Intramolecular transmission of the ATP regulatory signal in Escherichia coli aspartate transcarbamylase: specific involvement of a clustered set of amino acid interactions at an interface between regulatory and catalytic subunits.

Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP. Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites. The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP. These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain. Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding. These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model. This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.

The activation of Escherichia coli aspartate transcarbamylase by ATP. Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains.

The regulatory chain of E. coli aspartate transcarbamylase (E.C. 2.1.3.2) is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.

Heterotropic interactions in Escherichia coli aspartate transcarbamylase. Subunit interfaces involved in CTP inhibition and ATP activation.

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

Allosteric regulation in a family of enterobacterial aspartate transcarbamylases: intramolecular transmission of regulatory signals in chimeric enzymes.

Several enterobacterial aspartate transcarbamylases (ATCases) exhibit a [2(C3):3(r2)] quaternary structure analogous to that of the Escherichia coli enzyme. Despite their conserved quaternary structures, these enzymes present substantial differences in the co-operativity of substrate binding and in their allosteric regulation by nucleotide effectors. A comparison between different enzymatic species provides an opportunity to expand our understanding of the molecular basis of allostery in ATCase. Chimeric ATCases were constructed by exchanging subdomain regions involved in quaternary structural features, such as the r1-c4 regulatory-catalytic subunit interface analyzed in this study, in order to define the involvement of this interface in the several components of allosteric regulation. The r1-c4 interface was found to constitute an essential element for the recognition and the transmission of the ATP regulatory signal in the Serratia marcescens and the Proteus vulgaris ATCases, as it does in the E. coli ATCase. Besides, the specific amino acid composition of the C-terminal region of the regulatory chain and its interactions with the amino acid residues in the 240s loop of the catalytic chain (r1-c4 interactions) were found to modulate the amplitude of the enzyme's response to ATP. The C-terminal region of the regulatory chain did not appear to participate directly in the regulation of the three native ATCases by CTP. Even when CTP acts as an activator, as in the P. vulgaris and S. marcescens ATCases, its signal follows a route distinct from that of the general activator ATP. Synergistic inhibition by CTP and UTP was found to involve the transmission of a specific UTP signal. This signal appeared different in the various ATCases, involving the C-terminal region of the regulatory chain in the E. coli and S. marcescens ATCases but not in the P. vulgaris ATCase.

Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains.

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

Evaluation of von Willebrand factor phenotypes and genotypes in Hemophilia A patients with and without identified F8 mutations.

BACKGROUND: Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA and pose a potential for misdiagnosis. OBJECTIVES: To investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities and VWF genotypes. PATIENTS/METHODS: Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence. RESULTS: Four cases had VWF:Ag < 3 IU dL(-1) and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n = five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n = 1 case). One control had VWF:Ag < 30 IU dL(-1) and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB. CONCLUSIONS: These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.

Differential responses of human umbilical and coronary artery endothelial cells to apoptosis.

Apoptosis, or programmed cell death, plays an important role not only in normal homeostasis but is increasingly implicated in a number of pathological processes. The contribution of apoptosis in the pathophysiology of coronary artery disease has been suggested. In this study, the authors compared the effects of inductive and suppressive signals as well as the two combined apoptotic signals during the early stages of apoptosis using two types of human endothelial cells isolated from the umbilical vein (HUVECs) and from the coronary artery (HCAECs) vascular beds as study targets. The authors demonstrated that HUVECs were more susceptible than HCAECs to apoptosis, as measured by a 2.5-fold increase in caspase-3 activity, which in turn may suggest a different pattern in association to the apoptotic mechanism within each cell type. Under inducing conditions of apoptosis, a significant increase in tissue factor (TF) expression at both the mRNA and protein level in HUVECs compared to the level of TF expression in HCAECs was observed. These different responses of endothelial cell types potentially indicate unequal susceptibility to apoptosis, depending on their vascular bed origins, and emphasize the importance of cell-type and -origin considerations when selecting a study model of apoptosis and pathophysiology.

New gene variants associated with venous thrombosis: a replication study in White and Black Americans.

BACKGROUND: We evaluated 10 single-nucleotide polymorphisms (SNPs) identified in three European case-control studies as risk factors for venous thrombosis. OBJECTIVES: We sought to replicate the positive findings from this report among Whites and to evaluate the association of these SNPs with venous thrombosis for the first time among Blacks. PATIENT/METHODS: These SNPs were evaluated in a case-control study of deep vein thrombosis and pulmonary embolism that included 1076 cases and 1239 controls. About 50% of subjects were African Americans. We measured plasma factor (F) XI on a subset of subjects. RESULTS: Among Whites, positive findings for rs13146272 in the CYP4V2 gene, for rs3087505 in the KLKB1 gene and for rs3756008 and rs2036914 in the F11 gene were found. We did not find significant associations for rs2227589 in the SERPINC1 gene and for rs1613662 in the GP6 gene. Among Blacks, rs2036914 in F11 and rs670659 in RGS7 were related to venous thrombosis, but the study had limited statistical power for many SNPs. Among Blacks, plasma FXI was related to two SNPs and the OR relating to the 90th percentile of the control distribution of plasma FXI was 2.6 (95% CI, 1.4, 5.0). CONCLUSIONS: Our study supports the finding that genetic variants in the F11 gene are risk factors for venous thrombosis among both Whites and Blacks, although the findings in Blacks require confirmation. A meta-analysis of five case-control studies indicates that rs2227589 in the SERPINC1 gene, rs13146272 in the CYP4V2 gene and rs1613662 in the GP6 gene are risk factors for venous thrombosis among Whites.

Heterotropic interactions in aspartate transcarbamoylase: turning allosteric ATP activation into inhibition as a consequence of a single tyrosine to phenylalanine mutation.

Aspartate transcarbamoylase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allostery. This enzyme shows cooperativity between the catalytic sites, and its activity is feedback inhibited by CTP and activated by ATP. These regulatory processes involve several interfaces between catalytic and regulatory chains as well as between domains within these two types of chains. As far as the regulatory chain is concerned, its two domains are in contact through a hydrophobic interface, in which a tyrosine residue is inserted in a pocket involving two leucine residues of the allosteric domain and a valine and a leucine residue of the zinc domain. To probe the possible implication of this hydrophobic core in the CTP and ATP regulatory effect, the tyrosine was replaced by a phenylalanine through oligonucleotide-directed mutagenesis. Interestingly, the resulting mutant shows a complete inversion of the ATP effect; it is now inhibited by ATP instead of being activated by this nucleotide triphosphate. This mutant remains normally sensitive to the feedback inhibitor CTP. This result shows that the hydrophobic interface between the two domains of the regulatory chain plays an important role in the discrimination between the regulatory signals promoted by the two allosteric effectors.