Intersubunit Contact Region Research Articles

Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface.

Ryanodine receptors are large channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain–domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region.

Evaluating Cooperativity in Dimeric Hemoglobins

A growing number of homo- and heterodimeric hemoglobins have been identified not only within several phyla of invertebrates and lower vertebrates, but also in unicellular micro-organisms, such as bacteria, algae, and fungi. Even though all these proteins share the typical globin fold (with the exception of truncated hemoglobins, in which two helices are missing), the quaternary assembly and the nature of the subunit interface differ widely. This observation, which implies that cooperative ligand binding arises from diverse molecular mechanisms, renders dimeric hemoglobins ideal molecular machineries for testing thermodynamic models and mechanistic hypotheses aimed at explaining the origin of cooperative phenomena. The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis (HbI) is by far the most extensively investigated protein within this class of proteins. Thus, cooperativity is ascribed to the direct communication between the two heme groups that are in close contact across the subunit interface. The heme propionate groups provide a direct pathway for the heme–heme interaction and ligand-linked structural changes are localized to the heme proximal side. A completely different mechanism is observed in hemoglobins from lower vertebrates, such as Cyclostomata, in which cooperative oxygen binding is the result of ligand-linked subunit dissociation. The behavior of lamprey (Petromyzon marinus) and hagfish (Myxine glutinosa) hemoglobins is prototypic: the ligand-bound species are monomeric, whereas the deoxygenated proteins are able to form both homodimers and heterodimers (and eventually higher order heteropolymers). This multifaceted behavior indicates that there is no unique structural basis at the origin of cooperativity in dimeric hemoglobins, both from the thermodynamic and mechanistic points of view. Cooperative ligand binding thus appears to have been selected evolutionarily, despite the mutational pressure on the intersubunit contact regions (not conserved), as a consequence of a conserved tertiary fold endowed with sufficient conformational plasticity to allow ligand-linked, heme iron-driven structural rearrangements that ultimately lead to cooperative behaviors. In this chapter, it is envisaged that cooperativity in dimeric hemoglobins can arise from two completely different mechanisms: (1) co- operative ligand binding driven by ligand-linked subunit dissociation (mainly heterotropic), or (2) cooperative ligand binding within a stable dimer (homotropic). In real systems, the presence of one mechanism does not exclude the other.

Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.

Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.

Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy–entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.

DNA-induced conformational changes in cyclic AMP receptor protein: detection and mapping by a protein footprinting technique using multiple chemical proteases

Cyclic AMP receptor protein (CRP) is a regulator of transcription in Escherichia coli which mediates its activity by binding specific DNA sequences in a cyclic AMP-dependent manner. The interaction of CRP with specific DNA was probed by a protein footprinting technique using chemical proteases of different charge, size, and hydrophobicity. The experimental data were compared with known crystal structures of cAMP-CRP and cAMP-CRP-DNA complexes to determine a correlation between the structure of the complexes, the nature of the chemical protease and protein cleavage patterns. In addition, such comparison allowed us to determine if DNA binding in solution induced conformational changes in the protein not apparent in the crystal structure. In the cAMP-CRP-DNA complex, both the protections and the enhancements of proteolytic cleavage were observed outside of the known CRP-DNA interface, suggesting that CRP undergoes a conformational change upon binding DNA. Among the observed changes, the most interesting were those around the B α-helix and β-strand 8, since this region overlaps with the activation region 2 which CRP uses for protein-protein interactions with RNA polymerase. DNA-induced changes were observed also in the region involved in CRP-CytR interaction and in CRP intersubunit contact regions. These data suggest that binding of DNA in solution induces conformational changes in CRP which can be transmitted via intersubunit contacts to regions of the protein involved in interactions with other members of transcriptional machinery.

Internal structure of ovomacroglobulin studied by electron microscopy.

As a model for the molecular structure of proteins belonging to the alpha 2-macroglobulin family, ovomacroglobulin of reptilian origin was studied by electron microscopy in the original tetrameric form as well as in the dissociated forms into half- and quarter molecules. The following aspects of the molecular internal structure which had previously not been known for the homologous human alpha 2-macroglobulin or chicken ovomacroglobulin were revealed. First, the negatively stained tetrameric native protein gave an appearance of a collection of four semi-circular strings placed on the four corners of a molecule. They were connected to each other in the center of a molecule through a set of globular domains which formed a cross-figured subunit contact region. Second, two kinds of active half-molecules prepared either by the reduction of intersubunit disulfide bonds or by the disruption of noncovalent subunit interface had similarly elongated forms having semi-circular units on the two ends, indicating quasi-equivalent subunit arrangement in the two kinds of half-molecules. We thus concluded that the structure of native ovomacroglobulin can be represented by four circular strings each equipped with an extra domain to form the central intersubunit contact region. The results may also be adapted to the internal structure of human alpha 2-macroglobulin because it was sometimes possible to observe similar ring-like internal structure in the human protein.

Functionally Distinct Agonist and Receptor-binding Regions in Human Chorionic Gonadotropin: Development of a tertiary structure model

Abstract The histidine residues in human chorionic gonadotropin (hCG) were chemically modified using diethyl pyrocarbonate. Derivatives of hCG with an average of 0.5-3.5 histidines modified (maximum of 4 per hCG) had reduced receptor-binding and cell-stimulating activities. Acylation of hCG at progressively lower pH values (conditions in which 1 of the 2 absolutely conserved histidines alpha His-83 is not titratable, whereas alpha His-94 becomes increasingly protonated and resistant to modification) produced hCG derivatives with a greater retention of receptor-binding activity than cell-stimulating activity. The involvement of alpha His-94 as part of the receptor-binding region of the hormone and of alpha His-83 as a putative active site residue was inferred. Proteinaceous protease inhibitors were shown to neutralize the agonist activity of hCG and to reduce the binding of hCG to its receptor and also to specific antisera. It was presumed that an inhibitor-hormone complex was formed which was analogous to the complexing of inhibitor with the of a serine protease. The discovery of primary sequence analogies between hCG and the serine protease chymotrypsin enabled the prediction of hCG structure using the enzyme as a folding template. Solvent-exposed and buried core regions of the peptide chain were delineated using smoothed hydrophobicity profiles in combination with Chou-Fasman secondary structure predictions. Hypervariable hydrophobicity indices between residues 38 and 80 of the human beta subunits reflected different folding arrangements which presumably conferred the individual receptor specificities. When mapped to the putative structure these receptor-determinant loops were adjacent to an area of the alpha subunit analogous to the substrate pocket of serine proteases. Disulfide bond assignments and intersubunit contact regions were identifiable. The proposed tertiary structure for hCG manifests the topographical epitopes defined using monoclonal antibodies and satisfies the currently available data on specific modification and its effects upon hormonal structure and function. This paper is considered to be the first report of a differential effect upon the agonist and receptor binding abilities of a glycoprotein hormone after modification of the proteinaceous, as opposed to the glycosylated, moiety of the molecule.

Effects of thiolation of amino groups of ovine lutropin on immunoreactivity, receptor binding and bioactivity

We have studied the reaction of ϵ-amino groups of ovine lutropin with N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and observed that modification of up to 1 3 of all available residues primarily occurs in the α-subunit. The 12 lysines available could be classified into three categories, as highly, moderately and less accessible. The three lysine residues of the hormone that appeared to resist modification may be in the intersubunit contact region. Sequential modifications of lysine residue in the a-subunit led to progressive reduction in receptor binding and immunological activities, but not in full steroidogenic potential. The discrepancy between reduced receptor binding and full steroidogenic activity may be related to the introduction of additional hydrophobicity to the ovine lutropin molecule. Total inactivation resulted only when the majority ( 9 12 ) of available amino groups were altered by this reaction. Thus, our study shows that by a limited reaction it is possible to create conjugation site(s) in ovine lutropin with significant retention of hormonal activity.

Three-state combinatorial switch models as applied to the binding of oxygen by human hemoglobin.

We have generated a series of all 6561 unique, discrete three-state combinatorial switch models to describe the partitioning of the cooperative oxygen-binding free change among the 10 variously ligated forms of human hemoglobin tetramers. These models were inspired by the experimental observation of Smith and Ackers that the cooperative free energy of the intersubunit contact regions of the 10 possible ligated forms of human hemoglobin tetramers can be represented by a particular distribution of three distinct energy levels [Smith, F. R., & Ackers, G. K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5347-5351]. A statistical thermodynamic formulation accounting for both dimer-tetramer equilibria and ligand binding properties of hemoglobin solutions as a function of oxygen and protein concentrations was utilized to exhaustively test these thermodynamic models. In this series of models each of the 10 ligated forms of the hemoglobin tetramer can exist in one, and only one, of three possible energy levels; i.e., each ligated form was assumed to be associated with a discrete energy state. This series of models includes all possible ways that the 10 ligation states of hemoglobin can be distributed into three distinct cooperative energy levels. The mathematical models, as presented here, do not permit equilibria between energy states to exist for any of the 10 unique ligated forms of hemoglobin tetramers. These models were analyzed by nonlinear least-squares estimation of the free energy parameters characteristic of this statistical thermodynamic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Dimerization of native and proteolytically modified neurophysins as monitored by proton magnetic resonance spectroscopy: proximity of tyrosine-49 to the subunit interface.

Neurophysin is a self-associating protein in which peptide-hormone binding and dimerization are thermodynamically linked. The structural basis of the linkage is unknown. We have studied the dimerization of bovine neurophysin I and two proteolytically modified derivatives by proton nuclear magnetic resonance spectroscopy in order to identify residues at the intersubunit contact regions and to evaluate the origin of the reported loss of dimerization associated with tryptic excision of residues 1-8. The concentration dependence at neural pH of the spectra of native neurophysin and des-90-92-neurophysin demonstrated a finite set of dimerization-sensitive resonances that included the ring protons of Tyr-49. Using these to monitor dimerization, we confirmed predictions of a large increase in the dimerization constant associated with carboxyl protonation. By the same criteria, dimerization of the des-1-8 protein, in disagreement with earlier reports, was found to be undiminished relative to that of the native protein. However, spectral changes in the Tyr-49 ring ortho proton region associated with dimerization of the des-1-8 protein differed significantly from those in the native protein and indicated an altered conformation of the des-1-8 dimer apparently restricted to the vicinity of Tyr-49. The results are shown to place Tyr-49 adjacent to both the intersubunit contact region and the 1-8 sequence in the native protein, loss of stabilizing interactions with 1-8 leading to altered interactions of Tyr-49 with the subunit interface. Because Tyr-49 is also close to the peptide-binding site, this arrangement spatially links the peptide-binding and dimerization sites of neurophysin.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of erythrocruorin from the earthworm Octolasium complanatum

The spontaneous assembly of the earthworm erythrocruorin molecule (60 S) from its 1/12 subunits (10 S) obtained by alkaline dissociation is a long debated problem, since the 60 S to 10 S dissociation step has been regarded as essentially irreversible or as only partially reversible when freshly dissociated solutions are used. Erythrocruorin from the earthworm Octolasium complanatum has been reassembled from its 10 S subunits. "Age" of the subunits, pH, and divalent cation concentration are the factors that influence the assembly reaction. Of primary importance is the age of the subunits, i.e. their exposure time to the alkaline dissociating pH. Parallel sedimentation velocity and sodium dodecyl sulfate/polyacrylamide gel electrophoresis experiments on the dissociated and reassembled solutions indicate that two processes take place at alkaline pH values: disulfide exchange and limited proteolysis. These processes, whose relative importance differs in the various preparations, might be responsible for the loss of reassociating capacity of the 10 S subunits. With freshly dissociated subunits, reassembly up to 80% may be achieved at pH 6.2 to 6.5 in the absence of divalent cations; the presence of 25 to 50 mM-Ca2+ renders the reaction essentially pH-independent in the range 6.2 to 8. The effect of Ca2+ is discussed in the light of the presence of structure-stabilizing binding sites for divalent cations at the 10 S intersubunit's contact regions.

Purification, structure, and properties of hybrid beta-galactosidase proteins.

Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.

Modification of the acceptor binding site of γ-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem. 254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem. 255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(α S, 5 S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [ 14C] p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M r 68,000, shown to result from crosslinking of the two subunits of transpeptidase ( M r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.

Binding of hydrophobic compounds to apoferritin subunits: Effects on the polymerization state

Apoferritin is a very stable polymer made up by 24 identical subunits forming a nearly spherical, hollow shell in which a ferric hydroxide core is enclosed. Dissociation of apoferritin into subunits occurs only at extremes of pH; thus, in the acid range, dissociation begins below pH 2.8 and the subunits reassociation begins above pH 3.5 [ 11. The importance of hydrophobic interactions at the intersubunit contact regions has been clearly established [ 1,2] and binding of hydrophobic molecules by the subunits could be anticipated. This work reports experiments on the binding of two hydrophobic compounds to apoferritin subunits. ANS was chosen because of its widespread use as a hydrophobic fluorescent probe; BZ, which is an antiinflammatory drug, since it has been shown to bind to hydrophobic sites in serum albumin [3]. Both compounds bind to apoferritin subunits, but not to the native protein, and induce their polymerization.

Kinetics of deoxyhemoglobin subunit dissociation determined by haptoglobin binding: estimation of the equilibrium constant from forward and reverse rates.

Deoxyhemoglobin tetramers dissociate into dimers very slowly, with half-times on the order of several hours. It is demonstrated that absorbance changes in the Soret region which accompany this dissociation and persist upon binding of haptoglobin 1-1 to the dissociated dimers can be used for accurate kinetic determinations over the necessarily long periods required for study. This method of study for the slow reactions depends upon long-term spectral integrity of the reaction mixtures and upon accurate measurement. The variation in rate constants determined by this procedure has been correlated with variations in structural constraints at the dimer-dimer contact region. In the presence of 2,3-diphosphoglycerate the rate constant is decreased, consistent with the role of this effector in binding to both beta chains and stabilizing the constrained deoxy tetramer against dissociation into alphabeta dimers. With hemoglobin specifically modified (des-Arg-141alpha) to eliminate half the constraining salt links within the dimer-dimer contact region, the dissociation rate is increased by approximately three orders of magnitude. In hemoglobin S where the amino acid substitution is not directly in the intersubunit contact region of interest, the dissociation rate is found to be approximately the same as that for hemoglobin A. Combination of the dissociation rate constants determined by haptoglobin binding with stopped-flow determinations of the rate constant for reassociation of dissociated dimers provides an estimate of the equilibrium constant, 0K2, for the deoxyhemoglobin dimer-tetramer equilibrium. This estimate is independent of any assumptions regarding other energetic quantities, and yields a value of 2.54 +/- 0.7 X 10(10)M-1 (heme) in 0.1 M Tris-HCl, 0.1 M NaCl, and 1 mM EDTA, pH 7.4, 21.5 degrees C. Thus the intersubunit contact energy is -14.0 +/- 0.2 kcal/mol of heme. The stabilization energy between deoxy and oxy tetramers is found to be approximately 6.4 kcal/mol, under these conditions.