Virtual patients: an effective educational intervention to improve paediatric basic specialist trainee education in the management of suspected child abuse?

Child abuse is a particularly difficult subject to teach at both undergraduate and postgraduate level. Most doctors are dissatisfied with their training in child abuse recognition and management. We developed an interactive video based Virtual Patient to provide formal training for paediatric Basic Specialist Trainees in the recognition of suspected child abuse. The Virtual Patient case revolves around the management of suspected physical abuse in a seven month old child, who initially presents to the Emergency Department with viral upper respiratory tract symptoms. This Virtual Patient was used to facilitate a case discussion with Basic Specialist Trainees. A questionnaire was developed to determine their perception of the value of the Virtual Patient as an educational tool. Twenty five Basic Specialist Trainees completed the questionnaire. Upon completion of the case, 23/25 (92%) participants reported greater self confidence in their ability to recognize cases of suspected child abuse and 24/25 (96%) of participants reported greater self confidence in their ability to report cases of suspected child abuse. Basic Specialist Trainees perceived the Virtual Patient to be a useful educational tool. Virtual Patients may have a role to play in enhancing postgraduate training in the recognition of suspected child abuse.

The 1.9 A resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY.

To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an alpha-thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 A resolution. We have also characterized this analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chemically modified derivative, phosphono-CheY, exhibits many of the chemical properties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the alpha-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10sigma. The molecule shows a number of localized conformational changes that are believed to constitute the postphosphorylation activation events. The most obvious of these changes include movement of the side chain of the active site base, Lys 109, and a predominately buried conformation of the side chain of Tyr 106. In addition, there are a number of more subtle changes more distant from the active site involving the alpha4 and alpha5 helices. These results are consistent with our previous structural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural changes away from the active site.

Identification of the binding interfaces on CheY for two of its targets, the phosphatase CheZ and the flagellar switch protein fliM.

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.

Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway.

The crystal structure at 2.0-A resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth beta-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.

Phosphohistidines in bacterial signaling.

The movement of Gram-negative bacteria in response to nutrients in the environment is driven by two interlinked chemotaxis systems, the methyl-accepting chemotaxis protein (MCP)-mediated pathway, and the phosphoenolpyruvate: sugar phosphotransferase (PTS)-mediated pathway. The physical link connecting the two systems is unclear, but the common utilization of histidine-containing phosphocarrier proteins is an intriguing similarity. The recent structure determinations of several proteins from the PTS-mediated pathway, the phosphotransfer domain from the kinase CheA of the MCP-mediated chemotaxis pathway, and a homologous kinase, ArcB, enable the comparison of the histidine active sites of these systems. Overall, the tertiary folds of the proteins are quite different, as are the structural details of the histidine active sites within the proteins, and therefore there is not an obvious structural homolog via which the two pathways communicate, despite their similar chemical mechanisms.

Structure and dynamics of a CheY-binding domain of the chemotaxis kinase CheA determined by nuclear magnetic resonance spectroscopy.

The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals. Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which binds to the downstream targets of phosphorylation, the response regulators CheY and CheB. This protein fragment contains the CheY-binding domain flanked on each side by regions that correspond to domain linkers in the intact protein. The structure of the domain was determined from 1429 restraints derived from heteronuclear multidimensional NMR experiments. Hybrid distance geometry--dynamical simulated annealing methods were used to calculate a family of structures that satisfy the experimental distance restraints and torsion angle restraints. The root mean square deviation of the 69 ordered residues in the domain is 0.52 A for the backbone heavy atoms and 0.99 A for all heavy atoms. The residues that have been implicated as important for CheY binding form a face consisting of several partially buried hydrophobic residues, framed by charged residues. The dynamic properties of this protein fragment were measured and analyzed using both isotropic and anisotropic models of molecular motion. The linker regions are very flexible and disordered, as evidenced by the very dynamics properties as compared to the CheY-binding domain. The CheY-binding domain of CheA is structurally similar to the histidine-containing phosphocarrier, HPr, which is a protein involved in the phosphoenolpyruvate:sugar phosphotransferase (PTS) pathway. This structural similarity suggests a possible evolutionary relationship of the PTS and chemotaxis pathways.

Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker.

Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA1-233) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA1-233 remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA124-257. Relaxation properties of the backbone 15N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T1 and T2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA1-233 were also compared with those of CheA1-134. Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.

Nuclear magnetic resonance assignments and global fold of a CheY-binding domain in CheA, the chemotaxis-specific kinase of Escherichia coli.

CheA is the histidine autokinase in the Escherichia coli chemotaxis signal transduction pathway responsible for coupling of signals received by transmembrane receptors to the response regulators CheY and CheB. Here NMR spectroscopy is used to study a 14 kDa fragment of CheA, residues 124-257, that binds the response regulator CheY. Backbone atom resonance assignments were obtained by analysis of 3D HNCACB, 3D CBCA(CO)NH, and HNCO spectra, whereas side-chain assignments were obtained primarily by analysis of 3D H(CCO)NH, 3D C(CO)NH, 3D HCCH-TOCSY, and 3D 1H, 15N TOCSY-HSMQC spectra. NOE cross peak patterns and intensities as well as torsion angle restraints were used to determine the secondary structure, and a low-resolution structure was calculated by hybrid distance-geometry simulated annealing methods. The CheA124-257 fragment consists of four antiparallel beta strands and two helices, arranged in an "open-faced beta-sandwich" motif, as well as two unstructured ends that correspond to domain linkers in the full-length protein. The 15N-1H correlation spectrum of 15N-labeled CheA124-257 bound to unlabeled CheY shows specific localized changes that may correspond to a CheY-binding face on CheA.

Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface.

Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway. The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY. When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site. A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA. The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis.

Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem.

In the cyanobacterium Anabaena, the precursor to tRNA(Leu) has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566-1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5' and 3' exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5' splice site, occurs with kcat congruent to 14 min-1 and kcat/Km = 5 x 10(4) M-1 min-1 (32 degrees C, 15 mM MgCl2), unexpectedly efficient for a small group I intron. (kcat/Km is comparable to that of the Tetrahymena pre-rRNA intron, and kcat is an order of magnitude higher than any previously reported for a group I intron). The second step, ligation of the exons, is so slow (k = 0.3 min-1) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5' splice site, is unstable without the additional base pairs in the anticodon stem.(ABSTRACT TRUNCATED AT 250 WORDS)