cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis.

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRPMTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRPMTB remain unclear. Here, we investigate the binding mechanism between CRPMTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRPMTB forms high-order CRPMTB-DNA oligomers through interactions with nonspecific DNA sequences or preformed CRPMTB-DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRPMTB-DNA oligomers, reduces the affinity of CRPMTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRPMTB-DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRPEcoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRPMTB-DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRPBCG). The functional behavior, thermodynamic stability, and dimerization constant of CRPBCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRPEcoli, illustrating that structural homology does not imply allosteric homology.

The Efficacy, Adverse Events, and Withdrawal Rates of the Pharmacological Management of Chronic Spinal Cord Injury Pain: A Systematic Review and Meta-Analysis.

OBJECTIVE: To establish the efficacy of medications, incidence of adverse events (AEs), and withdrawal rates associated with the pharmacological management of chronic spinal cord injury pain. METHODOLOGY: PubMed, MEDLINE, Embase, CINAHL, Web of Science, CENTRAL, and PsycINFO were searched (November 2017) and updated (January 2020). Two independent review authors screened and identified papers for inclusion. RESULTS: Twenty-one studies met inclusion requirements for efficacy analysis and 17 for AE and withdrawal rate analysis; no additional papers were included from the updated 2020 search. Treatments were divided into six categories: anticonvulsants (n = 6), antidepressants (n = 3), analgesics (n = 8), anti-spasticity medications (n = 2), cannabinoids (n = 1), and other (n = 2). Trials of anticonvulsants, antidepressants, and cannabinoids included long-term follow-up trials (2 weeks to 4 months), and trials of analgesics and anti-spasticity medications, among others, were short-term trials (0-2 days). Effectiveness for neuropathic pain was found for pregabalin (3/3 studies) and lidocaine (2/3 studies). Studies using ketamine also reported effectiveness (2/2), but the quality of these papers was rated as poor. The most frequently reported AEs included dizziness, dry mouth, nausea, and constipation. Pregabalin was associated with a higher risk of somnolence (risk ratio [RR] 3.15, 95% confidence interval [CI]: 2.00-4.98) and dizziness (RR 2.9, 95% CI: 1.58-5.30). Ketamine was associated with a higher risk of reduced vision (RR 9.00, 95% CI: 0.05-146.11), dizziness (RR 8.33, 95% CI: 1.73-40.10), and somnolence (RR 7.00, 95% CI: 1.73-40.1). Withdrawal rates ranged from 18.4% for antidepressants to 0-30% for anticonvulsants, 0-10% for anti-spasticity medications, 0-48% for analgesics, 28.6% for cannabinoids, and 0-22.2% for other medications. CONCLUSION: Pregabalin was found to be effective for neuropathic pain vs placebo. Cannabinoids were ineffective for neuropathic pain. AEs are a common cause for withdrawal. The nature of AEs was poorly reported, and AE reporting should be improved in future randomized controlled trials.

Integrated Method to Attach DNA Handles and Functionally Select Proteins to Study Folding and Protein-Ligand Interactions with Optical Tweezers.

Optical tweezers has emerged as a powerful tool to study folding, ligand binding, and motor enzymes. The manipulation of proteins with optical tweezers requires attaching molecular handles to the protein of interest. Here, we describe a novel method that integrates the covalent attachment of DNA handles to target proteins with a selection step for functional and properly folded molecules. In addition, this method enables obtaining protein molecules in different liganded states and can be used with handles of different lengths. We apply this method to study the cAMP binding domain A (CBD-A) of Protein kinase A. We find that the functional selection step drastically improves the reproducibility and homogeneity of the single molecule data. In contrast, without a functional selection step, proteins often display misfolded conformations. cAMP binding stabilizes the CBD-A against a denaturing force, and increases the folded state lifetime. Data obtained with handles of 370 and 70 base pairs are indistinguishable, but at low forces short handles provide a higher spatial resolution. Altogether, this method is flexible, selects for properly folded molecules in different liganded states, and can be readily applicable to study protein folding or protein-ligand interactions with force spectroscopy that require molecular handles.