RNA Dynamics by NMR Spectroscopy

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined—Lego like—than in proteins, hence the numerous revelations of RNA excited states.     

From Protein Structure to Function via Computational Tools and Approaches

The three-dimensional structures of proteins are often considered fundamental for understanding their function. Yet, because of the complexity of protein structure, extracting specific functional information from structures can be a considerable challenge. Here, we present selected approaches and tools that we have developed to study and connect protein sequence, structure, and function spaces. First, we consider a global perspective of structure space and view the protein data bank (PDB) as a database. We highlight challenges in searching protein structure space and in using the PDB as the starting point for computational structural studies. Then we describe a function-oriented view and show examples of how multiple protein structures can be used to extract insights about the function and specificity of proteins at the family level.     

Salivary proline-rich proteins in mammals: Roles in oral homeostasis and counteracting dietary tannin

We review information on the structure of proline-rich proteins (PRPs), their various functions related to oral homeostasis and dietary tannin, and the structural basis of these functions. Consideration of the multifunctional nature of these salivary proteins helps explain both the subtle and large variations found in structure and secretion rates both within individuals and between species. We propose that the ancestral function of PRPs is in maintaining oral homeostasis and that counteracting dietary tannins by binding with them is a derived function. PRPs are effective in oral homeostasis at low secretion levels, whereas counteracting tannin depends on high secretion levels. In the dietary habits ranging from carnivores through omnivores to exclusively planteaters, the dietary nitrogen level is progressively reduced, and plant allelochemical intake, including tannins, increases. We suggest that during this evolution from meat-eater to plant-eater, there was some point in omnivory at which selective pressure from nitrogen limitations, arising from a low nitrogen/high tannin diet, became sufficiently great for the evolution of increased secretion level and diversification of PRPs for dealing with tannin. If this hypothesis is correct, carnivorous mammals should secrete low levels of PRPs for oral homeostasis, but should never secrete high levels, unless they are secondarily carnivorous. Omnivores consuming a diet of very little animal tissue but higher levels of tannin-containing foliage or fruit should generally have the capacity to produce high levels of salivary PRPs. Browsers and frugivores should also produce high levels of PRPs, but grazers may have reduced secretion rates depending on the antiquity of the dietary habit. This hypothesis is consistent with the limited information available on the abundance, type, and distribution of PRPs in mammals. Studies are suggested which would test the functional and evolutionary arguments presented.     

Elongation Factors EFIA and EF-Tu: Their Role in Translation and Beyond

Protein biosynthesis is a central process in cell life. Although the ribosome provides the machinery for biosynthesis, successful translation of the information contained in mRNA also relies on a number of protein factors that transiently associate with this giant ribonucleic complex. Among them, the elongation factors EF-Tu (bacteria) and EFIA (eukarya and archaea), which carry the aa-tRNA to the ribosome, have earned a special consideration. Here we present an excursus of structural information collected in the last decade on these enzymes. A significant advance in this field has been the recent crystallographic characterization of the complex between ribosome and EF-Tu. These findings, along with structural studies performed on EF-Tu complexes with small molecules and proteins, have provided a picture of the interactions of these proteins with a differentiate variety of biological partners. In contrast to EF-Tu, t he structural characterization of the states assumed by archaeal and eukaryal EFIA is largely incomplete. Here we also review solution and computational studies that have been successfully applied to integrate and complement crystallographic data. Analogies and differences between EFs isolated from different organisms will be highlighted. The new challenges of this research area, including the structural interpretation of the role of these proteins in other biological processes, are also addressed.     

The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective

Around 3000 proteins are thought to bind zinc in vivo, which corresponds to ~10% of the human proteome. Zinc plays a pivotal role as a structural, catalytic, and signaling component that functions in numerous physiological processes. It is more widely used as a structural element in proteins than any other transition metal ion, is a catalytic component of many enzymes, and acts as a cellular signaling mediator. Thus, it is expected that zinc metabolism and homeostasis have sophisticated regulation, and elucidating the underlying molecular basis of this is essential to understanding zinc functions in cellular physiology and pathogenesis. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and their malfunction has been implicated in a number of inherited diseases such as acrodermatitis enteropathica. The present review updates our current understanding of the biological functions of MTs and ZIP and ZnT transporters from several new perspectives.     

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.     

Theoretical and computational chemistry

Computer-based and theoretical approaches to chemical problems can provide atomistic understanding of complex processes at the molecular level. Examples ranging from rates of ligand-binding reactions in proteins to structural and energetic investigations of diastereomers relevant to organo-catalysis are discussed in the following. They highlight the range of application of theoretical and computational methods to current questions in chemical research.     

Structural Bioinformatics and Deep Learning of Metalloproteins: Recent Advances and Applications

All living organisms require metal ions for their energy production and metabolic and biosynthetic processes. Within cells, the metal ions involved in the formation of adducts interact with metabolites and macromolecules (proteins and nucleic acids). The proteins that require binding to one or more metal ions in order to be able to carry out their physiological function are called metalloproteins. About one third of all protein structures in the Protein Data Bank involve metalloproteins. Over the past few years there has been tremendous progress in the number of computational tools and techniques making use of 3D structural information to support the investigation of metalloproteins. This trend has been boosted by the successful applications of neural networks and machine/deep learning approaches in molecular and structural biology at large. In this review, we discuss recent advances in the development and availability of resources dealing with metalloproteins from a structure-based perspective. We start by addressing tools for the prediction of metal-binding sites (MBSs) using structural information on apo-proteins. Then, we provide an overview of the methods for and lessons learned from the structural comparison of MBSs in a fold-independent manner. We then move to describing databases of metalloprotein/MBS structures. Finally, we summarizing recent ML/DL applications enhancing the functional interpretation of metalloprotein structures.     

Atomic Structure of a 36.8 (210) Tilt Grain Boundary in TiO2

An understanding of the atomic structure at internal interfaces is of crucial importance for the electronic and structural properties of most advanced materials. Here, we present a detailed study of the atomic structure at a [001] tilt grain boundary of σ5(210) in Tio₂ (rutile). Z-contrast imaging is used to obtain a 2-D atomic map of the cation positions at the interface. Details of the charge state of cations and atomic structure around anion sites are then provided using electron energy loss spectroscopy. In particular, the spectroscopic data for oxygen is interpreted using multiple scattering theory to give 3-D structural information. These combined techniques allow a unique grain boundary structure to be defined.     

Glial Modulation of Energy Balance: The Dorsal Vagal Complex Is No Exception

The avoidance of being overweight or obese is a daily challenge for a growing number of people. The growing proportion of people suffering from a nutritional imbalance in many parts of the world exemplifies this challenge and emphasizes the need for a better understanding of the mechanisms that regulate nutritional balance. Until recently, research on the central regulation of food intake primarily focused on neuronal signaling, with little attention paid to the role of glial cells. Over the last few decades, our understanding of glial cells has changed dramatically. These cells are increasingly regarded as important neuronal partners, contributing not just to cerebral homeostasis, but also to cerebral signaling. Our understanding of the central regulation of energy balance is part of this (r)evolution. Evidence is accumulating that glial cells play a dynamic role in the modulation of energy balance. In the present review, we summarize recent data indicating that the multifaceted glial compartment of the brainstem dorsal vagal complex (DVC) should be considered in research aimed at identifying feeding-related processes operating at this level.     

Functional molecules and assemblies in controlled environments: formation and measurements

The local environment of a functional molecule or nanoscale assembly has tremendous impact on it and thus can be used for functional control. In addition, the local environment is critical in the interface to the physical, chemical, and biological worlds beyond the assemblies that are the most common applications targeted. Functional measurements without local structural information lack key insight into both the details and the roles of the environment. This Account focuses on progress toward and challenges in the controlled assembly and measurements of functional nanostructures in well-defined environments. The study of single precise supramolecular assemblies in well-defined environments offers unique insights into both interactions and function. By designing interactions between molecules and controlling assembly conditions, we can create and place atomically precise nanostructures. The tools to test the structures targeted and to measure the function of these assemblies are just now being developed and becoming available. Advances in this field have depended on gaining access to measurements at this scale. In particular, we recognize but do not yet understand the critical role of the chemical and physical environment of the assemblies. Likewise, we are just now realizing the important role that the substrates to which the assemblies are attached play in these processes. In order to develop a predictive understanding and the ability to design and to optimize functional assemblies, we must elucidate the physical, chemical, and electronic couplings among the molecules in the assemblies and with their substrates. With a suite of atomic- and molecular-resolution analytical tools, we are able both to ascertain whether the targeted structures have been formed and to measure their function. One of the keys to our ability to determine structure and measure function has been the development and application of methods for the automated acquisition, analysis, and associations of thousands or tens of thousands of single-molecule/particle/assembly structural, dynamic, spectroscopic, and functional data points.     

The systems biology of glycosylation

Glycosylation can have a profound influence on the function of a variety of eukaryotic cells. In particular, it can affect signal transduction and cell-cell communication properties and thus shape critical cell decisions, including the regulation of differentiation and apoptosis. Regulation of glycosylation has multiple layers of complexity, both structural and functional, which make its experimental and theoretical analysis difficult to perform and interpret. Novel research methodologies provided by systems biology can help to address many outstanding issues and integrate glycosylation with other metabolic and cell regulation processes. Here we review the toolbox available for biochemical systems analysis of glycosylation.     

STIM Proteins: An Ever-Expanding Family

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.     

Communication pathways between the nucleotide pocket and distal regulatory sites in protein kinases

Protein kinases control many cellular processes via the ATP-dependent phosphorylation of specific amino acids on target proteins. Despite the availability of the three-dimensional structures of a variety of protein kinases, it has been particularly difficult to explain how noncatalytic domains removed from the active site regulate catalytic function. In this review, we describe how solution methodologies complement the available structural data and help explain how protein kinases may utilize medium-to-long-range effects to regulate substrate phosphorylation. For illustration, two protein kinases, cAMP-dependent protein kinase and the C-terminal Src kinase, are presented as paradigms for the serine/threonine- and tyrosine-specific families. While active-site residues provide an optimal environment for fast phosphoryl group transfer in these and other kinases, the overall rate of protein phosphorylation is limited by nucleotide binding and associated structural changes. Hydrogen-deuterium exchange studies reveal that nucleotide binding induces changes that radiate from a central structural assembly composed of the catalytic loop, glycine-rich loop, and helix alpha C to unique peripheral regions inside and outside the kinase core. This collection of conserved and unique elements delivers information from the active site to distal regions and possibly provides information flow back to the active site. This "push-pull" hypothesis offers a means for understanding how protein kinases can be regulated by protein-protein interactions far from the active site.     

Computational Studies of Allosteric Regulation in the Hsp90 Molecular Chaperone: From Functional Dynamics and Protein Structure Networks to Allosteric Communications and Targeted Anti-Cancer Modulators

Computational studies of allosteric interactions have witnessed a recent renaissance fueled by growing interest in the modeling of complex molecular assemblies and biological networks. Allosteric interactions of the molecular chaperone Hsp90 with a diverse array of cochaperones and client proteins allow for molecular communication in signal transduction networks. In this review, recent developments in the understanding of allosteric interactions in the context of structural, functional, and computational studies of the Hsp90 chaperone are discussed. A comprehensive analysis of structural and network-based models of protein allostery is provided. Computational and experimental approaches and advances in the understanding of Hsp90 interactions and regulatory mechanisms are reviewed to provide a systematic and critical view of the current progress and most challenging questions in the field. The current status and future prospects for translational research, bridging the basic science of chaperones with the discovery of anti-cancer therapies, are also highlighted.     

Molecular Recognition in C-Type Lectins: The Cases of DC-SIGN,Langerin, MGL,and L-Sectin

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.     

Advances in Strategies and Tools Available for Interrogation of Protein O-GlcNAcylation

The attachment of a single O-linked β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of numerous proteins in the nucleus, cytoplasm, and mitochondria is a reversible post-translational modification (PTM) and plays an important role as a regulator of various cellular processes in both healthy and disease states. Advances in strategies and tools that allow for the detection of dynamic O-GlcNAcylation on cellular proteins have helped to enhance our initial and ongoing understanding of its dynamic effects on cellular stimuli and given insights into its link to the pathogenesis of several chronic diseases. Furthermore, chemical genetic strategies and related tools have been successfully applied to a myriad of biological systems with a new level of spatiotemporal and molecular precision. These strategies have started to be used in studying and controlling O-GlcNAcylation both in vivo and in vitro. In this minireview, overviews of recent advances in molecular tools being applied to the detection and identification of O-GlcNAcylation on cellular proteins as well as on individual proteins are provided. In addition, chemical genetic strategies that have already been applied or are potentially usable in O-GlcNAc functional are also discussed.     

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic,Prognostic, and Therapeutic Molecular Biomarkers

Gastrointestinal (GI) cancer remains one of the common causes of morbidity and mortality. A high number of cases are diagnosed at an advanced stage, leading to a poor survival rate. This is primarily attributed to the lack of reliable diagnostic biomarkers and limited treatment options. Therefore, more sensitive, specific biomarkers and curative treatments are desirable. Functional proteomics as a research area in the proteomic field aims to elucidate the biological function of unknown proteins and unravel the cellular mechanisms at the molecular level. Phosphoproteomic and glycoproteomic studies have emerged as two efficient functional proteomics approaches used to identify diagnostic biomarkers, therapeutic targets, the molecular basis of disease and mechanisms underlying drug resistance in GI cancers. In this review, we present an overview on how functional proteomics may contribute to the understanding of GI cancers, namely colorectal, gastric, hepatocellular carcinoma and pancreatic cancers. Moreover, we have summarized recent methodological developments in phosphoproteomics and glycoproteomics for GI cancer studies.