Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E. coli aspartate transcarbamoylase.

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.

Tryptophan residues at subunit interfaces used as fluorescence probes to investigate homotropic and heterotropic regulation of aspartate transcarbamylase.

The homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) are accompanied by various structure modifications. The large quaternary structure change associated with the T to R transition, promoted by substrate binding, is accompanied by different local conformational changes. These tertiary structure modifications can be monitored by fluorescence spectroscopy, after introduction of a tryptophan fluorescence probe at the site of investigation. To relate unambiguously the fluorescence signals to structure changes in a particular region, both naturally occurring Trp residues in positions 209c and 284c of the catalytic chains were previously substituted with Phe residues. The regions of interest were the so-called 240's loop at position Tyr240c, which undergoes a large conformational change upon substrate binding, and the interface between the catalytic and regulatory chains in positions Asn153r and Phe145r supposed to play a role in the different regulatory processes. Each of these tryptophan residues presents a complex fluorescence decay with three to four independent lifetimes, suggesting that the holoenzyme exists in slightly different conformational states. The bisubstrate analogue N-phosphonacetyl-L-aspartate affects mostly the environment of tryptophans at position 240c and 145r, and the fluorescence signals were related to ligand binding and the quaternary structure transition, respectively. The binding of the nucleotide activator ATP slightly affects the distribution of the conformational substates as probed by tryptophan residues at position 240c and 145r, whereas the inhibitor CTP modifies the position of the C-terminal residues as reflected by the fluorescence properties of Trp153r. These results are discussed in correlation with earlier mutagenesis studies and mechanisms of the enzyme allosteric regulation.

The allosteric activator Mg-ATP modifies the quaternary structure of the R-state of Escherichia coli aspartate transcarbamylase without altering the T<-->R equilibrium.

The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues. We show here that, in the presence of the nucleotide effector ATP, known to stimulate the enzyme activity, the scattering profiles show a marked dependence on the metal bound to ATP. Whereas ATP has no major effect on the scattering pattern of ATCase, a saturating concentration of Mg-ATP notably modifies the scattering profile of the enzyme, either in the absence or in the presence of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA). The transition with PALA in the presence of this metal-nucleotide complex remains concerted. Furthermore, Mg-ATP, as already observed with ATP, has no detectable direct effect on the T to R transition. The experimental scattering curves in the presence of Mg-ATP were fitted by a modeling approach using rigid body movements of the regulatory subunits and the catalytic trimers in the crystal structures. While the differences observed in the T-state in the presence of Mg-ATP are essentially attributed to the binding per se of the nucleotide, the solution structure of the R-state complexed to Mg-ATP is even more extended along the 3-fold axis than the previously described R solution structure, which is already more stretched out along the same axis than the crystal R structure. Based on the crystal structure of the enzyme in the R-state complexed with free ATP, a proposal is made to account for the effect of magnesium.

Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state.

Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T-->R transition observed in the crystals. Taking the crystal R structure as a-reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11 degrees in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9 degrees around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis.

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.

Unlike the quaternary structure transition, the tertiary structure change of the 240s loop in allosteric aspartate transcarbamylase requires active site saturation by substrate for completion.

The quaternary structural change associated with the homotropic cooperative interactions in Escherichia coli aspartate transcarbamylase (ATCase) is accompanied by various tertiary structural modifications; the most notable one involves the 240s loop formed by residues 230--245 of the catalytic chain. In order to monitor local conformational changes in this region by fluorescence spectroscopy, Tyr-240 has been replaced by a Trp residue, in a mutant enzyme, in which both naturally occurring Trp residues in positions 209 and 284 of the catalytic chains had previously been substituted by Phe residues. This F209F284W240-ATCase still displays homotropic cooperativity for aspartate and undergoes the same T to R quaternary structure change as does the wild-type enzyme. Upon binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate, the fluorescence emission spectrum of this mutant shows a red shift directly proportional to the fraction of catalytic sites occupied by this compound, a maximum value of 4 nm being attained when all six active sites are ligated. An identical shift is observed with the catalytic subunits of this modified enzyme, when all three active sites are occupied. In contrast, the quaternary structural change of the F209F284W240-ATCase, monitored by small-angle X-ray scattering, is complete when only four out of six catalytic sites are occupied. Thus, the 240s loop adopts its final conformation only when the neighboring active site is bound.

X-ray scattering titration of the quaternary structure transition of aspartate transcarbamylase with a bisubstrate analogue: influence of nucleotide effectors.

The regulation of aspartate transcarbamylase (ATCase) involves various conformational changes, including a large quaternary structure rearrangement. This is directly related to a major change in its solution X-ray scattering curve upon binding the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA), allowing us to monitor directly the amount of the different quaternary structures present in solution. Data were analysed by singular vector decomposition without any prior assumption as to the number of quaternary structure states. Scattering curves in the presence of variable concentrations of PALA, alone or with saturating CTP or ATP, can be accounted for with only two states. Consequently the method gives the fraction of molecules in either state. Whereas CTP slightly decreases the proportion of molecules in the R state, ATP has no detectable effect, whatever the amount of PALA ligated to ATCase. The requirement for only two quaternary structures, suggesting a concerted transition, promoted us to test the ability of the classical model, proposed by Monod, Wyman and Changeux, to account for our data. By and large, it is satisfactory as regards the homotropic effect of PALA and the observed effect of CTP, although it remains incompatible with some other observations, which support the involvement of more indirect mechanisms in the inhibitory properties of CTP. But ATP does not directly influence the T to R transition and consequently must act by a totally different mechanism.

Doppler echocardiography in familial hypertrophic cardiomyopathy: the French Cooperative Study.

UNLABELLED: Familial hypertrophic cardiomyopathy (HCM) has been poorly studied, although it may represent 50% of all HCM. We studied 346 subjects belonging to 20 unrelated families. Patients were considered affected in view of left ventricular (LV) wall thickness. One hundred twenty-seven adults were considered affected, id est. had a left ventricular wall thickness (LVWT) > 13 mm, whereas 123 had a LVWT > 15 mm, suggesting that the cut-off value is usually not critical. Within affected patients, 95% had an asymmetrical HCM (interventricular septum/left posterior wall thickness > 1.3 mm), whereas 84% had a ratio > 1.5. Distribution of the affected patients according with Maron's classification are in keeping with published studies about sporadic forms. Doppler derived isovolumetric relaxation time was prolonged in HCM (105 +/- 23 vs 88 +/- 16 msec, P < 0.001), and the ratio peak velocity of A wave over peak velocity of E wave was significantly lower in affected individuals (0.99 +/- 0.56 vs 0.83 +/- 0.46, P < 0.05). None of the 24 children studied (10 +/- 3 years) were considered affected according to echocardiographic criteria. CONCLUSION: Echocardiography is the obligatory first step during genetic study for recognizing familial HCM. It allows classification in adults but not in children. Doppler estimate of diastolic function may be helpful in the future to recognize genetically affected subjects with normal or subnormal echocardiographic examination.

[Clinico-pathological polymorphism of hypertrophic cardiomyopathy in echocardiography]. / Polymorphisme anatomo-clinique de la cardiomyopathie hypertrophique en échocardiographie.

An echocardiographic Study of 322 adults (age: 40 +/- 16 years), belonging to 20 families with hypertrophic cardiomyopathy (HCM), was undertaken. Affected subjects had a LV diastolic wall thickness > 13 mm. The patients were classified according to the distribution of left ventricular hypertrophy (LVH) and by Maron's classification: 189 subjects were normal, 127 were affected and 6 could not been classified. By Maron's classification: 3% were type I, 33% were type II, 58% were type III and 6% were type IV. LVH was asymmetrical in 95% of cases (septum/posterior wall ratio > 1.3). The familial distribution of LVH of the 4 families in which HCM was genetically related to different loci (chromosome 11, 14 exon 13, 14 exon 8, fifth locus); the LVH was analysed from two short axis LV parasternal views and each plane was divided into 5 segments. The distribution of LVH was said to be identical between two first degree relations when all the same segments were affected, similar when they differed by only 1 or 2 segments and different when they differed by 3 or more segments. In the 26 pairs studied, LVH was identical in 2/26 (8%), similar in 11/26 (42%) and different in 13/26 (50%). Familial HCM usually gives rise to asymmetrical LVH affecting the septum and free wall. An identical distribution in 50% of affected first degree relatives.

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.

[Demonstration of a fifth locus implicated in familial hypertrophic cardiomyopathies]. / Mise en évidence d'un cinquième locus impliqué dans les cardiomyopathies hypertrophiques familiales.

Hypertrophic cardiomyopathy is familial in about 50% of cases and is transmitted in the autosomal dominant mode. The first morbid gene implicated in the disease was the gene coding the beta myosin heavy chain (beta MHC) on chromosome 14. However, only 30% of families have this genetic abnormality. Recently, three new loci have been identified on chromosomes 1q3, 11p13-q13 and 15q2. In order to determine whether other genes could be implicated in the disease a linkage analysis study was performed in a West Indian family. The method is based on the analysis of the distribution of the disease in the family and the microsatellite markers. The microsatellites used were those which recognised the 4 loci previously mentioned and 4 new markers situated and arranged with respect to known microsatellites. The results show that in the family studied, the disease did not concord with the markers of the beta MHC gene or with those recognising the loci on chromosomes 1q3, 11p13-q13 and 15q2. There is, therefore, a fifth gene implicated in familial HCM. The heterogeneity of the disease seems even greater than originally thought.

Identification of a mutation near a functional site of the beta cardiac myosin heavy chain gene in a family with hypertrophic cardiomyopathy.

Several mutations within the gene coding for the cardiac beta myosin heavy chain (designed MYH7) have been shown to be responsible for Familial Hypertrophic Cardiomyopathy (FHC) in several families, and evidence of genetic heterogeneity has been reported. To investigate the MYH7 gene as the cause of the disease in a small family with FHC, inheritance of the disease and chromosome 14 q11-q12 markers haplotype were studied, exons coding for the head domain of the cardiac beta myosin heavy chain (beta MHC) were analysed for mutations by MDE gel electrophoresis, and sequenced. We report a mutation within exon eight of the MYH7 gene at a very conserved amino acid at position 232, which results in the conversion of an asparagine to serine. This residue Asn-232 is located in a MHC area that has been recently identified as a critical site for ATPase activity. According to recent results on the three-dimensional structure of the myosin head or subfragment-1 (S1), Asn-232 is located in an alpha-helix which forms part of the nucleotide binding pocket. Although this mutation affects an active site, it seems to be associated with a favourable prognosis and a weak penetrance in this family.

Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.

Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striatal astroglia.

Embryonic rat mesencephalic neurons were plated at low density in a chemically defined medium (CDM) or in CDM conditioned on either mesencephalic or striatal astrocytes (CM Gmes and CM Gstr). It was found that "axon-like" neurites, in general long with few branching points, could be initiated in CM Gmes and CM Gstr, whereas "dendrite-like" neurites (shorter and with a high branching capacity) were preferentially initiated in CM Gmes. The effects of CM Gmes and CM Gstr on the morphology of mesencephalic neurons were abolished by protein denaturating treatments. Comparisons with basic FGF, laminin, or fibronectin demonstrated that these three molecules were also able to modify the morphological traits of the neurons. However the different morphologies observed in CM Gmes and CM Gstr could not be explained only by the presence of these proteins in the conditioned media. Our results therefore indicate that different factors may regulate the initiation of different categories of neurites and that in contrast to several molecules able to promote neurite elongation these "initiation" factors may show important regional specificity.

Further studies on the role of astroglia in brain neurons maturation and morphogenesis.

Using in vitro cultures of dissociated brain neurons and astrocytes, we have compared the morphologies of mesencephalic and striatal neurons cultured for two days on mesencephalic and striatal astrocytes in the four possible combinations. From these comparisons, it appears that: 1. Neurons grown on co-regionalized (homotopic) astrocytes have more primary neurites and branching points than neurons grown on heterotopic astrocytes. 2. The total neuritic length is only slightly affected by the type of co-culture. 3. The branched arborization which develop faster on homotopic astrocytes present several dendritic features. Following these morphological observations, we have been able to demonstrate: 1. That mesencephalic astrocytes (but not striatal astrocytes) secrete trypsin sensitive factors different from laminin and FGF that increase the number of primary neurites and branching points but have no or little effect on total neuritic length. 2. That mesencephalic astrocytes (but not striatal astrocytes) present at their surface a 190 KD glycoprotein specifically recognized by the fucose-specific lectin UEA.