The 50 most-cited articles in Biochemistry, 2019

The assignment of functions to uncharacterized proteins discovered in genome projects requires easily accessible tools and computational resources for large-scale, user-friendly leveraging of the protein, genome, and metagenome databases by experimentalists. This article describes the web resource developed by the Enzyme Function Initiative (EFI; accessed at https://efi.igb.illinois.edu/ ) that provides "genomic enzymology" tools ("web tools") for (1) generating sequence similarity networks (SSNs) for protein families (EFI-EST); (2) analyzing and visualizing genome context of the proteins in clusters in SSNs (in genome neighborhood networks, GNNs, and genome neighborhood diagrams, GNDs) (EFI-GNT); and (3) prioritizing uncharacterized SSN clusters for functional assignment based on metagenome abundance (chemically guided functional profiling, CGFP) (EFI-CGFP). The SSNs generated by EFI-EST are used as the input for EFI-GNT and EFI-CGFP, enabling easy transfer of information among the tools. The networks are visualized and analyzed using Cytoscape, a widely used desktop application;…

Abstract Rubisco is the primary carboxylase of the Calvin cycle, the most abundant enzyme in the biosphere, and one of the best-characterized enzymes. On the basis of correlations between Rubisco kinetic parameters, it is widely posited that constraints embedded in the catalytic mechanism enforce trade-offs between CO2 specificity, SC/O, and maximum carboxylation rate, kcat,C. However, the reasoning that established this view was based on data from ≈20 organisms. Here, we re-examine models of trade-offs in Rubisco catalysis using a data set from ≈300 organisms. Correlations between kinetic parameters are substantially attenuated in this larger data set, with the inverse relationship between kcat,C and SC/O being a key example. Nonetheless, measured kinetic parameters display extremely limited variation, consistent with a view of Rubisco as a highly constrained enzyme. More than 95% of kcat,C values are between 1 and 10 s–1, and no measured kcat,C exceeds 15 s–1. Similarly, SC/O varies by only 30% among Form I Rubiscos and <10% among C3 plant enzymes. Limited variation in SC/O forces…

Abstract Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. The active enzyme harbors the nickel(I) hydrocorphin coenzyme F-430 as a prosthetic group and catalyzes the reversible reduction of methyl-coenzyme M (CH3–S-CoM) with coenzyme B (HS-CoM) to methane and CoM-S–S-CoB. MCR is also involved in anaerobic methane oxidation in reverse of methanogenesis and most probably in the anaerobic oxidation of ethane, propane, and butane. The challenging question is how the unreactive CH3–S thioether bond in methyl-coenzyme M and the even more unreactive C–H bond in methane and the other hydrocarbons are anaerobically cleaved. A key to the answer is the negative redox potential (Eo′) of the Ni(II)F-430/Ni(I)F-430 couple below −600 mV and the radical nature of Ni(I)F-430. However, the negative one-electron redox potential is also the Achilles heel of MCR; it makes the nickel enzyme one of the most O2-sensitive enzymes known to date. Even under physiological conditions, the Ni(I) in MCR is oxidized to the Ni(II) or Ni(III) states, e.g., when in the…

Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N 2 reduction to 2NH 3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal ( M ) in their active site metallocofactors (metal-ion composition, M Fe 7 ). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N 2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E 4 (4H) (“Janus”) state. N 2 binding/reduction in this state is coupled to reductive elimination ( re ) of the two hydrides as H 2, the forward direction of a reductive-elimination/oxidative-addition ( re/oa ) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N 2 /D 2 . Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H 2 formation by hydride protonolysis ( k HP ) but significant differences in the…

Chemical probing is an important tool for characterizing the complex folded structures of RNA molecules, many of which play key cellular roles. Electrophilic SHAPE reagents create adducts at the 2'-hydroxyl position on the RNA backbone of flexible ribonucleotides with relatively little dependence on nucleotide identity. Strategies for adduct detection such as mutational profiling (MaP) allow accurate, automated calculation of relative adduct frequencies for each nucleotide in a given RNA or group of RNAs. A number of alternative reagents and adduct detection strategies have been proposed, especially for use in living cells. Here we evaluate five SHAPE reagents: three previously well-validated reagents 1M7 (1-methyl-7-nitroisatoic anhydride), 1M6 (1-methyl-6-nitroisatoic anhydride), and NMIA ( N-methylisatoic anhydride), one more recently proposed NAI (2-methylnicotinic acid imidazolide), and one novel reagent 5NIA (5-nitroisatoic anhydride). We clarify the importance of carefully designed software in reading out SHAPE experiments using massively parallel sequencing approaches. We…

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations,…

γ-Secretase is a membrane-embedded protease complex, with presenilin as the catalytic component containing two transmembrane aspartates in the active site. With more than 90 known substrates, the γ-secretase complex is considered "the proteasome of the membrane", with central roles in biology and medicine. The protease carries out hydrolysis within the lipid bilayer to cleave the transmembrane domain of the substrate multiple times before releasing secreted products. For many years, elucidation of γ-secretase structure and function largely relied on small-molecule probes and mutagenesis. Recently, however, advances in cryo-electron microscopy have led to the first detailed structures of the protease complex. Two new reports of structures of γ-secretase bound to membrane protein substrates provide great insight into the nature of substrate recognition and how Alzheimer's disease-causing mutations in presenilin might alter substrate binding and processing. These new structures offer a powerful platform for elucidating enzyme mechanisms, deciphering effects of disease-causing…

Covalent inhibitors are experiencing a growing resurgence in drug design and are an increasingly useful tool in molecular biology. The ability to attach inhibitors to their targets by a covalent linkage offers pharmacodynamic and pharmacokinetic advantages, but this can also be a liability if undesired off-target reactions are not mitigated. The discovery of new electrophilic groups that react selectively with specific amino acid residues is therefore highly desirable in the design of targeted covalent inhibitors (TCIs). Additionally, the ability to control the reactivity through exploitation of the target enzyme's machinery, as in mechanism-based inhibitors (MBIs), greatly benefits from the discovery of new strategies. This Perspective showcases recent advances in electrophile development and their application in TCIs and MBIs, exhibiting high selectivity for their targets.

We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LD-specific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and Lipi-Green allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.

An increasing body of evidence suggests that aggregation-prone proteins associated with various neurodegenerative diseases synergistically promote their mutual aggregation, leading to the co-occurrence of multiple neurodegenerative diseases in the same patient. Here we investigated teh molecular basis of synergistic interactions between the two pathological proteins, tau and α-synuclein, using various biophysical techniques including transmission electron microscopy (TEM), circular dichroism (CD), and solution and solid-state NMR. Our biophysical analyses of α-synuclein aggregation in the absence and presence of tau reveal that tau monomers promote the formation of α-synuclein oligomers and subsequently fibril formation. Solution NMR results also indicate that monomeric forms of tau selectively interact with the C-terminal region of the α-synuclein monomer, accelerating α-synuclein aggregation. In addition, a combined use of TEM and solid-state NMR spectroscopy reveals that the synergistic interactions lead to the formation of toxic α-synuclein aggregates with a distinct morphology…

Base editing is a genome editing strategy that induces specific single-nucleotide changes within genomic DNA. Two major DNA base editors, cytosine base editors and adenine base editors, that consist of a Cas9 protein linked to a deaminase enzyme that catalyzes targeted base conversion directed by a single-guide RNA have been developed. This strategy has been used widely for precise genome editing because, unlike CRISPR-Cas nuclease-based genome editing systems, this strategy does not create double-strand DNA breaks that often result in high levels of undesirable indels. However, recent papers have reported that DNA base editors can cause substantial off-target editing in both genomic DNA and RNA. The off-target editing described in these studies is primarily independent of guide RNA and arises from the promiscuous reactivity of the deaminase enzymes used in DNA base editors. In this Perspective, we discuss the development of DNA base editors, the guide RNA-independent off-target activity reported in recent studies, and strategies that improve the selectivity of DNA base editors.

Cancer drug resistance has become the major problem facing current clinical treatment via different kinds of therapies. Proteolysis targeting chimeras (PROTACs) as a novel and powerful strategy have attracted a great deal of attention both from academia and from industry for their sensitivity to drug-resistant targets relying on their unique characteristics compared to those of traditional inhibitors. PROTACs exert their function by degrading the target protein instead of inhibiting targets. Thus, different kinds of resistance could be conquered by PROTACs such as target mutation or overexpression. Various resistant targets have been overcome by PROTACs, including AR, ER, BTK, BET, and BCR-ABL. Though PROTACs have achieved some significant advances in combating drug resistance, more cases are needed to prove the efficiency of PROTACs in addressing the hurdle of resistance in the near future.

Histone deacetylase 6 (HDAC6) is emerging as a target for inhibition in therapeutic strategies aimed at treating cancer, neurodegenerative disease, and other disorders. Among the metal-dependent HDAC isozymes, HDAC6 is unique in that it contains two catalytic domains, CD1 and CD2. CD2 is a tubulin deacetylase and a tau deacetylase, and the development of HDAC6-selective inhibitors has focused exclusively on this domain. In contrast, there is a dearth of structural and functional information regarding CD1, which exhibits much narrower substrate specificity in comparison with CD2. As the first step in addressing the CD1 information gap, we now present X-ray crystal structures of seven inhibitor complexes with wild-type, Y363F, and K330L HDAC6 CD1. These structures broaden our understanding of molecular features that are important for catalysis and inhibitor binding. The active site of HDAC6 CD1 is wider than that of CD2, which is unexpected in view of the narrow substrate specificity of CD1. Amino acid substitutions between HDAC6 CD1 and CD2, as well as conformational differences in…

ADVERTISEMENT RETURN TO ISSUEPREVViewpointNEXTCas14: Big Advances from Small CRISPR ProteinsDavid F. Savage*David F. SavageDepartment of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States*E-mail: [email protected]More by David F. Savagehttp://orcid.org/0000-0003-0042-2257Cite this: Biochemistry 2019, 58, 8, 1024–1025Publication Date (Web):February 11, 2019Publication History Received14 January 2019Published online11 February 2019Published inissue 26 February 2019https://pubs.acs.org/doi/10.1021/acs.biochem.9b00035https://doi.org/10.1021/acs.biochem.9b00035editorialACS PublicationsCopyright © 2019 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views8202Altmetric-Citations39LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage…

Mitochondria are multifunctional subcellular organelles whose operations encompass energy production, signal transduction, and metabolic regulation. Given their wide range of roles, they have been studied extensively as a potential therapeutic target for the treatment of various diseases, including cancer, diabetes, and neurodegenerative diseases. Mitochondrion-mediated pathways have been identified as promising targets in the context of these diseases. However, the delivery of specific probes and drugs to the mitochondria is one of the major problems that remains to be solved. Over the past decade, much effort has been devoted to developing mitochondrion-targeted delivery methods based on the membrane characteristics and the protein import machinery of mitochondria. While various methods utilizing small molecules to polymeric particles have been introduced, it is notable that many of these compounds share common structural elements and physicochemical properties for optimal selectivity and efficiency. In this Perspective, we will review the most recently developed…

Abstract We recently reported the initiation of a Phase I clinical trial with AB680, a potent human CD73 inhibitor, being developed for the treatment of solid tumors (NCT03677973). We undertook a detailed kinetic analysis of the interaction between human CD73 and AB680 to determine the mode of inhibition. We found AB680 to be a reversible, slow-onset competitive inhibitor of human CD73 with a Ki of 5 pM. Clinical candidates of this potency are uncommon and deserve special consideration during lead optimization.

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H 2 O 2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are…

The pharmaceutical and chemical industries depend on additives to protect enzymes and other proteins against stresses that accompany their manufacture, transport, and storage. Common stresses include vacuum-drying, freeze-thawing, and freeze-drying. The additives include sugars, compatible osmolytes, amino acids, synthetic polymers, and both globular and disordered proteins. Scores of studies have been published on protection, but the data have never been analyzed systematically. To spur efforts to understand the sources of protection and ultimately develop more effective formulations, we review ideas about the mechanisms of protection, survey the literature searching for patterns of protection, and then compare the ideas to the data.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix-loop-helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate…

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771–785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-Ras G12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-Ras G12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that…

The global transcription factor, p53, is a master regulator of gene expression in cells. Mutations in the TP53 gene promote unregulated cell growth through the inactivation of downstream effectors of the p53 pathway. In fact, mutant p53 is highly prone to misfolding and frequently resides inside the cell as large aggregates, causing loss of physiological function of the tumor-suppressor protein. Here, we review the plausible reasons for functional loss of p53, including amyloid formation leading to unhindered cancer progression. We discuss previous as well as recent findings regarding the amyloid formation of p53 in vitro and in vivo . We elaborate on prion-like properties of p53 amyloids and their possible involvement in cancer progression. Because the p53 pathway is historically most targeted for the development of anticancer therapeutics, we have also summarized some of the recent approaches and advances in reviving the antiproliferative activities of wild-type p53. In this Perspective, we provide insight into understanding p53 as a prion-like protein and propose cancer to be…

Carbohydrates make up one of the four major classes of biomolecules, often conjugated with proteins as glycoproteins or with lipids as glycolipids, and participate in many important biochemical functions in living species. However, glycoproteins or glycolipids often exist as mixtures, and as a consequence, it is difficult to isolate individual glycoproteins or glycolipids as pure forms to understand the role carbohydrates play in the glycoconjugate. Currently, the only feasible way to obtain pure glycoconjugates is through synthesis, and of the many methods developed for the synthesis of oligosaccharides, those with automatic and programmable potential are considered to be more effective for addressing the issues of carbohydrate diversity and related functions. In this Perspective, we describe how data science, including algorithm and machine learning, can be used to assist the chemical synthesis of oligosaccharide in a programmable and one-pot manner and how the programmable method can be used to accelerate the construction of diverse oligosaccharides to facilitate our…

p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The…

Life is an emergent property of transient interactions between biomolecules and other organic and inorganic molecules that somehow leads to harmony and order. Measurement and quantitation of these biological interactions are of value to scientists and are major goals of biochemistry, as affinities provide insight into biological processes. In an organism, these interactions occur in the context of forces and the need for a consideration of binding affinities in the context of a changing mechanical landscape necessitates a new way to consider the biochemistry of protein-protein interactions. In the past few decades, the field of mechanobiology has exploded, as both the appreciation of, and the technical advances required to facilitate the study of, how forces impact biological processes have become evident. The aim of this review is to introduce the concept of force dependence of biomolecular interactions and the requirement to be able to measure force-dependent binding constants. The focus of this discussion will be on the mechanotransduction that occurs at the integrin-mediated…

The occurrence of 8-oxo-7,8-dihydroguanine (OG) in the genome, as one of the major DNA oxidative damages, has been implicated in an array of biological processes, ranging from mutagenesis to transcriptional regulation. Genome-wide mapping of oxidative damages could shed light on the underlying cellular mechanism. In the present study, we engineered the hOGG1 enzyme, a primary 8-oxoguanine DNA glycosylase, into a guanine oxidation-profiling tool. Our method, called enTRAP-seq, successfully identified more than 1400 guanine oxidation sites in the mouse embryonic fibroblast genome. These OG peaks were enriched in open chromatin regions and regulatory elements, including promoters, 5' untranslated regions, and CpG islands. Collectively, we present a simple and generalizable approach for the genome-wide profiling of DNA damages with high sensitivity and specificity.

Abstract The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-β32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling…

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not…

Direct biocatalytic conversion of CO 2 to formic acid is an attractive means of reversibly storing energy in chemical bonds. Formate dehydrogenases (FDHs) are a heterogeneous group of enzymes that catalyze the oxidation of formic acid to carbon dioxide, generating two protons and two electrons. Several FDHs have recently been reported to catalyze the reverse reaction, i.e., the reduction of carbon dioxide to formic acid, under appropriate conditions. The main challenges with these enzymes are relatively low rates of CO 2 reduction and high oxygen sensitivity. Our earlier studies (Yu et al. (2017) J. Biol. Chem. 292, 16872–16879) have shown that the FdsABG formate dehydrogenase from Cupriavidus necator is able to effectively catalyze the reduction of CO 2, using NADH as a source of reducing equivalents, with a good oxygen tolerance. On the basis of this result, we have developed a highly thermodynamically efficient and cost-effective biocatalytic process for the transformation of CO 2 to formic acid using FdsABG. We have cloned the full-length soluble formate dehydrogenase (FdsABG)…

The diversity of the cellular proteome substantially exceeds the number of genes coded by the DNA of an organism because one or more residues in a majority of eukaryotic proteins are post-translationally modified (PTM) by the covalent conjugation of specific chemical groups. We now know that PTMs alter protein conformation and function in ways that are not entirely understood at the molecular level. NMR spectroscopy has been particularly successful as an analytical tool in elucidating the themes underlying the structural role of PTMs. In this Perspective, we focus on the NMR-based characterization of three abundant PTMs: phosphorylation, acetylation, and glycosylation. We detail NMR methods that have found success in detecting these modifications at a site-specific level. We also highlight NMR studies that have mapped the conformational changes ensuing from these PTMs as well as evaluated their relation to function. The NMR toolbox is expanding rapidly with experiments available to probe not only the average structure of biomolecules but also how this structure changes with time on…

When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochemically characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liquid chromatography analysis and a fluorescent analogue of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were…

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l -cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l -cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro- l -tyrosine (F 2 -Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide ( • NO), an oxygen surrogate, similarly binds to uncross-linked F 2 -Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F 2 -Tyr157 CDO and mature wild-type CDO in complex with both l -cysteine and…

Type 1 diabetes (T1D) is an autoimmune disease characterized by an insulin deficiency. Ever since the discovery of insulin almost 100 years ago, patients with T1D have relied on multiple daily insulin injections to survive an otherwise deadly disease. Despite decades of research and clinical trials, no treatment exists yet to prevent or cure T1D. A recent prevention trial using the anti-CD3 antibody teplizumab in individuals at a high risk of developing T1D has provided the first piece of evidence that a safe and transient intervention may be able to delay disease. In this Perspective, we review the 40-year long history of anti-CD3 and discuss how this antibody became a candidate for the treatment of autoimmune diabetes. The path that leads to its use in this latest clinical trial for T1D has been winding and strewn with setbacks. The molecular actions of the anti-CD3 antibody that target T lymphocytes are well-understood, but its systemic effect on immune function has proven more difficult to unravel. Moreover, preclinical data suggested that the utility of anti-CD3 for the…

CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA…

A fundamental question in biology is how biological patterns emerge. Because of the presence of numerous confounding factors, it is tremendously challenging to elucidate the mechanisms underlying pattern formation solely on the basis of studies of natural biological systems. Synthetic biology provides a complementary approach to investigating pattern formation by creating systems that are simpler and more controllable than their natural counterparts. In this Perspective, we summarize recent work on synthetic systems that generate spatial patterns, review the tools for building synthetic patterns, and discuss future directions of studying pattern formation with synthetic biology.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro . However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro . We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen…

Microbial rhodopsins, a photoactive membrane protein family, serve as fundamental tools for optogenetics, an innovative technology for controlling biological activities with light. Microbial rhodopsins are widely distributed in nature and have a wide variety of biological functions. Regardless of the many different known types of microbial rhodopsins, only a few of them have been used in optogenetics to control neural activity to understand neural networks. The efforts of our group have been aimed at identifying and characterizing novel rhodopsins from nature and also at engineering novel variant rhodopsins by rational design. On the basis of the molecular and functional characteristics of those novel rhodopsins, we have proposed new rhodopsin-based optogenetics tools to control not only neural activities but also “non-neural” activities. In this Perspective, we introduce the achievements and summarize future challenges in creating optogenetics tools using rhodopsins. The implementation of optogenetics deep inside an in vivo brain is the well-known challenge for existing rhodopsins.…

ABSTRACT KRAS4b is a small GTPase involved in cellular signaling through receptor tyrosine kinases. Activation of KRAS4b is achieved through the interaction with nucleotide exchange factors while inactivation is regulated by through interaction with GTPase activating proteins. The activation of KRAS4b only occurs after recruitment of the regulatory proteins to the plasma membrane thus making the role of the phospholipid bilayer an integral part of the activation mechanism. The phospholipids, primarily with anionic head groups, interact with both the membrane anchoring hypervariable region and the G-domain, thus influencing the orientation of KRAS at the membrane surface. The orientation of the G-domain at the membrane surface is believed to play a role in the regulation of KRAS activation. Much of the research has focused on the role of phosphatidyl serine but little has been done regarding the important signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We report here the use of fluorescence anisotropy decay, atomic force microscopy, and molecular dynamic simulations to…

During the catalytic step immediately prior to O–O bond formation in Photosystem II, a water molecule deprotonates and moves next to the water-splitting Mn 4 CaO 5 cluster’s O5 oxo bridge. Considerable evidence identifies O5 as one of the two substrate waters that ultimately form O 2 . The relocated oxygen, known as O6 or O x, may be the second. It is currently debated whether O6 or O x originates as the Mn-bound water denoted W2 or as the Ca 2+ -bound water denoted W3. To distinguish between these two possibilities, we analyzed the D–O–D bending mode of the water molecule that deprotonates/relocates to become O6/O x . We show that this D–O–D bending mode is not altered by the D1-S169A mutation. Previously, we showed that this D–O–D bending mode is altered substantially when Sr 2+ is substituted for Ca 2+ . Because Sr 2+ /Ca 2+ substitution alters this D–O–D bending mode but the D1-S169A mutation does not, we conclude that the water-derived oxygen that relocates and becomes O6/O x derives from the Ca 2+ -bound W3. This conclusion provides an important constraint for proposed…

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone…

The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping,…

Parkinson’s disease etiology involves amyloid formation by α-synuclein (αSyn). In vivo, αSyn is constitutively acetylated at the α-amino N-terminus. Here, we find N-terminally acetylated αSyn (Ac-αSyn) aggregates more slowly than non-acetylated αSyn (NH 3 -αSyn) with significantly reduced sensitivity to thioflavin T (ThT). Fibril differences were characterized by transmission electron microscopy, circular dichroism spectroscopy, and limited proteolysis. Interestingly, the low-ThT Ac-αSyn fibrils seed both acetylated and non-acetylated αSyn and faithfully propagate the low-ThT character through several generations, indicating a stable fibril polymorph. In contrast, the high-ThT NH 3 -αSyn seeds lose fidelity over subsequent generations. Despite it being outside of the amyloid core, the chemical nature of the N-terminus modulates αSyn aggregation and fibril polymorphism.

Progranulin (PGRN) is an evolutionarily conserved glycoprotein associated with several disease states, including neurodegeneration, cancer, and autoimmune disorders. This protein has recently been implicated in the regulation of lysosome function, whereby PGRN may bind to and promote the maturation and activity of the aspartyl protease cathepsin D (proCTSD, inactive precursor; matCTSD, mature, enzymatically active form). As the full-length PGRN protein can be cleaved into smaller peptides, called granulins, we assessed the function of these granulin peptides in binding to proCTSD and stimulating matCTSD enzyme activity in vitro. Here, we report that full-length PGRN and multi-granulin domain peptides bound to proCTSD with low to submicromolar binding affinities. This binding promoted proCTSD destabilization, the magnitude of which was greater for multi-granulin domain peptides than for full-length PGRN. Such destabilization correlated with enhanced matCTSD activity at acidic pH. The presence and function of multi-granulin domain peptides have typically been overlooked in previous…

Abstract Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, UT and UD for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint…

Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In this work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods. We used HADDOCK to perform protein-protein docking experiments on all 20 combinations and subsequently to select four complexes for extensive molecular dynamics simulations. The potential of mean force is computed for a rotation of the cytochrome domain relative to LPMO. We find that the LPMO loops are largely responsible for the preferred orientations of the cytochrome domains. This leads us to postulate a hybrid version of NcLPMO9F, with exchanged loops and predicted altered cytochrome binding preferences for this variant. Our…

Development of small molecule inhibitors of protein-protein interactions (PPIs) is hampered by our poor understanding of the druggability of PPI target sites. Here, we describe the combined application of alanine-scanning mutagenesis, fragment screening, and FTMap computational hot spot mapping to evaluate the energetics and druggability of the highly charged PPI interface between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), an important drug target. FTMap identifies four binding energy hot spots at the active site. Only two of these are exploited by Nrf2, which alanine scanning of both proteins shows to bind primarily through E79 and E82 interacting with KEAP1 residues S363, R380, R415, R483, and S508. We identify fragment hits and obtain X-ray complex structures for three fragments via crystal soaking using a new crystal form of KEAP1. Combining these results provides a comprehensive and quantitative picture of the origins of binding energy at the interface. Our findings additionally reveal non-native interactions that might be…

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no…

value of 1 μM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.

With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.

Several diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (HD), are associated with specific proteins aggregating and depositing within tissues and/or cellular compartments. The aggregation of these proteins is characterized by the formation of extended, β-sheet rich fibrils, termed amyloid. In addition, a variety of other aggregate species also form, including oligomers and protofibrils. Specifically, HD is caused by the aggregation of the huntingtin (htt) protein that contains an expanded polyglutamine domain. Due to the link between protein aggregation and disease, small molecule aggregation inhibitors have been pursued as potential therapeutic agents. Two such small molecules are epigallocatechin 3-gallate (EGCG) and curcumin, both of which inhibit the fibril formation of several amyloid-forming proteins. However, amyloid formation is a complex process that is strongly influenced by the protein's environment, leading to distinct aggregation pathways. Thus, changes in the protein's environment may alter the effectiveness of aggregation…