Prenatal monitoring of ornithine transcarbamoylase deficiency in two families by DNA analysis

This study examined the variation in weight, height and body mass index of 1048 mothers living in a rural area of Bangladesh in relation to age, education, number of previous pregnancies, number of dead children, religion, family type, family size, and amount of land owned by the household. Multiple regression analysis revealed a positive relationship of education with all three variables. Moslem mothers were on average in better condition than Hindus. The number of dead children showed a negative relationship with height, weight and body mass index.     

Dynamin undergoes a GTP-dependent conformational change causing vesiculation

BACKGROUND: We assessed the value of dobutamine stress echocardiography (DSE) in predicting cardiac events in patients with acute or chronic myocardial infarction (MI), and we studied the association between DSE and these events. METHODS AND RESULTS: Two hundred sixty-six patients (mean [+/-SD] age 65.3 +/- 11.4 years) with acute (n = 139) or chronic (n = 127) MI were recruited from March 1995 through April 1997. Both groups underwent DSE and were followed up for an average of 14.1 +/- 8.0 months. DSE was positive in 111 (79.9%) patients with acute MI and 65 (51.2%) patients with chronic MI (P <.0001 ). Positive DSE results were associated with a higher rate of all cardiac events (cardiac mortality rate, reinfarction, and unstable angina) than negative DSE results in both patients with acute MI and patients with chronic MI (44 in 111 patients vs 6 in 28 patients, P =.052, and 31 in 65 patients vs 10 in 62 patients, P <.0001, respectively). Among patients with acute MI, the positive and negative predictive values of DSE for all cardiac events were 39.6% (95% confidence interval [CI] 30.5% to 48. 7%) and 78.6% (95% CI 63.4% to 93.8%), respectively. In chronic MI, the positive and negative predictive values were 47.7% (95% CI 35.5% to 59.8%) and 83.9% (95% CI 74.7% to 93.0%), respectively. In both acute (P =.03) and chronic (P <.0001 ) MI, positive DSE findings were independent predictors of all cardiac events. CONCLUSIONS: DSE is useful for predicting cardiac events. A positive finding on DSE is an independent predictor of cardiac events after both acute and chronic MI, whereas a negative DSE result predicts a low likelihood of subsequent cardiac events.     

Ligand-induced conformational change in penicillin acylase

Wild-type and constitutively active mutants of human MAP kinase kinase-1 (MKK1) were analyzed by deuterium exchange mass spectrometry using a protocol that minimized loss of deuterium during analysis due to back exchange. The observed peptides accounted for 335 out of 393 residues. Not counting overlap peptides, three peptides showed decreased exchange in constitutively active compared to wild-type MKK1 and nine showed increased exchange. Backbone amides in which exchange rates decreased upon kinase activation were observed near the regulatory phosphorylation sites Ser218 and Ser222 and the adjacent beta9 strand. These decreases are consistent with electrostriction or reduced solvent access due to domain closure or formation of new hydrogen or salt bonds around the catalytic cleft and within the activation lip. Increased exchange upon activation was observed within six peptides derived from helix C and the five-stranded beta sheet from the N-proximal lobe of the conserved kinase domain and in one peptide located at the interface between the N- and C-proximal lobes. Two amides that underwent increased exchange were specifically localized between residues 68 and 69 in beta1 and 140 and 142 in beta5. These residues probably form contacts with each other on opposite sites of the beta sheet as well as with helix C. These increases appeared to represent localized fluctuations, rather than rigid body rearrangements, suggesting that MKK1 activation requires enhanced flexibility within the N-proximal lobe, perhaps to accommodate ATP binding, phosphotransfer, or ADP release.     

Binding of L-branched-chain amino acids causes a conformational change in BkdR

BACKGROUND: Necrotizing soft tissue infections of the chest wall are uncommon, and they have received little discussion in the medical literature. METHODS: We performed a collective review of the literature to summarize information on etiology, prevention, treatment, complications, and outcome of chest wall necrotizing soft tissue infections. Manual, Medline, and Current Contents searches of the English-language medical literature were done. RESULTS: There were 9 reported cases of necrotizing soft tissue infection of the chest wall. Eight were complications of invasive procedures and operations. Tube thoracostomy for empyema (4 patients) was the most common antecedent procedure. Excessive soft tissue dissection during chest tube insertion was implicated in the genesis of these infections. Necrotizing infections complicated esophageal operations in 2 patients. Overall mortality was 89%. Only 3 of the 9 patients underwent early and adequate debridement. Chest wall stability and wound reconstruction were problematic in patients who survived the initial septic illness. CONCLUSIONS: Necrotizing soft tissue infections of the chest wall are highly lethal infections that require urgent and aggressive debridement. Diagnostic delay and inadequate debridement are common reasons for treatment failure. Repetitive surgical debridement is often needed to control sepsis. Wound closure is challenging in patients who survive the initial septic phase of their illness.     

Allosteric regulation of a ribozyme activity through ligand-induced conformational change

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.     

Inhibitor-induced conformational change in cytochrome P-450CAM

The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].     

Tetracaine reports a conformational change in the pore of cyclic nucleotide-gated channels

Local anesthetics are a diverse group of clinically useful compounds that act as pore blockers of both voltage- and cyclic nucleotide-gated (CNG) ion channels. We used the local anesthetic tetracaine to probe the nature of the conformational change that occurs in the pore of CNG channels during the opening allosteric transition. When applied to the intracellular side of wild-type rod CNG channels expressed in Xenopus oocytes from the alpha subunit, the local anesthetic tetracaine exhibits state-dependent block, binding with much higher affinity to closed states than to open states. Here we show that neutralization of a glutamic acid in the conserved P region (E363G) eliminated this state dependence of tetracaine block. Tetracaine blocked E363G channels with the same effectiveness at high concentrations of cGMP, when the channel spent more time open, and at low concentrations of cGMP, when the channel spent more time closed. In addition, Ni2+, which promotes the opening allosteric transition, decreased the effectiveness of tetracaine block of wild-type but not E363G channels. Similar results were obtained in a chimeric CNG channel that exhibits a more favorable opening allosteric transition. These results suggest that E363 is accessible to internal tetracaine in the closed but not the open configuration of the pore and that the conformational change that accompanies channel opening includes a change in the conformation or accessibility of E363.     

1.85-A resolution crystal structure of human ornithine transcarbamoylase complexed with N-phosphonacetyl-L-ornithine. Catalytic mechanism and correlation with inherited deficiency

The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analog N-phosphonacetyl-L-ornithine has been solved at 1.85-A resolution by molecular replacement. Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency). The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds L-ornithine. The active site is located in the cleft between domains and contains additional residues from an adjacent subunit. Binding of N-phosphonacetyl-L-ornithine promotes domain closure. The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined. The side chain of Cys-303 is positioned so as to be able to interact with the delta-amino group of L-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzyme-catalyzed reaction. This sulfhydryl group forms a charge relay system with Asp-263 and the alpha-amino group of L-ornithine, instead of with His-302 and Glu-310, as previously proposed. In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of approximately 20 residues between helix H10 and beta-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed. The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane.     

Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA

Site-directed mutagenesis studies have shown that the isopenicillin N synthase of Cephalosporium acremonium (cIPNS) requires two essential histidine residues (H216, H272) for activity. The determination of iron bound to the wildtype cIPNS and its absence in the mutants lacking histidine at positions 216 and 272 clearly supports the essential role these two histidines play in iron binding. However, nuclear magnetic resonance (NMR) studies have indicated that there could be three histidine residues that possibly coordinate the essential iron at the active site. To search for a presumed third histidine ligand, mutant cIPNS genes containing mutations at two histidine codons were created by in vitro cloning of fragments from the expression vectors bearing the respective cIPNS genes each with a single histidine mutation at positions H49, H64, H116, H126 and H137. All ten possible double histidine mutant cIPNS constructs were subsequently expressed in Escherichia coli. If a third histidine had a participatory role in the iron active centre of cIPNS, then one of the constructed double histidine mutants would have lost its enzymatic activity. However, analysis of the cIPNS activities of these recombinant double histidine mutants indicated that none of them was totally inactivated. Thus, the involvement of a third histidine can be repudiated.     

Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin-myosin-ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1-SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1-SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.     

Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor

TNF-alpha may play a role in mediating insulin resistance associated with obesity. This concept is based on studies of obese rodents and humans, and cell culture models. TNF elicits cellular responses via two receptors called p55 and p75. Our purpose was to test the involvement of TNF in glucose homeostasis using mice lacking one or both TNF receptors. C57BL/6 mice lacking p55 (p55(-)/-), p75, (p75(-)/-), or both receptors (p55(-)/-p75(-)/-) were fed a high-fat diet to induce obesity. Marked fasting hyperinsulinemia was seen for p55(-)/-p75(-)/- males between 12 and 16 wk of feeding the high-fat diet. Insulin levels were four times greater than wild-type mice. In contrast, p55(-)/- and p75(-)/- mice exhibited insulin levels that were similar or reduced, respectively, as compared with wild-type mice. In addition, high-fat diet-fed p75(-)/- mice had the lowest body weights and leptin levels, and improved insulin sensitivity. Obese (db/db) mice, which are not responsive to leptin, were used to study the role of p55 in severe obesity. Male p55(-)/-db/db mice exhibited threefold higher insulin levels and twofold lower glucose levels at 20 wk of age than control db/db expressing p55. All db/db mice remained severely insulin resistant based on fasting plasma glucose and insulin levels, and glucose and insulin tolerance tests. Our data do not support the concept that TNF, acting via its receptors, is a major contributor to obesity-associated insulin resistance. In fact, data suggest that the two TNF receptors work in concert to protect against diabetes.     

Activation by autophosphorylation or cGMP binding produces a similar apparent conformational change in cGMP-dependent protein kinase

Binding of cyclic nucleotide to or autophosphorylation of cGMP-dependent protein kinase (PKG) activates this kinase, but the molecular mechanism of activation for either process is unknown. Activation of PKG by cGMP binding produces a conformational change in the enzyme (Chu, D.-M., Corbin, J. D., Grimes, K. A., and Francis, S. H. (1997) J. Biol. Chem. 272, 31922-31928; Zhao, J., Trewhella, J., Corbin, J., Francis, S., Mitchell, R., Brushia, R., and Walsh, D. (1997) J. Biol. Chem. 272, 39129-31936). In the present studies, activation of type Ibeta PKG by either autophosphorylation or cGMP-binding alone causes (i) an electronegative charge shift on ion exchange chromatography, (ii) a similar increase ( approximately 3.5 A) in the Stokes radius as determined by gel filtration chromatography, and (iii) a similar decrease in the mobility of the enzyme on native gel electrophoresis. Consistent with these results, cGMP binding increases the rate of phosphoprotein phosphatase-1 catalyzed dephosphorylation of PKG which is autophosphorylated only at Ser-63 (not activated); however, dephosphorylation of PKG that is highly autophosphorylated (activated) is not stimulated by cGMP. The combined results suggest that activation of PKG by either autophosphorylation or cGMP binding alone produces a similar apparent elongation of the enzyme, implying that either process activates the enzyme by a similar molecular mechanism.     

Recombinant human O6-alkylguanine-DNA alkyltransferase induces conformational change in bound DNA

We aimed to assess the impact of a preconceptional clinic (PC) on the perinatal outcome (PO) of diabetic pregnancies attended in our centre. We studied 185 pregnancies attended in the 1986-1996 period (152 in women with insulin dependent diabetes mellitus (IDDM) and 33 with non insulin-dependent diabetes mellitus (NIDDM)) and we analysed the perinatal outcome for both mother and fetus. Sixty-six women (36.1%) had enrolled in the PC, 41.4% for IDDM and 9.1% for NIDDM pregnancies, p < 0.01. First pregnancy HbA1c (in SD around the mean) was 3.98 +/- 3.00 in non-attenders (NA) vs 2.57 +/- 2.41 in attenders (A), p < 0.01. The final HbA1c was in the normal range in both groups. D-R class according to White classification was 33.0% for NA vs 54.5% for A, p < 0.01. There were no differences in the rates of abortion and major malformations (8.8% NA vs 3.6% A, ns). Both groups differed in the rate of cesarean sections (54.9% NA vs 71.0% A, p < 0.05) and in the rate of small for gestational age infants (SGA) (8.7% NA vs 1.8% A, p < 0.05). There were no differences between groups in maternal or neonatal outcomes. In this group of diabetic women with a moderate although less than optimal metabolic control at the beginning of pregnancy, the impact of PC on PO is less evident than described.     

The conformational change and active site structure of tetrahydrodipicolinate N-succinyltransferase

Tetrahydrodipicolinate (THDP) N-succinyltransferase catalyzes the conversion of tetrahydrodipicolinate and succinyl-CoA to L-2-(succinylamino)-6-oxopimelate and CoA. This reaction represents the committed step of the succinylase branch of the diaminopimelate/L-lysine biosynthetic pathway by which many bacteria synthesize meso-diaminopimelate, a component of peptidoglycan, and L-lysine from L-aspartate. The crystal structures of THDP succinyltransferase in complex with the substrate/cofactor pairs L-2-aminopimelate/coenzyme A and L-2-amino-6-oxopimelate/coenzyme A have been determined and refined to 2.0 A resolution. The active site of the enzyme is a long narrow groove located at the interface between two left-handed parallel beta-helix (LbetaH) structural domains of the trimeric enzyme. On binding the amino acid acceptor and cofactor, this groove is covered by residues from the C-terminus of one subunit and a flexible loop excluded from the LbetaH domain of an adjacent subunit to form a tunnel. This conformational change is directly related to interactions between the enzyme and the bound amino acid substrate and cofactor and serves to shield the ligands from bulk solvent and to orient the nucleophilic amino group of the amino acid acceptor toward the mercaptoethylamine group of the cofactor.     

Kinetics of conformational change of troponin C induced by calcium

The kinetics of conformational change of troponin C (TN-C) induced by binding and removal of calcium ions were studied by measuring the fluorescence of tyrosine by stopped-flow spectrofluorometry. When the concentration of free calcium ions in the solution [Ca2+] was increased rapidly from 4X10(-9)M to 1X10(-3)M at neutral pH, a first-order reaction with a rate constant of 13.7 sec-1, which was preceded by a much faster reaction, was observed. In contrast, when [Ca2+] was reduced from 2X10(-3)M to 4.4X10(-8)M, two first-order reactions with rate constants of 7.4 and 0.78 sec-1, preceded by a much faster reaction, were observed.     

Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51

The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg2+. In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30-37 degrees C but is stabilized by the presence of ATP and Mg2+. Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C. AMP-P-N-P lacks this property, while ADP protects partially but only at 5-10 times higher concentrations than ATP. These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis. Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-S. Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP. The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an alpha-helical protein. Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees C. We surmise that the active conformation is more resistant to inactivation at elevated temperature. Our data suggest that one of the roles of ATP and Mg2+ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates.     

A conformational change of the photoactive bacteriopheophytin in reaction centers from Rhodobacter sphaeroides

It is demonstrated by ENDOR and Special TRIPLE spectroscopy that two distinct radical anion states of the intermediate electron acceptor (I), a bacteriopheophytin, can be freeze-trapped in isolated photosynthetic reaction centers of Rhodobacter sphaeroides. The formation of these states depends on the illumination time prior to freezing and the temperature. The first state, I1.-, is metastable and relaxes irreversibly at T approximately 160 K to the second state, I2.-. Experiments on quinone depleted as well as mutant reaction centers help to exclude the possibility that other cofactors besides the bacteriopheophytin in the A-branch, PhiA, are reduced during the trapping procedure. In particular, two mutants are investigated, in which the hydrogen bonds to PhiA that exist in the wild type are removed. These mutants are EL(L104), in which Glu at position L104 near the 13(1)-keto group of PhiA is replaced by Leu, and WF(L100), in which Trp at position L100 near the 13(2)-methyl ester of PhiA is replaced by Phe. Both mutations have characteristic effects on both I.- states. In addition, the replacement of Thr at position M133 near the 13(1)-keto group of the inactive bacteriopheophytin and of Gly at position M203 near the 13(1)-keto group of the accessory bacteriochlorophyll in the A-branch by Asp causes no changes of the electronic structure of I.-. The two I.- states are interpreted in terms of a reorientation of the 3-acetyl group of PhiA after reduction. Possible implications for the initial charge separation process are discussed.