Purification and crystallization of a non-GluR2 AMPA-receptor ligand-binding domain: a case of cryo-incompatibility addressed by room-temperature data collection

Glutamate is the major excitatory neurotransmitter in the brain. Among the cognate ionotropic glutamate receptors, the subfamily selective for AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) is responsible for most fast excitatory synaptic signaling and plays key roles in synaptic plasticity. AMPA receptors (AMPA-Rs) have also been implicated in a number of neurological disorders. To investigate subunit-specific differences in the ligand binding and activation of AMPA-Rs, the GluR4 AMPA-R ligand-binding domain (LBD) was crystallized in complex with full and partial agonists. This is the first non-GluR2 AMPA-R LBD available for structural analysis. Standard cryoprotection protocols yielded high-resolution diffraction from flash-cooled crystals of the complex with the full agonist glutamate. However, for cocrystals with the partial agonist kainate, systematic screening and optimization of cryoprotection conditions yielded at best mosaic, weak diffraction at 100 K. In contrast, room-temperature data collection from capillary-mounted kainate cocrystals exhibited reproducible diffraction to better than 3 A resolution. Together, these crystals lay the foundation for a structural comparison of LBD-agonist interactions in distinct AMPA-R subunits

Domain Architecture of a Calcium-Permeable AMPA Receptor in a Ligand-Free Conformation

Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs), the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD), and of multiple conformations of the ligand-binding domain (LBD). Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPA receptors, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation

Small molecule conjugates with dimetal species for protein inhibition

Methods for targeting a protein by providing an inhibitor covalently linked to a rhodium(II) complex, introducing the inhibitor to the target protein and allowing the inhibitor and protein to interact. The rhodium(II) complex covalently linked to the inhibitor binds the target protein both inorganically and organically and forms stabilizing secondary contacts between the rhodium(II) complex and the protein

Cysteine Modifiers Suggest an Allosteric Inhibitory Site on the CAL PDZ Domain

Protein–protein interactions have become attractive targets for both experimental and therapeutic interventions. The PSD-95/Dlg1/ZO-1 (PDZ) domain is found in a large family of eukaryotic scaffold proteins that plays important roles in intracellular trafficking and localization of many target proteins. Here, we seek inhibitors of the PDZ protein that facilitates post-endocytic degradation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR): the CFTR-associated ligand (CAL). We develop and validate biochemical screens and identify methyl-3,4-dephostatin (MD) and its analog ethyl-3,4-dephostatin (ED) as CAL PDZ inhibitors. Depending on conditions, MD can bind either covalently or non-covalently. Crystallographic and NMR data confirm that MD attacks a pocket at a site distinct from the canonical peptide-binding groove, and suggests an allosteric connection between target residue Cys319 and the conserved Leu291 in the GLGI motif. MD and ED thus appear to represent the first examples of small-molecule allosteric regulation of PDZ:peptide affinity. Their mechanism of action may exploit the known conformational plasticity of the PDZ domains and suggests that allosteric modulation may represent a strategy for targeting of this family of protein–protein binding modules

Crystal Structure of a Charge Engineered Human Lysozyme Having Enhanced Bactericidal Activity

Human lysozyme is a key component of the innate immune system, and recombinant forms of the enzyme represent promising leads in the search for therapeutic agents able to treat drug-resistant infections. The wild type protein, however, fails to participate effectively in clearance of certain infections due to inherent functional limitations. For example, wild type lysozymes are subject to electrostatic sequestration and inactivation by anionic biopolymers in the infected airway. A charge engineered variant of human lysozyme has recently been shown to possess improved antibacterial activity in the presence of disease associated inhibitory molecules. Here, the 2.04 Å crystal structure of this variant is presented along with an analysis that provides molecular level insights into the origins of the protein's enhanced performance. The charge engineered variant's two mutated amino acids exhibit stabilizing interactions with adjacent native residues, and from a global perspective, the mutations cause no gross structural perturbations or loss of stability. Importantly, the two substitutions dramatically expand the negative electrostatic potential that, in the wild type enzyme, is restricted to a small region near the catalytic residues. The net result is a reduction in the overall strength of the engineered enzyme's electrostatic potential field, and it appears that the specific nature of this remodeled field underlies the variant's reduced susceptibility to inhibition by anionic biopolymers

Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis

X-ray crystallographic structural analyses of the bacterial transmembrane receptor LapD identify conserved molecular mechanisms that control biofilm formation in response to changes in the intracellular levels of the second messenger c-di-GMP

Computational Design of a PDZ Domain Peptide Inhibitor that Rescues CFTR Activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators (“potentiators” and “correctors”), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called “stabilizers”) that rescue ΔF508-CFTR activity. To design the “stabilizers”, we extended our structural ensemble-based computational protein redesign algorithm to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods

MLSys: The New Frontier of Machine Learning Systems

Machine learning (ML) techniques are enjoying rapidly increasing adoption. However, designing and implementing the systems that support ML models in real-world deployments remains a significant obstacle, in large part due to the radically different development and deployment profile of modern ML methods, and the range of practical concerns that come with broader adoption. We propose to foster a new systems machine learning research community at the intersection of the traditional systems and ML communities, focused on topics such as hardware systems for ML, software systems for ML, and ML optimized for metrics beyond predictive accuracy. To do this, we describe a new conference, MLSys, that explicitly targets research at the intersection of systems and machine learning with a program committee split evenly between experts in systems and ML, and an explicit focus on topics at the intersection of the two

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]