Membrane lipids are both the substrates and a mechanistically responsive environment of TMEM16 scramblase proteins

Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo-EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function-related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer-tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs. © 2019 Wiley Periodicals, Inc.     

Inhibiting the NLRP3 Inflammasome

Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1β and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1β and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.     

Determinants for α4β2 vs. α3β4 Subtype Selectivity of Pyrrolidine-Based nAChRs Ligands: A Computational Perspective with Focus on Recent cryo-EM Receptor Structures

The selectivity of α4β2 nAChR agonists over the α3β4 nicotinic receptor subtype, predominant in ganglia, primarily conditions their therapeutic range and it is still a complex and challenging issue for medicinal chemists and pharmacologists. Here, we investigate the determinants for such subtype selectivity in a series of more than forty α4β2 ligands we have previously reported, docking them into the structures of the two human subtypes, recently determined by cryo-electron microscopy. They are all pyrrolidine based analogues of the well-known α4β2 agonist N-methylprolinol pyridyl ether A-84543 and differ in the flexibility and pattern substitution of their aromatic portion. Indeed, the direct or water mediated interaction with hydrophilic residues of the relatively narrower β2 minus side through the elements decorating the aromatic ring and the stabilization of the latter by facing to the not conserved β2-Phe119 result as key distinctive features for the α4β2 affinity. Consistently, these compounds show, despite the structural similarity, very different α4β2 vs. α3β4 selectivities, from modest to very high, which relate to rigidity/extensibility degree of the portion containing the aromatic ring and to substitutions at the latter. Furthermore, the structural rationalization of the rat vs. human differences of α4β2 vs. α3β4 selectivity ratios is here proposed.     

Folding DNA into a Lipid‐Conjugated Nanobarrel for Controlled Reconstitution of Membrane Proteins

Building upon DNA origami technology, we introduce a method to reconstitute a single membrane protein into a self‐assembled DNA nanobarrel that scaffolds a nanodisc‐like lipid environment. Compared with the membrane‐scaffolding‐protein nanodisc technique, our approach gives rise to defined stoichiometry, controlled sizes, as well as enhanced stability and homogeneity in membrane protein reconstitution. We further demonstrate potential applications of the DNA nanobarrels in the structural analysis of membrane proteins.     

Electron Cryo‐microscopy as a Tool for Structure‐Based Drug Development

For decades, X‐ray crystallography and NMR have been the most important techniques for studying the atomic structure of macromolecules. However, as a result of size, instability, low yield, and other factors, many macromolecules are difficult to crystallize or unsuitable for NMR studies. Electron cryo‐microscopy (cryo‐EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that cannot be crystallized, but atomic resolution has mostly been beyond its reach. A new generation of detectors that are capable of sensing directly the incident electrons has recently revolutionized the field, with structures of macromolecules now routinely being solved to near‐atomic resolution. In this review, we summarize some of the most recent examples of high‐resolution cryo‐EM structures. We put particular emphasis on proteins with pharmacological relevance that have traditionally been inaccessible to crystallography. Furthermore, we discuss examples where interactions with small molecules have been fully characterized at atomic resolution. Finally, we stress the current limits of cryo‐EM, and methodological issues related to its usage as a tool for drug development.     

结构生物学技术在线粒体钙离子单向转运体结构功能研究中的运用

线粒体钙离子单向转运体是一种位于线粒体内膜上的转运蛋白,具有选择和传导钙离子的功能,参与多种细胞的生理过程。目前已经确定了该单向转运体的组成成员,其中包括离子转运蛋白MCU、MCU的负调控亚基MCUb、2个Ca²⁺结合蛋白MICU1、MICU2以及MCU的调控亚基EMRE蛋白。近十多年来已经有大量的研究提出并构建了多种线粒体通过该单向转运体调节Ca²⁺摄取的模型。最近对其研究的重点转向单向转运体单个组分的结构以及对它们复合体的结构表征。这些问题的解决得益于结构生物学中核磁共振技术、X射线衍射技术的持续发展和冷冻电镜技术的突破。回顾了结构生物学技术在有关单向转运体结构问题过程中的综合运用。这一内容将有助于我们了解核磁共振、X射线晶体学以及冷冻电镜技术各自的优缺点,同时深入探究线粒体钙离子单向转运体各部分的结构及其发挥功能的分子机制。