Unique Structural Fold Research Articles

Evolution of a bispecific G-quadruplex-forming circular aptamer to block IL-6/sIL-6R interaction for inflammation inhibition.

IL-6 (interleukin-6) is an essential cytokine that participates in many inflammatory and immune responses, and disrupting the interaction between IL-6 and its receptor sIL-6R (soluble form of IL-6 receptor) represents a promising treatment strategy for inflammation and related diseases. Herein we report the first-ever effort of evolving a bispecific circular aptamer, named CIL-6A6-1, that is capable of binding both IL-6 and sIL-6R with nanomolar affinities and is stable in serum for more than 48 hours. CIL-6A6-1 can effectively block the IL-6/sIL-6R interaction and significantly inhibit cell inflammation. Most importantly, this bispecific aptamer is much more effective than aptamers that bind IL-6 and sIL-6R alone as well as tocilizumab, a commercially available humanized monoclonal antibody against sIL-6R, highlighting the advantage of selecting bispecific circular aptamers as molecular tools for anti-inflammation therapy. Interestingly, CIL-6A6-1 is predicted to adopt a unique structural fold with two G-quadruplex motifs capped by a long single-stranded region, which differs from all known DNA aptamers. This unique structural fold may also contribute to its excellent functionality and high stability in biological complex media. We anticipate that our study will represent a significant step forward towards demonstrating the practical utility of bispecific DNA aptamers for therapeutic applications.

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.

Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues*.

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.

Evolution of a highly functional circular DNA aptamer in serum.

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem–loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.

Receptor Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Signaling via Protein‐Protein Cross Talk

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+by increasing concentration of acP yielded lower KM [~21mM] compared to PhoB [~8mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking and GST-assay using various length of ASR [full length, 1–261, and truncated (1–229, 1–239, 1–249)]. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. Recent results of STK 3169 cloning, optimized expression, purification and subsequent characterization in phosphorylation assay will be presented. Support or Funding Information Supported by NIH-NIGMS Support for Competitive Research, SCORE SC3 award GM113803 to VT This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

“Ligand Binding and Phosphorylation of Anabaena Sensory Rhodopsin Transducer towards Putative Signaling State in Sensory Rhodopsin Mediated Protein‐Protein Cross Talk”

Anabaena sensory rhodopsin transducer, ASRT [125 amino acid sequence] is present under the same promoter of its sensory rhodopsin, ASR in Anabaena PCC 7120 a freshwater, filamentous, photosynthetic cynobacteria. The ASRT exists as tetrameric soluble protein indicated to function as downstream signaling partner to sensory rhodopsin. Both solution and crystal structures has reveled that this primarily beta stranded protein exhibit a helical face at carboxyl terminus. Besides receptor binding, ASRT is been shown to serve as a novel eukaryotic-like interaction with DNA. We have demonstrated that carboxyl helical face of tetramer is involved in unusual tetrameric stability. A blast search matches the ASRT to others of unknown function, with 84% similarity and 74% identity to a protein in the myxobacterium Polyangium cellulosum and with 72% similarity and 58% identity to a protein in the legume symbiont Sinorhizobium meliloti. Often the homologues of this family are termed as “DUF”, Domain of Unknown Function member. This DUF1362 family is wide spread over microbial population including pathogens as well. The gene neighbor hood, along with structural fold analysis termed this family as novel small carbohydrate binding protein. We have observed unique xylan binding via electrostatic interaction. Additional bioinformatics analysis has revealed a phosphor transfer along with unique structural fold that may transform it as a unique carbohydrate binding module. However, the signaling state/mechanism of ASRT is obscure. Our initial data with efficient donor, acetyl phosphate, acP using fluorescence quenching of lone Trp-9 suggests that ASRT is involved in phosphorylation. We noticed two notable consensus phosphor transfer residue motif in ASRT, SDKE [53–56] and TRLD [105–108]. Acetyl phosphate has been used as phosphoryl donor in vitro to numerous response regulators [CheY/PhoB/OmpR]. The extent of quenching in presence of Mg2+ by increasing concentration of acP yielded lower KM [~21 mM] compared to PhoB [~8 mM]. It is likely that proximity of phosphor accepting residue (s) to the lone Trp is not comparable to others. Interestingly the ASRT mutant E56Q and D108N indicate the loss of phosphor transfer. We hypothesize that putative position shown as red in figure is involved in ASRT phosphorylation. This motif is in close proximity to receptor binding deduced by CABS docking. Phosphor transfer data along with plausible impact on tetrameric stability will be discussed in this study. We have identified a Serine Threonine Kinase, STK 3169 gene as a likely candidate in phosphor relay. We have cloned and optimizing the functional expression, purification for subsequent characterization in phosphorylation assay. Support or Funding Information Supported by NIH-NIGMS SCORE SC3GM113803 & BCU CURE 2017 award to VT Helical Face of ASRT with Phosphor Transfer Site This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Asp133 Residue in NhaA Na+/H+ Antiporter Is Required for Stability Cation Binding and Transport

Na+/H+ antiporters have a crucial role in pH and Na+ homeostasis in cells. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and revealed a previously unknown structural fold, which has since been identified in several secondary active transporters. This unique structural fold is very delicately electrostatically balanced. Asp133 and Lys 300 have been ascribed essential roles in this balance and, more generally, in the structure and function of the antiporter. In this work, we show the multiple roles of Asp133 in NhaA: (i) The residue's negative charge is critical for the stability of the NhaA structure. (ii) Its main chain is part of the active site. (iii) Its side chain functions as an alkaline-pH-dependent gate, changing the protein's conformation from an inward-facing conformation at acidic pH to an outward-open conformation at alkaline pH, opening the periplasm funnel. On the basis of the experimental data, we propose a tentative mechanism integrating the structural and functional roles of Asp133.

The ring residue proline 8 is crucial for the thermal stability of the lasso peptide caulosegnin II.

Lasso peptides are fascinating natural products with a unique structural fold that can exhibit tremendous thermal stability. Here, we investigate factors responsible for the thermal stability of caulosegnin II. By employing X-ray crystallography, mutational analysis and molecular dynamics simulations, the ring residue proline 8 was proven to be crucial for thermal stability.

Sodium-Proton (Na(+)/H(+)) Antiporters: Properties and Roles in Health and Disease.

The transmembranal Na(+)/H(+) antiporters transport sodium (or several other monovalent cations) in exchange for H(+) across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na(+)/H(+) exchangers (NHEs) in many species from prokaryotes to eukaryotes. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na(+)/H(+) antiporter is Ec-NhaA, the main Na(+)/H(+) antiporter of Escherichia coli.The crystal structure of down-regulated Ec-NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na(+)/H(+) antiporter. It reveals a unique structural fold (called the NhaA fold) in which transmembrane segments (TMs) are organized in inverted-topology repeats, including two antiparallel unfolded regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. This unique structural fold (The NhaA fold) contributes to the cation binding site and facilitates the rapid conformational changes expected for Ec-NhaA. The NhaA fold has now been recognized to be shared by four Na(+)/H(+) antiporters (bacterial and archaeal) and a Na(+) symporter. Remarkably, no crystal structure of any of the human Na(+)/H(+) antiporters exists. Nevertheless, the Ec-NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, three human plasmalemmal proteins that are members of the SLC9 family that are involved in human pathophysiology. Moreover, as outlined in this review, developments in the field, including cellular and biophysical methods that enable ion levels and fluxes to be measured in intact cells as well as in knockout mice, have led to striking advances in the identification and characterization of plasma membrane NHEs and NHA.Very little is known about the endomembrane isoforms of NHE. These intracellular exchangers may serve a function in cation homeostasis and/or osmoregulation, and not in pH regulation as is the case for the plasmalemmal isoforms. This intriguing possibility should be borne in mind when designing future studies. Future progress towards gaining an understanding of the SLC9 gene family, including its structure-function relationships and regulatory mechanisms in health and in disease, is likely to include insights into the pathophysiology of multiple diseases.

NhaA: A Unique Structural Fold of Secondary Active Transporters

AbstractThe Na+/H+ antiporters transport sodium (or several other monovalent cations) in exchange for H+ across lipid bilayers in all kingdoms of life. They are critical in the pH homeostasis of cytoplasm and/or organelles. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na+/H+ antiporter is Ec‐NhaA, the main Na+/H+ antiporter of Escherichia coli. The crystal structure of an inactive, down‐regulated Ec‐NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na+/H+ antiporter. It reveals a unique structural fold (called the NhaA fold) in which helical transmembrane segments (TMs) are organized in inverted‐topology repeats, including two antiparallel non‐helical extended chain regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. The NhaA fold contributes to the cation binding site and facilitates rapid conformational changes of Ec‐NhaA. The NhaA fold has now been recognized to be shared by four Na+/H+ antiporters (bacterial and archaeal) and two Na+ symporters. Thus far, no crystal structure of any of the human Na+/H+ antiporters has been determined. Nevertheless, the Ec‐NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, which are involved in human pathophysiology. Future elucidation of structure‐function relationships of eukaryotic and prokaryotic Na+/H+ antiporters are likely to provide insights into the human pathophysiology. Here, we will focus on the NhaA structural fold which underpins the antiporter functionality of Ec‐NhaA and the secondary active transporters.

1.55 Å resolution X-ray crystal structure of Rv3902c from Mycobacterium tuberculosis.

The crystallographic structure of the Mycobacterium tuberculosis (TB) protein Rv3902c (176 residues; molecular mass of 19.8 kDa) was determined at 1.55 Å resolution. The function of Rv3902c is unknown, although several TB genes involved in bacterial pathogenesis are expressed from the operon containing the Rv3902c gene. The unique structural fold of Rv3902c contains two domains, each consisting of antiparallel β-sheets and α-helices, creating a hand-like binding motif with a small binding pocket in the palm. Structural homology searches reveal that Rv3902c has an overall structure similar to that of the Salmonella virulence-factor chaperone InvB, with an r.m.s.d. for main-chain atoms of 2.3 Å along an aligned domain.

A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.

Crystal Structure of a Heat-inducible Transcriptional Repressor HrcA from Thermotoga maritima: Structural Insight into DNA Binding and Dimerization

All cells have a defense mechanism against a sudden heat-shock stress. Commonly, they express a set of proteins that protect cellular proteins from being denatured by heat. Among them, GroE and DnaK chaperones are representative defending systems, and their transcription is regulated by a heat-shock repressor protein HrcA. HrcA repressor controls the transcription of groE and dnaK operons by binding the palindromic CIRCE element, presumably as a dimer, and the activity of HrcA repressor is modulated by GroE chaperones. Here, we report the first crystal structure of a heat-inducible transcriptional repressor, HrcA, from Thermotoga maritima at 2.2A resolution. The Tm_HrcA protein crystallizes as a dimer. The monomer is composed of three domains: an N-terminal winged helix-turn-helix domain (WH), a GAF-like domain, and an inserted dimerizing domain (IDD). The IDD shows a unique structural fold with an anti-parallel beta-sheet composed of three beta-strands sided by four alpha-helices. The Tm_HrcA dimer structure is formed through hydrophobic contact between the IDDs and a limited contact that involves conserved residues between the GAF-like domains. In the overall dimer structure, the two WH domains are exposed, but the conformation of these two domains seems to be incompatible with DNA binding. We suggest that our structure may represent an inactive form of the HrcA repressor. Structural implication on how the inactive form of HrcA may be converted to the active form by GroEL binding to a conserved C-terminal sequence region of HrcA is discussed.

Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle

Hyaluronan binding protein 1 (HABP1) is a negatively charged multifunctional mammalian protein with a unique structural fold. Despite the fact that HABP1 possesses mitochondrial localization signal, it has also been localized to other cellular compartments. Using indirect immunofluorescence, we examined the sub-cellular localization of HABP1 and its dynamics during mitosis. We wanted to determine whether it distributes in any distinctive manner after mitotic nuclear envelope disassembly or is dispersed randomly throughout the cell. Our results reveal the golgi localization of HABP1 and demonstrate its complete dispersion throughout the cell during mitosis. This distinctive distribution pattern of HABP1 during mitosis resembles its ligand hyaluronan, suggesting that in concert with each other the two molecules play critical roles in this dynamic process.

Protein structure in anisotropic environments: Unique structural fold from orientational constraints

Protein structure in anisotropic environments: Unique structural fold from orientational constraints

Protein structure in anisotropic environments: Unique structural fold from orientational constraints

The mathematical foundation of the determination of protein structure from orientational constraints is described. The tools used are vector algebra, gram matrices, and determinants. The discussion begins in the general abstract setting and proceeds to a discussion of how the methods can be applied to the determination of protein structure using solid state nuclear magnetic resonance. Examples are given relating to the structure of the peptide gramicidin A. © 2000 John Wiley & Sons, Inc. Concepts Magn Reson 12: 71–82, 2000.