Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

Ca2+ binding and conformational changes in a calmodulin domain.

Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k.

The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.

Kinetics of DNA hydration.

The hydration of the d(CGCGAATTCGCG) B-DNA duplex in solution was studied by nuclear magnetic relaxation dispersion (NMRD) of the water nuclei 1H, 2H, and 17O, and by nuclear Overhauser effects (NOEs) in high-resolution two-dimensional 1H NMR spectra. By comparing results from the free duplex with those from its complex with netropsin, water molecules in the "spine of hydration" in the AATT region of the minor groove could be distinguished from hydration water elsewhere in the duplex. The 2H and 17O relaxation dispersions yield a model-independent residence time of 0.9(+/-0.1) ns at 4 degrees C for five highly ordered water molecules in the spine. When corrected for frequency offset effects, the NOE data yield the same residence time as the NMRD data, giving credence to both methods. At 27 degrees C, the residence time is estimated to 0.2 ns, a factor of 40 shorter than the tumbling time of the duplex. The NMRD data show that all water molecules associated with the duplex, except the five molecules in the spine, have residence times significantly shorter than 1 ns at 4 degrees C. There is thus no long-lived hydration structure associated with the phosphate backbone. In contrast to 2H and 17O, the 1H relaxation dispersion is dominated by labile DNA protons and therefore provides little information about DNA hydration.

NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state.

In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+-induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild-type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 +/- 15% of the open conformation and an exchange rate of (1-7) x 10(4) s(-1) were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2+ titration of the 15N-labeled mutant, the macroscopic binding constants [log(K1) = 4.9 +/- 0.3 and log(K2) = 3.15 +/- 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+-binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.

1H NMR assignments of apo calcyclin and comparative structural analysis with calbindin D9k and S100 beta.

The homodimeric S100 protein calcyclin has been studied in the apo state by two-dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence-specific 1H NMR assignments were obtained for all but one backbone and > 90% of the side-chain resonances. To our knowledge, the 2 x 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homonuclear methods. Sequential and medium-range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel beta-type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 x 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100 beta, and monomeric apo calbindin D9k has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three-dimensional structures of the calcyclin monomer subunit and calbindin D9k, which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D9k is monomeric, but full-length S100 proteins form homodimers.

Sequence dependence and direct measurement of crossover isomer distribution in model Holliday junctions using NMR spectroscopy.

A 32-base-pair model of the Holliday junction (HJ) intermediate in genetic recombination has been prepared and analyzed in-depth by 2D and 3D (1)H NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permutation of the base pairs at the junction relative to a previously studied HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11459], designed to probe the effect of the sequence at the n - 1 position (where n is the residue directly at the branch point) on the stacking geometry. Observation of several interbase nuclear Overhauser effects (NOEs) clearly indicates a strong preference for the isomer opposite that observed for J2, confirming the dependence of stacking isomer preference on the sequence at the junction. As for other model HJs studied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single (15)N-labeled thymine residue at the branch point. 1D (15)n-filtered (1)H-detected experiments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more direct and accurate measure of the crossover isomer distribution. The comparative analysis of our immobile HJs and a model cruciform structure [Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the relevance of crossover isomer preference in vivo.

Two-dimensional 1H nuclear magnetic resonance studies of the half-saturated (Ca2+)1 state of calbindin D9k. Further implications for the molecular basis of cooperative Ca2+ binding.

Calbindin D9k exhibits cooperative binding of two calcium ions, hence study of the half-saturated states of the protein is critical to understanding the binding process. However, the half-saturated states are not significantly populated under equilibrium conditions. To circumvent this problem, an absolutely conserved glutamic acid residue in the C-terminal binding site (site II) has been mutated to glutamine (E65Q), causing a substantial reduction in calcium affinity and permitting detailed two-dimensional 1H NMR analysis of calbindin D9k with a calcium ion bound only in the N-terminal EF-hand. Complete 1H resonance assignments have been obtained for (Ca2+)1 E65Q, as well as near complete assignments for the apo and (Ca2+)2 states. A value of 1.1(+/- 0.2) x 10(3) M-1 has been determined for the calcium binding constant in site II, from an analysis of the chemical shift changes in response to titration with calcium. The elements of secondary structure and global folding patterns were identified from nuclear Overhauser effects, backbone spin-spin coupling constants and the exchange rates of backbone amide protons. Although the mutation has only very small effects on the secondary structure and global fold of the protein, it so drastically lowers affinity for Ca2+ in the C-terminal site that (Ca2+)2 E65Q does not correspond to a standard (Ca2+)2 state. From the analysis of the half-saturated state, it is apparent that some reorganization of the structure and changes in the internal dynamics of calbindin D9k does occur for each step of the apo-->(Ca2+)1(I)-->(Ca2+)2 binding pathway. When the first ion is bound to the N-terminal EF-hand, that half of the molecule adopts a conformation and dynamic state similar to the fully calcium-loaded protein state, whereas only minor changes occur in the C-terminal EF-hand. It is only upon binding of the second calcium ion that the C-terminal EF-hand switches over to the fully calcium-loaded state. Together with the results from our earlier study of the apo-->(Ca2+)1(II)-->(Ca2+)2 binding pathway, these findings indicate that changes in protein conformation and dynamics associated with Ca2+ binding contribute to the observed positive cooperativity, and that the molecular details of the cooperative binding events are different for the two binding pathways.

Hyperkinetic disorders in seven-year-old children with perceptual, motor and attentional deficits.

One hundred and forty-one seven-year-old Swedish children took part in an extensive neuropsychiatric study involving total population samples of children who had shown perceptual, motor and attentional deficits in pre-school, and blindly examined comparison children. The present paper reports on generalized hyperkinesis, i.e. hyperactivity in the child in all of three different assessment settings. About a third of children diagnosed according to strict criteria as suffering from 'Minimal Brain Dysfunction' showed generalized hyperkinesis. In children without perceptual-motor deficits, hyperkinesis was very much rarer. A total population frequency for generalized hyperkinesis in the range of 1-3% is reported as probable. Heredity for delayed maturation, non-optimal pre-, peri- and neonatal factors and adverse psychosocial factors were found to interact in the shaping of the disorder.

Perceptual, motor and attentional deficits in seven-year-old children. Neurological screening aspects.

In an extensive neuropsychiatric study of seven-year-old children, operational criteria for diagnosing minimal brain dysfunction (MBD) syndrome were used. Detailed behavioural assessment and meticulous neurological examination provided the basis for the MBD diagnosis. The time-consuming specialist examination by the child neurologist was considered too sophisticated for use in everyday clinical practice. Therefore, the results obtained at a short neurodevelopmental screening assessment performed by a child psychiatrist were analysed with the aim of finding a limited set of neurological examination items with high discriminating capacity detecting for MBD syndromes. A set of six such items (diadochokinesis, hopping on one leg, standing on one leg, cutting out a paper circle, associated movements when walking on lateral sides of feet and the labyrinth test of the WISC) produced a minimal rate of misclassified cases. It is argued that this discriminant set may be useful in everyday child psychiatric and pediatric assessment of children who raise suspicion of suffering from MBD.

Mental retardation in Swedish urban children: some epidemiological considerations.

The total population of children born in 1971 and living in Gothenburg, Sweden, by the end of 1977 was screened in order to estimate prevalence figures for various neurodevelopmental disorders. Ninety-four percent of all children assessed attended public preschools. Questionnaires aimed at detecting perceptual, conceptual, motor, and attentional deficits were completed by preschool teachers for 72% of children in these schools. Samples of children with and without problems on the questionnaire were given neuropsychiatric examinations. National registers were searched to identify mentally retarded children not in public preschools. The total population frequency figure for unequivocal mental retardation was almost 1% with an additional 1% of the total population deemed to be of borderline intelligence.

Perceptual, motor and attentional deficits in six-year-old children. Epidemiological aspects.

A total population study of 4797 six-year-old children attending the public preschools in the city of Göteborg (Gothenburg) has been carried out. A questionnaire with 34 questions about MBD-related problems was distributed to all pre-school teachers. Three thousand four hundred and forty-eight questionnaires were completed. Factor analysis of the questionnaire and empirical results from a pilot study provided the basis for selecting for further study children with pre-school signs and symptoms suggestive of MBD. Neurological, psychiatric and psychological assessment of 82 children with, and 59 children without, pre-school symptoms of MBD revealed that in the index groups 41% of the children, and in the control group 3% of the children, had MBD. Extrapolation procedures gave a total population frequency of 1.2% with severe MBD and a further 5.9% with mild-moderate MBD. A very large questionnaire refusal rate (28%) is discussed. The relevance of the calculated frequency figures, especially as regards the mild-moderate MBD category, cannot be properly evaluated until long-term follow-up has been completed.

Immune reactions and long-term therapy with human leukocyte interferon.

Twenty patients with osteosarcoma were treated with exogenous human leukocyte interferon for periods ranging from 6 to 18 months. Eleven of them remained free from detectable tumour growth during this treatment. Blood samples from all patients were tested for antibodies against interferon and against impurities in the interferon preparations. No patient developed detectable levels of neutralizing antibodies against interferon. All patients formed antibodies against contaminants in the concentrated crude interferon and the partially purified interferon preparation which had been used for treatment.

Virus infections and recurrence of osteosarcoma in patients receiving human leukocyte interferon.

Human leukocyte interferon was given as adjuvant treatment for osteosarcoma in 26 patients. Blood from these patients was studied at regular intervals for antibodies to various micro-organisms. A continuous record of clinically manifest infections during the course of interferon therapy was made in 23 of the 26 patients. Distant metastases arose in 14 patients - the metastasis group. The report presents comparisons between these patients and the non-metastasis group. Seroconversion in tests with any of the studied micro-organisms was not common. Most of the seroconversions were not associated with clinical symptoms. Seroconversion during interferon therapy was confined to patients in the metastasis group. In some cases there was a chronologic link with early evidence of metastasis. The results of the serologic investigations are discussed and are correlated to the course of interferon therapy and of the neoplastic disease.

Microbiological findings in pregnant women with premature rupture of the membranes.

From 30 consecutive cases of premature rupture of the membranes (PROM) and matched controls, specimens from urine, cervix, amniotic fluid, and placenta as well as neonatal nose and throat swabs were investigated bacteriologically and virologically. In addition virus serological investigation was done. Among the PROM cases the anaerobic cervical isolates outnumbered the aerobic ones, and the total number of aerobic as well as anaerobic isolates was less in the control group. The anaerobes B. fragilis and Strept. intermedius were isolated from the cervix, amniotic fluid, or placenta in 23% and 30% of the PROM patients, respectively. None of the controls harboured B. fragilis, while Strept. intermedius was isolated from 6.7% of the controls. Group B streptococci were recovered from the mother's cervix in 20% of the PROM patients and in 6.7% among the controls. Four cases of neonatal septicaemia were encountered, and another two cases were clinically suspected, but not microbiologically verified, contributing to a high perinatal mortality rate (17,6%). Of the PROM patients, 27% developed puerperal infection, while none of the control mothers had such complications. The significance of the anaerobic bacteria as well as group B streptococci for the maternal and neonatal outcome in cases of PROM is discussed, and a possible aetiological role of ascending infection in this complication of pregnancy is postulated.

Congenital rubella after maternal reinfection.

This report concerns a boy with congenital rubella virus infection. The diagnosis was confirmed by virus isolation, demonstration of rubella-specific serum IgM and by persistence of serum antibody at the age of 9 months. In 2 sera from the mother sampled 2 weeks apart 20 months before the birth of the boy, low titers of rubella antibody were demonstrated by hemagglutination-inhibition, hemolysis-in-gel and complement fixation tests, but not by neutralization. Significant rises in titer were demonstrable by all serologic reactions--including neutralization--at the time of birth of the infected child. The mother was not aware of any rubella-like illness or exposure to such disease during pregnancy. The case is discussed against findings of neutralizing activity in sera from natural immunes and rubella vaccinees.