Thus, RegA protein appears to use a unique structural motif in binding RNA, which may be related to its unusual RNA recognition properties.