Structure of the molybdate/tungstate binding protein mop from Sporomusa ovata

BACKGROUND: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE. RESULTS: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4). CONCLUSIONS: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.     

Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.     

Analysis of the dominance of mutations in cAMP-binding sites of murine type I cAMP-dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells.

Structural lesions in cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide-dependent kinase activation in preparations from cells that were hemizygous or heterozygous for mutant R1 subunit expression. No wild-type kinase activation was observed in extracts from heterozygous mutant cells. This "dominance" was investigated by characterizing expression of wild-type and mutant R1 subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild-type R1 subunits and R1 subunits with a lesion (Glu200) that inactivates cAMP-binding site A. By both studies of cAMP dissociation and two-dimensional gel analysis, wild-type R subunits comprised about 35% of total R1 subunits in heterozygous mutants. Synthesis of wild-type and mutant R1 subunits was equivalent, but wild-type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel R1 subunit-containing species from heterozygous mutant preparations whose elution behavior suggested a trimeric kinase consisting of an R1 subunit dimer and one catalytic (C) subunit. Wild-type R1 subunit was found only in dimer and "trimer" peaks; the tetrameric kinase peak contained only mutant R1 subunit. It is concluded that C subunit binds preferentially to mutant R1 subunit in heterozygous cells forming either tetrameric kinase with mutant R1 subunit homodimers or trimeric kinase with R1 subunit heterodimers. This preferential binding results both in suppression of wild-type kinase activation and differential stabilization of mutant R1 subunits.     

Asymmetric contributions to RNA binding by the Thr(45) residues of the MS2 coat protein dimer.

A prominent feature of the interaction of MS2 coat protein with RNA is the quasi-symmetric insertion of a bulged adenine (A-10) and a loop adenine (A-4) into conserved pockets on each subunit of the coat protein dimer. Because of its presence in both of these adenine-binding pockets, Thr(45) is thought to play an important role in interaction with RNA on both subunits of the dimer. To test the significance of Thr(45), we introduced all 19 amino acid substitutions. However, we were initially unable to determine the effects of the mutations on RNA binding because every substitution compromised the ability of coat protein to fold correctly. Genetic fusion of coat protein subunits reverted these protein structural defects, allowing us to show that the RNA binding activity of coat protein tolerates substitution of Thr(45), but only on one or the other subunit of the dimer. Single-chain heterodimer complementation experiments suggest that the primary site of Thr(45) interaction with RNA is with A-4 in the translational operator. Either contact of Thr(45) with A-10 makes little contribution to stability of the RNA-protein complex, or the effects of Thr(45) substitution are offset by conformational adjustments that introduce new, favorable contacts at nearby sites.     

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.     

Complete sequence and model for the C1 subunit of the carotenoprotein, crustacyanin, and model for the dimer, beta-crustacyanin, formed from the C1 and A2 subunits with astaxanthin

The complete sequence has been determined for the C1 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus (L.). The polypeptide, 181 residues long, is similar (38% identity) to the other main subunit, A2 and to plasma retinol-binding protein. The tertiary structure of the C1 subunit has been modelled on that derived for the A2 subunit from the coordinates of retinol-binding protein. Residues lining the putative binding cavities and at the putative carotenoid binding sites of the two subunits are highly conserved. The carotenoid environments are characterized by a preponderance of aromatic and polar residues and the absence of charged side-chains. A tentative model for the dimer, beta-crustacyanin, formed between the two subunits with their associated carotenoid ligands, is discussed. The model is based on the crystal structure of the dimer of bilin-binding protein, a member of the same superfamily. This structure has enabled us to examine mechanisms for the bathochromic spectral shift of the protein-bound carotenoid and to identify likely contact regions between dimers in octameric alpha-crustacyanin.     

Isolation and characterization of a dimeric cAMP-dependent protein kinase from the fungus Saccobolus platensis

A cAMP-dependent protein kinase from mycelia of Saccobolus platensis was characterized. The holoenzyme seems to be a dimer (i.e., regulatory subunit--catalytic subunit) of 78,000 Da, slightly activated by cAMP but susceptible to dissociation into its subunits by cAMP, or by kemptide and protamine, the best substrates for Saccobolus protein kinase. The regulatory subunit was purified to homogeneity by affinity chromatography. It is highly specific for cAMP and has two types of binding sites but failed to inhibit the phosphotransferase activity of the homologous or the heterologous (bovine heart) catalytic components. The activity of the catalytic subunit was completely abolished by the regulatory component of the bovine heart protein kinase as well as by a synthetic peptide corresponding to the active site of the mammalian protein kinase inhibitor. The data suggest that interaction between the subunits of the S. platensis protein kinase is different than that found in cAMP-dependent protein kinases from other sources. Similarities and differences between the Saccobolus protein kinase and enzymes from low eucaryotes and mammalian tissues are discussed.     

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.     

Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel.

The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.     

Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants

Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid. A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2). Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) [Koyama, T., et al. (1995) Biochem. Biophys. Res. Commun. 212, 681-686], respectively. These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit.