The 50 most-cited articles in Biochemistry, 2018

Processing bodies (P-bodies) are cytoplasmic ribonucleoprotein (RNP) granules primarily composed of translationally repressed mRNAs and proteins related to mRNA decay, suggesting roles in post-transcriptional regulation. P-bodies are conserved in eukaryotic cells and exhibit properties of liquid droplets. However, the function of P-bodies in translational repression and/or mRNA decay remains contentious. Here we review recent advances in our understanding of the molecular composition of P-bodies, the interactions and processes that regulate P-body liquid-liquid phase separation (LLPS), and the cellular localization of mRNA decay machinery, in the context of how these discoveries refine models of P-body function.

Inhibition of Bruton's tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the…

Liquid-liquid phase separation seems to play critical roles in the compartmentalization of cells through the formation of biomolecular condensates. Many proteins with low-complexity regions are found in these condensates, and they can undergo phase separation in vitro in response to changes in temperature, pH, and ion concentration. Low-complexity regions are thus likely important players in mediating compartmentalization in response to stress. However, how the phase behavior is encoded in their amino acid composition and patterning is only poorly understood. We discuss here that polymer physics provides a powerful framework for our understanding of the thermodynamics of mixing and demixing and for how the phase behavior is encoded in the primary sequence. We propose to classify low-complexity regions further into subcategories based on their sequence properties and phase behavior. Ongoing research promises to improve our ability to link the primary sequence of low-complexity regions to their phase behavior as well as the emerging miscibility and material properties of the resulting…

Hydrogen bonds play integral roles in biological structure, function, and conformational dynamics and are fundamental to life as it has evolved on Earth. However, our understanding of these fundamental and ubiquitous interactions has seemed fractured and incomplete, and it has been difficult to extract generalities and principles about hydrogen bonds despite thousands of papers published on this topic, perhaps in part because of the expanse of this subject and the density of studies. Fortunately, recent hydrogen bond proposals, discussions, and debates have stimulated new tests and models and have led to a remarkably simple picture of the structure of hydrogen bonds. This knowledge also provides clarity concerning hydrogen bond energetics, limiting and simplifying the factors that need be considered. Herein we recount the advances that have led to this simpler view of hydrogen bond structure, dynamics, and energetics. A quantitative predictive model for hydrogen bond length can now be broadly and deeply applied to evaluate current proposals and to uncover structural features of…

Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green cutinase-catalyzed PET recycling methods. Leaf and branch compost cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature ( T g, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC’s tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence…

Intracellular environments are heterogeneous milieus comprised of macromolecules, osmolytes, and a range of assemblies that include membrane-bound organelles and membraneless biomolecular condensates. The latter are nonstoichiometric assemblies of protein and RNA molecules. They represent distinct phases and form via intracellular phase transitions. Here, we present insights from recent studies and provide a perspective on how phase transitions that lead to biomolecular condensates might contribute to cellular functions.

Liquid-liquid phase separation and related condensation processes of intrinsically disordered proteins (IDPs), proteins with intrinsically disordered regions, and nucleic acids underpin various condensed-liquid droplets or gel-like assemblies in the cellular environment. Collectively referred to as condensates, these bodies provide spatial/temporal compartmentalization, often serving as hubs for regulated biomolecular interactions. Examples include certain extracellular materials, transcription complexes, and membraneless organelles such as germ and stress granules and the nucleolus. They are critically important to cellular function; thus misregulation of their assembly is implicated in many diseases. Biomolecular condensates are complex entities. Our understanding of their inner workings is only in its infancy. Nonetheless, insights into basic biophysical principles of their assembly can be gained by applying analytical theories to elucidate how IDP phase behaviors are governed by the properties of the multivalent, solvent-mediated interactions entailed by the proteins' amino acid…

In eukaryotic cells, structures called heterochromatin play critical roles in nuclear processes ranging from gene repression to chromosome segregation. Biochemical and in vivo studies over the past several decades have implied that the diverse functions of heterochromatin rely on the ability of these structures to spread across large regions of the genome, to compact the underlying DNA, and to recruit different types of activities. Recent observations have suggested that heterochromatin may possess liquid droplet-like properties. Here, we discuss how these observations provide a new perspective on the mechanisms for the assembly, regulation, and functions of heterochromatin.

Covalent enzyme inhibitors are widely applied as biochemical tools and therapeutic agents. As a complement to categorization of these inhibitors by reactive group or modification site, we present a categorization by mechanism, which highlights common advantages and disadvantages inherent to each approach. Established categories for reversible and irreversible covalent inhibition are reviewed with representative examples given for each class, including covalent reversible inhibitors, slow substrates, residue-specific reagents, affinity labels (classical, quiescent, and photoaffinity), and mechanism-based inactivators. The relationships of these categories to proteomic profiling probes (activity-based and reactivity-based) as well as complementary approaches such as prodrug and soft drug design are also discussed. A wide variety of strategies are used to balance reactivity and selectivity in the design of covalent enzyme inhibitors. Use of a shared terminology is encouraged to clearly convey these mechanisms, to relate them to prior use of covalent inhibitors in enzymology, and to…

Interest in how proline contributes to cancer biology is expanding because of the emerging role of a novel proline metabolic cycle in cancer cell survival, proliferation, and metastasis. Proline biosynthesis and degradation involve the shared intermediate Δ 1 -pyrroline-5-carboxylate (P5C), which forms l -glutamate-γ-semialdehyde (GSAL) in a reversible non-enzymatic reaction. Proline is synthesized from glutamate or ornithine through GSAL/P5C, which is reduced to proline by P5C reductase (PYCR) in a NAD(P)H-dependent reaction. The degradation of proline occurs in the mitochondrion and involves two oxidative steps catalyzed by proline dehydrogenase (PRODH) and GSAL dehydrogenase (GSALDH). PRODH is a flavin-dependent enzyme that couples proline oxidation with reduction of membrane-bound quinone, while GSALDH catalyzes the NAD + -dependent oxidation of GSAL to glutamate. PRODH and PYCR form a metabolic relationship known as the proline–P5C cycle, a novel pathway that impacts cellular growth and death pathways. The proline–P5C cycle has been implicated in supporting ATP production,…

The central dogma of molecular biology introduced by Crick describes a linear flow of information from DNA to mRNA to protein. Since then it has become evident that RNA undergoes several maturation steps such as capping, splicing, 3′-end processing, and editing. Likewise, nucleotide modifications are common in mRNA and are present in all organisms impacting on the regulation of gene expression. The most abundant modification found in mRNA is N6-methyladenosine (m 6 A). Deposition of m 6 A is a nuclear process and is performed by a megadalton writer complex primarily on mRNAs, but also on microRNAs and lncRNAs. The m 6 A methylosome is composed of the enzymatic core components METTL3 and METTL14, and several auxiliary proteins necessary for its correct positioning and functioning, which are WTAP, VIRMA, FLACC, RBM15, and HAKAI. The m 6 A epimark is decoded by YTH domain-containing reader proteins YTHDC and YTHDF, but METTLs can act as “readers” as well. Eraser proteins, such as FTO and ALKBH5, can remove the methyl group. Here we review recent progress on the role of m 6 A in…

This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and…

Even after a century of investigation, our understanding of how enzymes work remains far from complete. In particular, several factors that enable enzymes to achieve high catalytic efficiencies remain only poorly understood. A number of theories have been developed, which propose or reaffirm that enzymes work as structural scaffolds, serving to bring together and properly orient the participants so that the reaction can proceed; therefore, leading to enzymes being viewed as only passive participants in the catalyzed reaction. A growing body of evidence shows that enzymes are not rigid structures but are constantly undergoing a wide range of internal motions and conformational fluctuations. In this Perspective, on the basis of studies from our group, we discuss the emerging biophysical model of enzyme catalysis that provides a detailed understanding of the interconnection among internal protein motions, conformational substates, enzyme mechanisms, and the catalytic efficiency of enzymes. For a number of enzymes, networks of conserved residues that extend from the surface of the…

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with Ca II binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for Ln III s and Y III over Ca II . Here we present the nuclear magnetic resonance solution structure of LanM complexed with Y III . This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein’s picomolar affinity for Ln III s, and it suggests a role of unusual N i +1 –H···N i hydrogen bonds, in which LanM’s unique EF-hand proline residues are engaged, in selective Ln III recognition. This work sets the stage for a detailed mechanistic understanding of LanM’s Ln selectivity, which may…

Extracellular electron transfer (EET) in microbial cells is essential for certain biotechnological applications and contributes to the biogeochemical cycling of elements and syntrophic microbial metabolism in complex natural environments. The Gram-positive lactic acid bacterium Enterococcus faecalis, an opportunistic human pathogen, is shown to be able to transfer electrons generated in fermentation metabolism to electrodes directly and indirectly via mediators. By exploiting E. faecalis wild-type and mutant cells, we demonstrate that reduced demethylmenaquinone in the respiratory chain in the bacterial cytoplasmic membrane is crucial for the EET. Heme proteins are not involved, and cytochrome bd oxidase activity was found to attenuate EET. These results are significant for the mechanistic understanding of EET in bacteria and for the design of microbial electrochemical systems. The basic findings infer that in dense microbial communities, such as in biofilm and in the large intestine, metabolism in E. faecalis and similar Gram-positive lactic acid bacteria might be electrically…

Abstract The construction of more stable proteins is important in biomolecular engineering, particularly in the design of biologics-based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid m-chlorotyrosine (mClY) increases both the thermal stability (increasing the melting temperature by ∼1 °C and the melting enthalpy by 3 kcal/mol) and the enzymatic activity at elevated temperatures (15% higher than that of the parent enzyme at 40 °C) of this classic enzyme. The chlorine of mClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chemical calculations to be significantly enhanced by polarization via an intramolecular hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side chain, resulting in a distinctive synergistic HB-enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated…

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2's role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR-Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf-TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to…

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is…

Lytic polysaccharide monooxygenases (LPMOs) are major players in biomass conversion, both in Nature and in the biorefining industry. How the monocopper LPMO active site is positioned relative to the crystalline substrate surface to catalyze powerful, but potentially self-destructive, oxidative chemistry is one of the major questions in the field. We have adopted a multidisciplinary approach, combining biochemical, spectroscopic, and molecular modeling methods to study chitin binding by the well-studied LPMO from Serratia marcescens Sm AA10A (or CBP21). The orientation of the enzyme on a single-chain substrate was determined by analyzing enzyme cutting patterns. Building on this analysis, molecular dynamics (MD) simulations were performed to study interactions between the LPMO and three different surface topologies of crystalline chitin. The resulting atomistic models showed that most enzyme–substrate interactions involve the polysaccharide chain that is to be cleaved. The models also revealed a constrained active site geometry as well as a tunnel connecting the bulk solvent to the…

Recent technological advances led to the discovery of hundreds to thousands of peptides and small proteins (microproteins) encoded by small open reading frames (smORFs). Characterization of new microproteins demonstrates their role in fundamental biological processes and highlights the value in discovering and characterizing more microproteins. The elucidation of microprotein-protein interactions (MPIs) is useful for determining the biochemical and cellular roles of microproteins. In this study, we characterize the protein interaction partners of mitochondrial elongation factor 1 microprotein (MIEF1-MP) using a proximity labeling strategy that relies on APEX2. MIEF1-MP localizes to the mitochondrial matrix where it interacts with the mitochondrial ribosome (mitoribosome). Functional studies demonstrate that MIEF1-MP regulates mitochondrial translation via its binding to the mitoribosome. Loss of MIEF1-MP decreases the mitochondrial translation rate, while an elevated level of MIEF1-MP increases the translation rate. The identification of MIEF1-MP reveals a new gene involved in this…

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the degradation of polysaccharides in biomass by catalyzing powerful oxidative chemistry using only a single copper ion as a cofactor. Despite the natural abundance and importance of these powerful monocopper enzymes, the structural determinants of their functionality have remained largely unknown. We have used site-directed mutagenesis to probe the roles of 13 conserved amino acids located on the flat substrate-binding surface of CBP21, a chitin-active family AA10 LPMO from Serratia marcescens, also known as SmLPMO10A. Single mutations of residues that do not interact with the catalytic copper site, but rather are involved in substrate binding had remarkably strong effects on overall enzyme performance. Analysis of product formation over time showed that these mutations primarily affected enzyme stability. Investigation of protein integrity using proteomics technologies showed that loss of activity was caused by oxidation of essential residues in the enzyme active site. For most enzyme variants, reduced enzyme…

Compartmentalization of biochemical components, interactions, and reactions is critical for the function of cells. While intracellular partitioning of molecules via membranes has been extensively studied, there has been an expanding focus in recent years on the critical cellular roles and biophysical mechanisms of action of membraneless organelles (MLOs) such as the nucleolus. In this context, a substantial body of recent work has demonstrated that liquid-liquid phase separation plays a key role in MLO formation. However, less is known about MLO dissociation, with phosphorylation being the primary mechanism demonstrated thus far. In this Perspective, we focus on another mechanism for MLO dissociation that has been described in recent work, namely a reentrant phase transition (RPT). This concept, which emerges from the polymer physics field, provides a mechanistic basis for both formation and dissolution of MLOs by monotonic tuning of RNA concentration, which is an outcome of cellular processes such as transcription. Furthermore, the RPT model also predicts the formation of dynamic…

In recent years, there has been dramatic growth in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA-small molecule interactions. While a host of techniques exist to measure RNA-small molecule interactions, many of these have drawbacks that make them intractable for routine use and are often not broadly applicable. A newer technology called microscale thermophoresis (MST), which measures the directed migration of a molecule and/or molecule-ligand complex along a temperature gradient, can be used to measure binding affinities using very small amounts of sample. The high sensitivity of this technique enables measurement of affinity constants in the nanomolar and micromolar range. Here, we demonstrate how MST can be used…

The rapidly expanding number of protein sequences found in public databases can improve our understanding of how protein functions evolve. However, our current knowledge of protein function likely represents a small fraction of the diverse repertoire that exists in nature. Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions through the observation and investigation of the complex sequence-structure-function relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) to identify previously unexplored sequence and function space. We exemplify this approach using the nitroreductase (NTR) superfamily. We demonstrate that SSN investigations can provide a rapid and effective means to classify groups of proteins, therefore exposing experimentally unexplored sequences that may exhibit novel functionality. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes and their associated physiological…

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p K a values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H 2 O 2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over…

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In Escherichia coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our data set using a simple log-linear statistical model.…

The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes (A-D). Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes (…

ADVERTISEMENT RETURN TO ISSUEViewpointNEXTTargeting Dependency on the GPX4 Lipid Peroxide Repair Pathway for Cancer TherapyHengrui LiuHengrui LiuDepartment of Chemistry, Columbia University, New York, New York 10027, United StatesMore by Hengrui Liuhttp://orcid.org/0000-0002-5214-1572, Stuart L. Schreiber*Stuart L. SchreiberDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United StatesBroad Institute, Cambridge, Massachusetts 02142, United States*E-mail: [email protected]More by Stuart L. Schreiberhttp://orcid.org/0000-0003-1922-7558, and Brent R. Stockwell*Brent R. StockwellDepartment of Chemistry, Columbia University, New York, New York 10027, United StatesDepartment of Biological Sciences, Columbia University, 550 West 120th Street, MC 4846, New York, New York 10027, United States*E-mail: [email protected]More by Brent R. Stockwellhttp://orcid.org/0000-0002-3532-3868Cite this: Biochemistry 2018, 57, 14, 2059–2060Publication Date (Web):March 27, 2018Publication History Received13 March 2018Published online27 March 2018Published…

Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave…

Despite the common practice of presenting structures of biological molecules on an empty background and the assumption that interactions between biological macromolecules take place within the inert solvent, water represents an active component of various biological processes. This Perspective addresses indispensable, yet mostly ignored, roles of water in biological liquid-liquid phase transitions and in the biogenesis of various proteinaceous membrane-less organelles. We point out that changes in the structure of water reflected in the changes in its abilities to donate and/or accept hydrogen bonds and participate in dipole-dipole and dipole-induced dipole interactions in the presence of various solutes (ranging from small molecules to synthetic polymers and biological macromolecules) might represent a driving force for the liquid-liquid phase separation, define partitioning of various solutes in formed phases, and define the exceptional ability of intrinsically disordered proteins to be engaged in the formation of proteinaceous membrane-less organelles.

A flurry of research in recent years has revealed the molecular origins of many membraneless organelles to be the liquid phase separation of intrinsically disordered proteins (IDPs). Consequently, protein disorder has emerged as an important driver of intracellular compartmentalization by providing specialized microenvironments chemically distinct from the surrounding medium. Though the importance of protein disorder and its relationship to intracellular phase behavior are clear, a detailed understanding of how such phase behavior can be predicted and controlled remains elusive. While research in IDPs has largely focused on the implications of structural disorder on cellular function and disease, another field, that of artificial protein polymers, has focused on the de novo design of protein polymers with controllable material properties. A subset of these polymers, specifically those derived from structural proteins such as elastin and resilin, are also disordered sequences that undergo liquid-liquid phase separation. This phase separation has been used in a variety of biomedical…

A complete understanding of enzyme catalysis requires knowledge of both transition state features and the detailed motions of atoms that cause reactant molecules to form and traverse the transition state. The seeming intractability of the problem arises from the femtosecond lifetime of chemical transition states, preventing most experimental access. Computational chemistry is admirably suited to short time scale analysis but can be misled by inappropriate starting points or by biased assumptions. Kinetic isotope effects provide an experimental approach to transition state structure and a method for obtaining transition state analogues but, alone, do not inform how that transition state is reached. Enzyme structures with transition state analogues provide computational starting points near the transition state geometry. These well-conditioned starting points, combined with the unbiased computational method of transition path sampling, provide realistic atomistic motions involved in transition state formation and passage. In many, but not all, enzymatic systems, femtosecond local…

Replication of nucleic acids in the absence of genetically encoded enzymes represents a critical process for the emergence of cellular life. Repeated separation of complementary RNA strands is required to achieve multiple cycles of chemical replication, yet thermal denaturation under plausible prebiotic conditions is impaired by the high temperatures required to separate long RNA strands and by concurrent degradation pathways, the latter accelerated by divalent metal ions. Here we show how the melting temperature of oligoribonucleotide duplexes can be tuned by changes in pH, enabling the separation of RNA strands at moderate temperatures. At the same time, the risk of phosphodiester bond cleavage is reduced under the acid denaturation conditions herein described, both in the presence and in the absence of divalent metal ions. Through a combination of ultraviolet and circular dichroism thermal studies and gel electrophoresis, we demonstrate the relevance of geological pH oscillations in the context of the RNA strand separation problem. Our results reveal new insights in the field of…

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape…

The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S-20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different…

As one of the most abundant elements on earth, sulfur is part of many small molecular metabolites and is key to their biological activities. Over the past few decades, some general strategies have been discovered for the incorporation of sulfur into natural products. In this review, we summarize recent efforts in elucidating the biosynthetic details for two sulfur-containing metabolites, ergothioneine and ovothiol. Their biosyntheses involve an unprecedented trans-sulfur strategy, a combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation reaction and a PLP enzyme-mediated C-S lyase reaction.

The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes. The binding mode of an inhibitor and a reaction intermediate further substantiate a hypothesis by Seefeldt, Hoffman, and Dean that the activation of N 2 is made possible by a reductive elimination of H 2 that leaves the cofactor in a super-reduced state that can bind and reduce the inert N 2 molecule. Here we discuss the immediate implications of the structurally observed mode of binding of small molecules to the enzyme with respect to the early…

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca 2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca 2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond…

The study of enzyme reaction mechanisms is fundamentally important to our understanding of biochemistry, cellular metabolism, and drug development. This Perspective focuses on the use of kinetic solvent viscosity effects (KSVEs) to study enzyme reactions. This technique is easily implemented and uses steady-state kinetic analyses to probe whether substrate binding is diffusion-controlled and whether product release is the rate-limiting step in the catalytic cycle. In addition, KSVEs can identify isomerization steps that are important for catalysis. The use of KSVEs in combination with other techniques, such as kinetic isotope effects, pH effects, and site-directed mutagenesis, can provide a detailed view of the mechanism of enzyme action. We present the basic theory, important experimental considerations, and potential outcomes and briefly discuss some examples from the literature. The derivation of the equations that are important for data analysis is also presented.

Amide bonds at the proline nitrogen are particularly susceptible to rotation, affording cis and trans isomers. Installation of a stereochemically defined electron-withdrawing fluorine atom or fluorinated groups has the power to influence the cis – trans conformational preferences of the amide bond in X–(F)Pro (where X = any other amino acid). Advantageously, this also provides a sensitive reporter for 19 F nuclear magnetic resonance (NMR) studies of protein conformation, interactions, and dynamics. We deliberately use the term “fluorinated prolines” as an all-encompassing term to describe proline analogues containing one or more fluorine atoms and to avoid confusion with the more well-known 4-fluoroprolines. This review presents a critical discussion of the growing repertoire of fluorinated prolines that have been described and, importantly, provides a comparison of their uses and relative influence on amide-bond conformation and discusses the significant potential of using 19 F NMR as a tool to probe conformational changes in polypeptides.

ABSTRACT Actin Depolymerizing Factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein, and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, over four different values of pH ranging from pH 6.6 to pH 7.8, using single filament microfluidics techniques. We show that lowering pH reduces the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing…

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.

Abstract We recently characterized a new class of pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs from Methanomassiliicocales that are active and orthogonal in Escherichia coli. The aminoacyl-tRNA synthetases (aaRSs) of these pairs lack the N-terminal domain that is essential for tRNA recognition and in vivo activity in the Methanosarcina mazei (Mm) PylRS but share a homologous active site with MmPylRS; this facilitates the transplantation of mutations discovered with existing PylRS systems into the new PylRS systems to reprogram their substrate specificity for the incorporation of noncanonical amino acids (ncAAs). Several of the new PylRS/PyltRNA pairs, or their evolved variants [including Methanomethylophilus alvus (Ma) PylRS/MaPyltRNA(6)CUA], are mutually orthogonal to the MmPylRS/MmPyltRNA pair, and the active sites of the Mm pair and Ma pair can be diverged to enable the incorporation of distinct ncAAs in response to distinct codons via orthogonal translation in E. coli. Here we demonstrate that MaPylRS/MaPyltRNA(6)CUA is orthogonal to the aaRSs and tRNAs in mammalian cells…

Abstract There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes that hydrolyze β-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3–7.2 μM; Ki values of 0.03–1.5 μM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide.…

Enzymes show two distinct transport behaviors in the presence of their substrates in solution. First, their diffusivity enhances with an increasing substrate concentration. In addition, enzymes perform directional motion toward regions with a high substrate concentration, termed as chemotaxis. While a variety of enzymes has been shown to undergo chemotaxis, there remains a lack of quantitative understanding of the phenomenon. Here, we derive a general expression for the active movement of an enzyme in a concentration gradient of its substrate. The proposed model takes into account both the substrate-binding and catalytic turnover step, as well as the enhanced diffusion of the enzyme. We have experimentally measured the chemotaxis of a fast and a slow enzyme: urease under catalytic conditions and hexokinase for both full catalysis and for simple noncatalytic substrate binding. There is good agreement between the proposed model and the experiments. The model is general, has no adjustable parameters, and only requires three experimentally defined constants to quantify chemotaxis:…

Protein–protein interactions are fundamental to biology yet are rarely studied under physiologically relevant conditions where the concentration of macromolecules can exceed 300 g/L. These high concentrations cause cosolute–complex contacts that are absent in dilute buffer. Understanding such interactions is important because they organize the cellular interior. We used 19 F nuclear magnetic resonance, the dimer-forming A34F variant of the model protein GB1, and the cosolutes bovine serum albumin (BSA) and lysozyme to assess the effects of repulsive and attractive charge–charge dimer–cosolute interactions on dimer stability. The interactions were also manipulated via charge-change variants and by changing the pH. Charge–charge repulsions between BSA and GB1 stabilize the dimer, and the effects of lysozyme indicate a role for attractive interactions. The data show that chemical interactions can regulate the strength of protein–protein interactions under physiologically relevant crowded conditions and suggest a mechanism for tuning the equilibrium thermodynamics of protein–protein…

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine. Molecular dynamics simulations reproduce the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 3 10 -helical H-bonds. These effects facilitate local helix bending at the glycine site and change the collective dynamics of the helix.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a ∼530 kDa complex of 8 large (RbcL) and 8 small subunits (RbcS), mediates the fixation of atmospheric CO 2 into usable sugars during photosynthesis. Despite its fundamental role, Rubisco is a remarkably inefficient enzyme and thus is produced by plants in huge amounts. It has long been a key target for bioengineering with the goal to increase crop yields. However, such efforts have been hampered by the complex requirement of Rubisco biogenesis for molecular chaperones. Recent studies have identified an array of auxiliary factors needed for the folding and assembly of the Rubisco subunits. The folding of plant RbcL subunits is mediated by the cylindrical chloroplast chaperonin, Cpn60, and its cofactor Cpn20. Folded RbcL requires a number of additional Rubisco specific assembly chaperones, including RbcX, Rubisco accumulation factors 1 (Raf1) and 2 (Raf2), and the Bundle sheath defective-2 (BSD2), to mediate the assembly of the RbcL 8 intermediate complex. Incorporation of the RbcS and displacement of the assembly factors…

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn 2+ -binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even “capless” inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1–4 . Each inhibitor hydroxamate group coordinates to the catalytic Zn 2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved…

The cellular response to oxidative stress includes transcriptional changes, particularly for genes involved in DNA repair. Recently, our laboratory demonstrated that oxidation of 2'-deoxyguanosine (G) to 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) in G-rich potential G-quadruplex sequences (PQSs) in gene promoters impacts the level of gene expression up or down depending on the position of the PQS in the promoter. In the present report, bioinformatic analysis found that the 390 human DNA repair genes in the genome ontology initiative harbor 2936 PQSs in their promoters and 5'-untranslated regions (5'-UTRs). The average density of PQSs in human DNA repair genes was found to be nearly 2-fold greater than the average density of PQSs in all coding and noncoding human genes (7.5 vs 4.3 per gene). The distribution of the PQSs in the DNA repair genes on the nontranscribed (coding) vs transcribed strands reflects that of PQSs in all human genes. Next, literature data were interrogated to select 30 PQSs to catalog their ability to adopt G-quadruplex (G4) folds in vitro using five different…