TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM’s specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models’ prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime in vitro and in vivo. Therefore, we expect this framework to have numerous applications in drug and protein design.

Transcranial direct current stimulation (tDCS) of the human motor cortex induces changes in excitability within cortical and spinal circuits that occur during and after the stimulation. Recently, transcutaneous spinal direct current stimulation (tsDCS) has been shown to modulate spinal conduction properties, as assessed by somatosensory-evoked potentials, and transynaptic properties of the spinal neurons, as tested by postactivation depression of the H reflex or by the RIII nociceptive component of the flexion reflex in the lower limb. To further explore tsDCS-induced plastic changes in spinal excitability, we examined, in a double-blind crossover randomized study, the stimulus-response curves of the soleus H reflex before, during, at current offset and 15 min after anodal, cathodal, and sham tsDCS delivered at the Th11 level (2.5 mA, 15 min, 0.071 mA/cm(2), 0.064 C/cm(2)) in 17 healthy subjects. Anodal tsDCS induced a progressive leftward shift of the recruitment curve of the soleus H reflex during the stimulation; the effects persisted for at least 15 min after current offset. In contrast, both cathodal and sham tsDCS had no significant effects. This exploratory study provides further evidence for the use of tsDCS as an expedient, noninvasive tool to induce long-lasting plastic changes in spinal circuitry. Increased spinal excitability after anodal tsDCS may have potential for spinal neuromodulation in patients with central nervous system lesions.

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Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved.hydrogen-deuterium exchange | domain-domain interaction | conserved residues | protein structure | apolipoprotein E A poE is a 34-kDa protein whose normal function in the brain is to transport lipids and cholesterol to neuronal cells. In humans, there are three common isoforms-apoE2, apoE3, and apoE4-that appear to differ in important functional properties such as the distinct differences between apoE isoforms with respect to lipid transport (1). ApoE4 is the most studied since, relative to apoE3, it is known to be the major risk factor for the development of late-onset Alzheimer's disease (2, 3), whereas apoE2, the less common form, is associated with type III hyperlipoproteinemia (4, 5). A great many papers have discussed possible mechanisms for lipid binding (i.e., refs. 6-11), but there is no proposal for the mechanism of lipid binding that would explain the differences between the isoforms. From studies using FRET, small-angle X-ray diffraction, and electron paramagnetic resonance spectroscopy, it has been shown that apoE undergoes a conformational change on lipid binding, with the protein adopting a hairpin-like structure (7,(11)(12)(13). Here again, however, specific details are lacking.In this paper, we assemble a number of experimental and computational observations to provide a model for the mechanism of lipid binding. This model includes a consideration of the fulllength apoE structure, results from hydrogen-deuterium exchange (HDX) experiments, information on conserved and nonconserved residues, an appreciation of the role of intrinsically disordered regions (IDRs), and molecular dynamics calculations.The proposed model explains why there are differences between apoE isoforms with respect to lipid binding and why lipid enhances apoE binding to receptors such as the low-density lipoprotein receptor (LDLR). As such, it opens the way to develop small molecular weight compounds that could preferentially inf...

Elderly patients with comorbidities are at a higher risk for complications and adverse outcome after lumbar spine surgery. The effects of age and comorbidities on patient outcomes have been quantified. This information is critical in counseling elderly patients about the risk of surgery in their age group.

We used multivariate analysis to identify significant risk factors for visual loss after spine surgery. National population-based estimate of visual impairment after spine surgery confirms that ophthalmic complications after spine surgery are rare. Since visual loss may be reversible in the early stages, awareness, evaluation and prompt management of this rare but potentially devastating complication is critical.

Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators.