Symmetric Allosteric Mechanism of Hexameric Escherichia coli Arginine Repressor Exploits Competition between L-Arginine Ligands and Resident Arginine Residues

An elegantly simple and probably ancient molecular mechanism of allostery is described for the Escherichia coli arginine repressor ArgR, the master feedback regulator of transcription in L-arginine metabolism. Molecular dynamics simulations with ArgRC, the hexameric domain that binds L-arginine with negative cooperativity, reveal that conserved arginine and aspartate residues in each ligand-binding pocket promote rotational oscillation of apoArgRC trimers by engagement and release of hydrogen-bonded salt bridges. Binding of exogenous L-arginine displaces resident arginine residues and arrests oscillation, shifting the equilibrium quaternary ensemble and promoting motions that maintain the configurational entropy of the system. A single L-arg ligand is necessary and sufficient to arrest oscillation, and enables formation of a cooperative hydrogen-bond network at the subunit interface. The results are used to construct a free-energy reaction coordinate that accounts for the negative cooperativity and distinctive thermodynamic signature of L-arginine binding detected by calorimetry. The symmetry of the hexamer is maintained as each ligand binds, despite the conceptual asymmetry of partially-liganded states. The results thus offer the first opportunity to describe in structural and thermodynamic terms the symmetric relaxed state predicted by the concerted allostery model of Monod, Wyman, and Changeux, revealing that this state is achieved by exploiting the dynamics of the assembly and the distributed nature of its cohesive free energy. The ArgR example reveals that symmetry can be maintained even when binding sites fill sequentially due to negative cooperativity, which was not anticipated by the Monod, Wyman, and Changeux model. The molecular mechanism identified here neither specifies nor requires a pathway for transmission of the allosteric signal through the protein, and it suggests the possibility that binding of free amino acids was an early innovation in the evolution of allostery

A Network of Sites and Upskilled Therapists to Deliver Best-Practice Stroke Rehabilitation of the Arm : Protocol for a Knowledge Translation Study

Implementation of evidence-informed rehabilitation of the upper limb is variable, and outcomes for stroke survivors are often suboptimal. We established a national partnership of clinicians, survivors of stroke, researchers, healthcare organizations, and policy makers to facilitate change. The objectives of this study are to increase access to best-evidence rehabilitation of the upper limb and improve outcomes for stroke survivors. This prospective pragmatic, knowledge translation study involves four new specialist therapy centers to deliver best-evidence upper-limb sensory rehabilitation (known as SENSe therapy) for survivors of stroke in the community. A knowledge-transfer intervention will be used to upskill therapists and guide implementation. Specialist centers will deliver SENSe therapy, an effective and recommended therapy, to stroke survivors in the community. Outcomes include number of successful deliveries of SENSe therapy by credentialled therapists; improved somatosensory function for stroke survivors; improved performance in self-selected activities, arm use, and quality of life; treatment fidelity and confidence to deliver therapy; and for future implementation, expert therapist effect and cost-effectiveness. In summary, we will determine the effect of a national partnership to increase access to evidence-based upper-limb sensory rehabilitation following stroke. If effective, this knowledge-transfer intervention could be used to optimize the delivery of other complex, evidence-based rehabilitation interventions

Affinity, Specificity, and Cooperativity of DNA Binding by Bacterial Gene Regulatory Proteins

Nearly all of biology depends on interactions between molecules: proteins with small molecules, proteins with other proteins, nucleic acids with small molecules, and nucleic acids with proteins that regulate gene expression, our concern in this Special Issue. All those kinds of interactions, and others, constitute the vast majority of biology at the molecular level. An understanding of those interactions requires that we quantify them to learn how they interact: How strongly? With which partners? How—and how well—are different partners distinguished? This review addresses the evolution of our current understanding of the molecular origins of affinity and specificity in regulatory protein–DNA interactions, and suggests that both these properties can be modulated by cooperativity

Reversible ligand binding: theory and experiment

Reversible Ligand Binding: Theory and Experiment discusses the physical background of protein-ligand interactions—providing a comprehensive view of the laws that govern reversible, as well as irreversible, ligand binding. Special consideration is devoted to enzymology, a field usually treated separately from ligand binding, but actually governed by identical thermodynamic relationships. Attention is given to the design of experiments, including how to uncover evidence of biochemical features that may otherwise escape notice. Classical experiments are reviewed in order to further highlight the importance of the experimental design. Overall, the book supplies students with understandingnecessary for interpreting ligand binding experiments, formulating plausible reaction schemes, and analyzing the data according to a chosen model. Topics covered include: theory of ligand binding to monomeric proteins; practical considerations and commonly encountered problems; oligomeric proteins with multiple binding sites; ligand binding kinetics; hemoglobin and its ligands; single-substrate enzymes and their inhibitors; two-substrate enzymes and their inhibitors; and rapid kinetic methods for studying enzyme reactions

Calmodulin Transduces Ca2+ Oscillations into Differential Regulation of Its Target Proteins

Diverse physiological processes are regulated differentially by Ca2+ oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca2+ binding affinities, and target exchange rates that depend on both target identity and Ca2+ occupancy. The binding dynamics among Ca2+ Mg2+, calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca2+-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca2+ transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin

Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing.

The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD-IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism

Calmodulin Transduces Ca²⁺ Oscillations into Differential Regulation of Its Target Proteins

Diverse physiological processes are regulated differentially by Ca²⁺ oscillations through the common regulatory hub calmodulin. The capacity of calmodulin to combine specificity with promiscuity remains to be resolved. Here we propose a mechanism based on the molecular properties of calmodulin, its two domains with separate Ca²⁺ binding affinities, and target exchange rates that depend on both target identity and Ca²⁺ occupancy. The binding dynamics among Ca²⁺, Mg²⁺, calmodulin, and its targets were modeled with mass-action differential equations based on experimentally determined protein concentrations and rate constants. The model predicts that the activation of calcineurin and nitric oxide synthase depends nonmonotonically on Ca²⁺-oscillation frequency. Preferential activation reaches a maximum at a target-specific frequency. Differential activation arises from the accumulation of inactive calmodulin-target intermediate complexes between Ca²⁺ transients. Their accumulation provides the system with hysteresis and favors activation of some targets at the expense of others. The generality of this result was tested by simulating 60 000 networks with two, four, or eight targets with concentrations and rate constants from experimentally determined ranges. Most networks exhibit differential activation that increases in magnitude with the number of targets. Moreover, differential activation increases with decreasing calmodulin concentration due to competition among targets. The results rationalize calmodulin signaling in terms of the network topology and the molecular properties of calmodulin

The effectiveness of somatosensory retraining for improving sensory function in the arm following stroke: a systematic review

Objective: The aim of this study was to evaluate if somatosensory retraining programmes assist people to improve somatosensory discrimination skills and arm functioning after stroke. Data sources: Nine databases were systematically searched: Medline, Cumulative Index to Nursing and Allied Health Literature, PsychInfo, Embase, Amed, Web of Science, Physiotherapy Evidence Database, OT seeker, and Cochrane Library. Review methods: Studies were included for review if they involved (1) adult participants who had somatosensory impairment in the arm after stroke, (2) a programme targeted at retraining somatosensation, (3) a primary measure of somatosensory discrimination skills in the arm, and (4) an intervention study design (e.g. randomized or non-randomized control designs). Results: A total of 6779 articles were screened. Five group trials and five single case experimental designs were included (N = 199 stroke survivors). Six studies focused exclusively on retraining somatosensation and four studies focused on somatosensation and motor retraining. Standardized somatosensory measures were typically used for tactile, proprioception, and haptic object recognition modalities. Sensory intervention effect sizes ranged from 0.3 to 2.2, with an average effect size of 0.85 across somatosensory modalities. A majority of effect sizes for proprioception and tactile somatosensory domains were greater than 0.5, and all but one of the intervention effect sizes were larger than the control effect sizes, at least as point estimates. Six studies measured motor and/or functional arm outcomes (n = 89 participants), with narrative analysis suggesting a trend towards improvement in arm use after somatosensory retraining. Conclusion: Somatosensory retraining may assist people to regain somatosensory discrimination skills in the arm after stroke