Influenza A M2 Protein Conformation Depends On Choice Of Model Membrane

While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein. Here we show using site-directed spin-label EPR spectroscopy (SDSL-EPR) that the conformations of M2 peptides in membrane bilayers are clearly influenced by the lipid composition of the bilayers. Altering the bilayer thickness or the lateral pressure profile within the bilayer membrane changes the M2 conformation observed. The multiple M2 peptide conformations observed here, and in other published studies, optimistically may be considered conformations that are sampled by the protein at various stages during influenza infectivity. However, care should be taken that the heterogeneity observed in published structures is not simply an artifact of the choice of the model membrane. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 405–411, 2015

The conformation of the pore region of the M2 proton channel depends on lipid bilayer environment

The M2 protein from influenza A virus is a 97-amino-acid protein with a single transmembrane helix that forms proton-selective channels essential to virus function. The hydrophobic transmembrane domain of the M2 protein (M2TM) contains a sequence motif that mediates the formation of functional tetramers in membrane environments. A variety of structural models have previously been proposed which differ in the degree of helix tilt, with proposed tilts ranging from ~15° to 38°. An important issue for understanding the structure of M2TM is the role of peptide–lipid interactions in the stabilization of the lipid bilayer bound tetramer. Here, we labeled the N terminus of M2TM with a nitroxide and studied the tetramer reconstituted into lipid bilayers of different thicknesses using EPR spectroscopy. Analyses of spectral changes provide evidence that the lipid bilayer does influence the conformation. The structural plasticity displayed by M2TM in response to membrane composition may be indicative of functional requirements for conformational change. The various structural models for M2TM proposed to date—each defined by a different set of criteria and in a different environment—might provide snapshots of the distinct conformational states sampled by the protein

Kinase Inhibitor Profiling Reveals Unexpected Opportunities to Inhibit Disease-Associated Mutant Kinases

Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases, including ALK, LRRK2, RET, and EGFR, as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development

A UDP-X Diphosphatase from <i>Streptococcus pneumoniae</i> Hydrolyzes Precursors of Peptidoglycan Biosynthesis

<div><p>The gene for a Nudix enzyme (<i>SP_1669</i>) was found to code for a UDP-X diphosphatase. The <i>SP_1669</i> gene is localized among genes encoding proteins that participate in cell division in <i>Streptococcus pneumoniae.</i> One of these genes, <i>MurF</i>, encodes an enzyme that catalyzes the last step of the Mur pathway of peptidoglycan biosynthesis. Mur pathway substrates are all derived from UDP-glucosamine and all are potential Nudix substrates. We showed that UDP-X diphosphatase can hydrolyze the Mur pathway substrates UDP-N-acetylmuramic acid and UDP-N-acetylmuramoyl-L-alanine. The 1.39 Å resolution crystal structure of this enzyme shows that it folds as an asymmetric homodimer with two distinct active sites, each containing elements of the conserved Nudix box sequence. In addition to its Nudix catalytic activity, the enzyme has a 3′5′ RNA exonuclease activity. We propose that the structural asymmetry in UDP-X diphosphatase facilitates the recognition of these two distinct classes of substrates, Nudix substrates and RNA. UDP-X diphosphatase is a prototype of a new family of Nudix enzymes with unique structural characteristics: two monomers, each consisting of an N-terminal helix bundle domain and a C-terminal Nudix domain, form an asymmetric dimer with two distinct active sites. These enzymes function to hydrolyze bacterial cell wall precursors and degrade RNA.</p></div

Conformational Analysis of the DFG-Out Kinase Motif and Biochemical Profiling of Structurally Validated Type II Inhibitors

Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations that have also been referred to as DFG-out in the literature but that do not have a fully formed allosteric pocket. We have completed a structural analysis of almost 200 small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that nonclassical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two data sets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity

Recognition of ADPR by Bd-NDPSase.

<p><b>A)</b> Ribbon representation of the catalytic site of the ADPR-bound E140Q Bd-NDPSase crystal structure (PDB ID 5C7T). <b>B)</b> Schematic representation of the recognition of ADPR by Bd-NDPSase. Catalytic helix (α1) residues are shown in cyan, catalytic loop L9 residues are shown in magenta. N-terminal domain residues (1–44) are shown in green as is the specificity loop L7. Hydrogen bonds are shown as orange dashes. The prime symbol (‘) denotes residues of the opposite monomer.</p

Bd-NDPSase wild type and E140Q substrate specificity.

<p><b>A)</b> Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. <b>B)</b> Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine k_cat (5.2 (ms)^-1) and K_m (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).</p

Nucleoside recognition by bacterial Nudix sugar hydrolases.

<p>In all cases, the nucleoside is stacked by an aromatic residues in loop L1 and an arginine in strand β3’. <b>A)</b> Adenosine recognition by the Bd-NDPSase hydrolase (PDB ID 5C7T). <b>B)</b> Guanosine recognition by the Ec-NDPSase hydrolase (PDB ID 3O61). <b>C)</b> Adenosine recognition by the Ec-ADPR hydrolase (PDB ID 1KHZ). Substrate carbons are shown in black, residue carbons are colored using the main chain color convention. Nitrogen and Oxygen are colored in blue and red respectively. Hydrogen bonds are shown as orange dashes. The prime symbol (‘) denotes elements of the opposite monomer.</p

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from <i>Bdellovibrio bacteriovorus</i>

<div><p>Given the broad range of substrates hydrolyzed by Nudix (<i>nu</i>cleoside <i>di</i>phosphate linked to <i>X</i>) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from <i>Bdellovibrio bacteriovorus–</i>that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.</p></div