Showing papers on "Cooperativity published in 1994"

Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities.

Abstract: There are now several well-documented SSBs from both prokaryotes and eukaryotes that function in replication, recombination, and repair; however, no "consensus" view of their interactions with ssDNA has emerged. Although these proteins all bind preferentially and with high affinity to ssDNA, their modes of binding to ssDNA in vitro, including whether they bind with cooperativity, often differ dramatically. This point is most clear upon comparing the properties of the phage T4 gene 32 protein and the E. coli SSB protein. Depending on the solution conditions, Eco SSB can bind ssDNA in several different modes, which display quite different properties, including cooperativity. The wide range of interactions with ssDNA observed for Eco SSB is due principally to its tetrameric structure and the fact that each SSB protomer (subunit) can bind ssDNA. This reflects a major difference between Eco SSB and the T4 gene 32 protein, which binds DNA as a monomer and displays "unlimited" positive cooperativity in its binding to ssDNA. The Eco SSB tetramer can bind ssDNA with at least two different types of nearest-neighbor positive cooperativity ("limited" and "unlimited"), as well as negative cooperativity among the subunits within an individual tetramer. In fact, this latter property, which is dependent upon salt concentration and nucleotide base composition, is a major factor influencing whether ssDNA interacts with all four or only two SSB subunits, which in turn determines the type of intertetramer positive cooperativity. Hence, it is clear that the interactions of Eco SSB with ssDNA are quite different from those of T4 gene 32 protein, and the idea that all SSBs bind to ssDNA as does the T4 gene 32 protein must be amended. Although it is not yet known which of the Eco SSB-binding modes is functionally important in vivo, it is possible that some of the modes are used preferentially in different DNA metabolic processes. In any event, the vastly different properties of the Eco SSB-binding modes must be considered in studies of DNA replication, recombination, and repair in vitro. Since eukaryotic mitochondrial SSBs as well as SSBs encoded by prokaryotic conjugative plasmids are highly similar to Eco SSB, these proteins are likely to show similar complexities. However, based on their heterotrimeric subunit composition, the eukaryotic nuclear SSBs (RP-A proteins) are significantly different from either Eco SSB or T4 gene 32 proteins. Further subclassification of these proteins must await more detailed biochemical and biophysical studies.

Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences

Abstract: The X-ray structure of an oxygenated hemocyanin molecule, subunit II of Limulus polyphemus hemocyanin, was determined at 2.4 A resolution and refined to a crystallographic R-factor of 17.1%. The 73-kDa subunit crystallizes with the symmetry of the space group R32 with one subunit per asymmetric unit forming hexamers with 32 point group symmetry. Molecular oxygen is bound to a dinuclear copper center in the protein's second domain, symmetrically between and equidistant from the two copper atoms. The copper-copper distance in oxygenated Limulus hemocyanin is 3.6 +/- 0.2 A, which is surprisingly 1 A less than that seen previously in deoxygenated Limulus polyphemus subunit II hemocyanin (Hazes et al., Protein Sci. 2:597, 1993). Away from the oxygen binding sites, the tertiary and quaternary structures of oxygenated and deoxygenated Limulus subunit II hemocyanins are quite similar. A major difference in tertiary structures is seen, however, when the Limulus structures are compared with deoxygenated Panulirus interruptus hemocyanin (Volbeda, A., Hol, W.G.J.J. Mol. Biol. 209:249, 1989) where the position of domain 1 is rotated by 8 degrees with respect to domains 2 and 3. We postulate this rotation plays an important role in cooperativity and regulation of oxygen affinity in all arthropod hemocyanins.

Interactions of human replication protein A with oligonucleotides

Abstract: Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding protein that is essential for eukaryotic DNA replication. In order to gain a better understanding of the interactions between RPA and DNA, we have examined the interactions of human RPA with single-stranded oligonucleotides. Our analysis of RPA.DNA complexes demonstrated that RPA binds as a heterotrimer. Stoichiometric binding reactions monitored by fluorescence quenching indicated that the binding site size of human RPA is 30 nucleotides and that between 20-30 nucleotides of DNA directly interact with RPA. The binding of RPA to DNA of different lengths was systematically examined using deoxythymidine-containing oligonucleotides. We found that the binding affinity of RPA for short oligonucleotides was length dependent. The apparent association constant of RPA varied over 200-fold from approximately 7 x 10(7) M-1 for oligo(dT)10 to approximately 1.5 x 10(10) M-1 for oligo(dT)50. Human RPA binds to oligonucleotides with low cooperativity; the cooperativity parameter (omega) for RPA binding was estimated to be approximately 15.

Cooperativity of the Coil-Globule Transition in a Homopolymer: Microcalorimetric Study of Poly(N-isopropylacrylamide)

Abstract: The temperature-induced intramolecular coil-globule transition in poly(N-isopropylacrylamide) has been studied by microcalorimetry to investigate the cooperativity of this transition. Measurements were performed in the presence of sodium dodecyl sulfate (SDS) which prevents the polymer from aggregation both in the coil and in the globule state. It has been shown that the effective (van't Hoff) enthalpy of transition of a cooperative unit is 120 times less than the calorimetric enthalpy of a polymer molecule. This means that a coil-globule transition in this polymer is not an «all-or-none» process and occurs independently in cooperative units («domains») which are 120 times smaller than the polymer molecule and have a molecular weight about 6×10 4

Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.

Abstract: Clearance of excess cholesterol from cells by HDL is facilitated by the interaction of HDL apolipoproteins with cell-surface binding sites or receptors, a process that may be important in preventing atherosclerosis. In this study, synthetic peptides containing 18-mer amphipathic helices of the class found in HDL apolipoproteins (class A) were tested for their abilities to remove cholesterol and phospholipid from cultured sterol-laden fibroblasts and macrophages and to interact with cell-surface HDL binding sites. Lipid-free peptides containing two identical tandem repeats of class A amphipathic helices promoted cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible for esterification by acyl CoA/cholesterol acyltransferase, similar to what was observed for purified apolipoprotein A-I. Peptide-mediated removal of plasma membrane cholesterol and depletion of acyl CoA/cholesterol acyltransferase-accessible cholesterol appeared to occur by separate mechanisms, as the latter process was less dependent on extracellular phospholipid. The dimeric amphipathic helical peptides also competed for high-affinity HDL binding sites on cholesterol-loaded fibroblasts and displayed saturable high-affinity binding to the cell surface. In contrast, peptides with a single helix had little or no ability to remove cellular cholesterol and phospholipid, or to interact with HDL binding sites, suggesting that cooperativity between two or more helical repeats is required for these activities. Thus, synthetic peptides comprising dimers of a structural motif common to exchangeable apolipoproteins can mimic apolipoprotein A-I in both binding to putative cell-surface receptors and clearing cholesterol from cells.

Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding.

Abstract: The crystal structure of unliganded dihydrofolate reductase (DHFR) from Escherichia coli has been solved and refined to an R factor of 19% at 2.3-A resolution in a crystal form that is nonisomorphous with each of the previously reported E. coli DHFR crystal structures [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, B. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Significant conformational changes occur between the apoenzyme and each of the complexes: the NADP+ holoenzyme, the folate-NADP+ ternary complex, and the methotrexate (MTX) binary complex. The changes are small, with the largest about 3 A and most of them less than 1 A. For simplicity a two-domain description is adopted in which one domain contains the NADP+ 2'-phosphate binding site and the binding sites for the rest of the coenzyme and for the substrate lie between the two domains. Binding of either NADP+ or MTX induces a closing of the PABG-binding cleft and realignment of alpha-helices C and F which bind the pyrophosphate of the coenzyme. Formation of the ternary complex from the holoenzyme does not involve further relative domain shifts but does involve a shift of alpha-helix B and a floppy loop (the Met-20 loop) that precedes alpha B. These observations suggest a mechanism for cooperativity in binding between substrate and coenzyme wherein the greatest degree of cooperativity is expressed in the transition-state complex. We explore the idea that the MTX binary complex in some ways resembles the transition-state complex.

Cooperativity between two NF-kappa B complexes, mediated by high-mobility-group protein I(Y), is essential for cytokine-induced expression of the E-selectin promoter.

Abstract: Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-140/-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-kappa B consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-kappa B-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-kappa B to both NF-kappa B sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-selectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-kappa B complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-kappa B complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-kappa B complexes are involved in specific gene activation.

Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity.

Abstract: Multiple heat shock transcription factors (HSFs) have been discovered in several higher eukaryotes, raising questions about their respective functions in the cellular stress response. Previously, we had demonstrated that the two mouse HSFs (mHSF1 and mHSF2) interacted differently with the HSP70 heat shock element (HSE). To further address the issues of cooperativity and the interaction of multiple HSFs with the HSE, we selected new mHSF1 and mHSF2 DNA-binding sites through protein binding and PCR amplification. The selected sequences, isolated from a random population, were composed primarily of alternating inverted arrays of the pentameric consensus 5'-nGAAn-3', and the nucleotides flanking the core GAA motif were nonrandom. The average number of pentamers selected in each binding site was four to five for mHSF1 and two to three for mHSF2, suggesting differences in the potential for cooperative interactions between adjacent trimers. Our comparison of mHSF1 and mHSF2 binding to selected sequences further substantiated these differences in cooperativity as mHSF1, unlike mHSF2, was able to bind to extended HSE sequences, confirming previous observations on the HSP70 HSE. Certain selected sequences that exhibited preferential binding of mHSF1 or mHSF2 were mutagenized, and these studies demonstrated that the affinity of an HSE for a particular HSF and the extent of HSF interaction could be altered by single base substitutions. The domain of mHSF1 utilized for cooperative interactions was transferable, as chimeric mHSF1/mHSF2 proteins demonstrated that sequences within or adjacent to the mHSF1 DNA-binding domain were responsible. We have demonstrated that HSEs can have a greater affinity for a specific HSF and that in mice, mHSF1 utilizes a higher degree of cooperativity in DNA binding. This suggests two ways in which cells have developed to regulate the activity of closely related transcription factors: developing the ability to fully occupy the target binding site and alteration of the target site to favor interaction with a specific factor.

Ab-initio study of cooperativity in water chains - binding-energies and anharmonic frequencies

Abstract: Many-body interaction energies and anharmonic OH stretching frequencies have been calculated for water in chain formations, in a ring structure, and in a tetrahedral arrangement. The calculations w ...

Aspartate receptors of Escherichia coli and Salmonella typhimurium bind ligand with negative and half-of-the-sites cooperativity

Abstract: The aspartate receptors of Escherichia coli and Salmonella typhimurium which mediate chemotactic responsiveness to aspartate have 79% amino acid sequence identity but exhibited apparently quite different aspartate binding plots. The Scatchard plot of the Salmonella receptor was concave upward whereas the E. coli receptor gave a straight line. Because the two binding sites in the Salmonella receptor lacking aspartate have a 2-fold crystallographic symmetry axis and do not overlap, the observation of more than one class of binding sites must be due to a ligand-induced conformational change giving negative cooperativity. The closely related E. coli receptor was found to bind with only one class of sites but with a stoichiometry of one aspartate per dimer. The E. coli receptor thus binds with half-of-sites reactivity, an extreme form of negative cooperativity in which the second ligand is not observed to bind at all. Comparison of the X-ray crystal structure of the ligand binding domain with and without bound aspartate revealed ligand-induced conformational changes that explain the two examples of negative cooperativity.

Molecular basis of cooperativity in protein folding. V. Thermodynamic and structural conditions for the stabilization of compact denatured states.

Abstract: The heat-denatured state of proteins has been usually assumed to be a fully hydrated random coil. It is now evident that under certain solvent conditions or after chemical or genetic modifications, the protein molecule may exhibit a hydrophobic core and residual secondary structure after thermal denaturation. This state of the protein has been called the "compact denatured" or "molten globule" state. Recently is has been shown that alpha-lactalbumin at pH < 5 denatures into a molten globule state upon increasing the temperature (Griko, Y., Freire, E., Privalov, P.L. Biochemistry 33:1889-1899, 1994). This state has a lower heat capacity and a higher enthalpy at low temperatures the stabilization of the molten globule state is of an entropic origin since the enthalpy contributes unfavorably to the Gibbs free energy. Since the molten globule is more structured than the unfolded state and, therefore, is expected to have a lower configurational entropy, the net entropic gain must originate primarily from solvent related entropy arising from the hydrophobic effect, and to a lesser extent from protonation or electrostatic effects. In this work, we have examined a large ensemble of partly folded states derived from the native structure of alpha-lactalbumin in order to identify those states that satisfy the energetic criteria of the molten globule. It was found that only few states satisfied the experimental constraints and that, furthermore, those states were part of the same structural family. In particular, the regions corresponding to the A, B, and C helices were found to be folded, while the beta sheet and the D helix were found to be unfolded. At temperatures below 45 degrees C the states exhibiting those structural characteristics are enthalpically higher than the unfolded state in agreement with the experimental data. Interestingly, those states have a heat capacity close to that observed for the acid pH compact denatured state of alpha-lactalbumin [980 cal (mol.K)-1]. In addition, the folded regions of these states include those residues found to be highly protected by NMR hydrogen exchange experiments. This work represents an initial attempt to model the structural origin of the thermodynamic properties of partly folded states. The results suggest a number of structural features that are consistent with experimental data.

Interaction of the Shiga-like toxin type 1 B-subunit with its carbohydrate receptor.

Abstract: A study of the binding of the Shiga-like toxin 1 (SLT-1) to the P(k) trisaccharide [methyl 4-O-(4-O-alpha-D-galactopyranosyl)-4-O-beta-D- glucopyranoside] and its constituent dissacharides was carried out. The trisaccharide represents the carbohydrate recognition domain of the neutral glycolipid receptor of the SLT-1, globotriosylceramide (GbOse3). The binding constant for soluble trisaccharide to the soluble pentameric B-subunit is weak, with a K(a) of (0.5-1) x 10(3) M-1 for B-subunit monomer. Scatchard analysis of the binding data indicates five identical non-interacting carbohydrate binding sites per B-subunit pentamer and no cooperativity in binding. Despite weak binding (delta G = -3.6 kcal mol-1), the enthalpy of binding (delta H = -12 kcal mol-1) and the change in molar heat capacity accompanying binding (delta C(p) = -40 eu) are comparable to other protein-carbohydrate interactions. Dynamic light scattering studies indicate that carbohydrate binding induces protein aggregation. At carbohydrate concentrations where > 90% of B-subunit monomers are bound, the far-UV CD spectra were unchanged, whereas a change in the near-UV CD, maximal near 270 nm, titrated to give an apparent binding constant in good agreement with that obtained by isothermal microcalorimetry. Steady-state fluorescence and fluorescence lifetime measurements indicated that the environments of the central tryptophans are perturbed during saccharide binding, and the changes correlate with the extent of protein aggregation. On the basis of the thermodynamics of binding, optical spectroscopy, and binding-induced aggregation, we propose a model of SLT-1-membrane interaction that relies on protein-carbohydrate interaction for specificity and protein-lipid interaction for tight binding.

Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties

Abstract: The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (omega < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.

Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor.

Abstract: The purpose of this study was to explore the role of acidic amino acids in the allosteric behavior of gallamine at the m2 receptor. This was achieved by first mutating the acidic residues to neutral residues by site-directed mutagenesis. Both the parent and mutated receptors were expressed in mouse fibroblast A9L cells and characterized pharmacologically. The two main methods used were (i) Schild analysis of equilibrium binding data and (ii) study of the effect of gallamine on the dissociation kinetics of N-methylscopolamine. The Schild analysis gave an estimate of the affinity of gallamine for the allosteric site (KdA) and also a measure of the level of cooperativity (alpha) between the allosteric and primary binding sites. For the receptors studied, a good agreement was found between the alpha KdA values calculated from the Schild analysis and the IC50 values for the effect of gallamine on the N-methylscopolamine off-rate. One mutated receptor, in which the acidic EDGE (Glu-Asp-Gly-Glu) sequence of the putative third outer domain was changed to the neutral LAGQ (Leu-Ala-Gly-Gin) sequence, displayed an 8-fold reduction in affinity for gallamine at the allosteric site, in comparison with the parent receptor. The level of cooperatively between the allosteric and primary binding sites in this mutant was 46% of that of the parent receptor. A second mutated receptor, in which Asp-97 (near the top of putative transmembrane domain 3) was changed to asparagine, was found to have a level of cooperativity between sites 58% of that of the parent but was found not to be affected with respect to the affinity of gallamine for the allosteric site. When all of the acidic groups on the outer side were changed to neutral residues, there was still only an 8.6-fold reduction in gallamine affinity for the allosteric site, but the level of cooperativity was reduced to 19% of that found in the parent receptor. The results suggest that the allosteric site for gallamine binding in the m2 receptor residues at or near the putative third outer domain and that both the EDGE motif and Asp-97 play an essential role in the interaction between the two sites. However, none of the acidic amino acids mutated were found to be critical for binding at the allosteric site.

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

Abstract: The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.

Ca2+ cooperativity in neurosecretion measured using photolabile Ca2+ chelators

Abstract: 1. The photolabile Ca2+ chelator DM-nitrophen was injected into crayfish motor neuron terminals and photolyzed with light flashes of different intensity to determine the cooperativity of Ca2+ action in releasing neurotransmitter. 2. Each flash elicited a phasic postsynaptic response resembling an excitatory junctional potential, apparently due to a presynaptic "spike" in intracellular calcium concentration ([Ca2+]i). 3. When postsynaptic currents were measured under voltage clamp, a Ca2+ cooperativity of approximately 3-4 was inferred from a supralinear dependence of responses on changes in peak [Ca2+]i caused by flashes differing in intensity by 32-46%. 4. A similar Ca2+ cooperativity was inferred from postsynaptic potentials in response to flashes of varying intensity. 5. The time course of transmitter release indicated by flash responses had slightly slower rising and falling phases than excitatory postsynaptic potentials. There was also a slow tail of transmitter release lasting for approximately 200 ms after a flash. 6. This time course was explained quantitatively by simulations of DM-nitrophen photolysis and binding reactions and a model of Ca2+ activation of transmitter release.

Three-State Thermodynamic Analysis of the Denaturation of Staphylococcal Nuclease Mutants

Abstract: Using microcalorimetry, we found an equilibrium intermediate state during the denaturation of the wild-type and five mutant staphylococcal nuclease proteins: V66L, V66W, G88V, D77A, and E75V. The presence of two distinct heat absorption peaks allowed direct measurement of the enthalpy differences between the native, intermediate, and denatured states. Conditions of low pH and high NaCl concentration facilitated observation of the intermediate, or I-state. We propose to consider the nuclease protein as composed of two subdomains, divided along the active-site cleft. The structure of the I-state apparently consists mainly of the folded beta-barrel subdomain, as does that of a nuclease fragment protein [Shortle, D., & Abeygunawardana, C. (1993) Structure 1, 121-134]. The cooperativity of folding of the subdomains is maintained by electrostatic bonds across the active-site cleft. Removal of these bonds by the mutation D77A or E75V results in decooperation of the protein's structure and a three-state mechanism of denaturation at pH 7.0. The origins of differences in the enthalpy change of denaturation and in the m value of guanidinium chloride-induced denaturation with mutant nucleases are discussed in terms of this three-state mechanism.

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

Abstract: The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.

Crystal structure of fructose-1,6-bisphosphatase complexed with fructose 2,6-bisphosphate, AMP, and Zn2+ at 2.0-A resolution: aspects of synergism between inhibitors

Abstract: The crystal structure of fructose-1,6-bisphosphatase (Fru-1,6-Pase; EC 3.1.3.11) complexed with Zn2+ and two allosteric regulators, AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) has been determined at 2.0-A resolution. In the refined model, the crystallographic R factor is 0.189 with rms deviations of 0.014 A and 2.8 degrees from ideal geometries for bond lengths and bond angles, respectively. A 15 degrees rotation is observed between the upper dimer C1C2 and the lower dimer C3C4 relative to the R-form structure (fructose 6-phosphate complex), consistent with that expected from a T-form structure. The major difference between the structure of the previously determined Fru-2,6-P2 complex (R form) and that of the current quaternary T-form complex lies in the active site domain. A zinc binding site distinct from the three binding sites established earlier was identified within each monomer. Helix H4 (residues 123-127) was found to be better defined than in previously studied ligated Fru-1,6-Pase structures. Interactions between monomers in the active site domain were found involving H4 residues from one monomer and residues Tyr-258 and Arg-243 from the adjacent monomer. Cooperativity between AMP and Fru-2,6-P2 in signal transmission probably involves the following features: an AMP site, the adjacent B3 strand (residues 113-118), the metal site, the immediate active site, the short helix H4 (residues 123-127), and Tyr-258 and Arg-243 from the adjacent monomer within the upper (or lower) dimer. The closest distance between the immediate active site and that on the adjacent monomer is only 5 A. Thus, the involvement of H4 in signal transmission adds another important pathway to the scheme of the allosteric mechanism of Fru-1,6-Pase.