scholarly journals Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Lucas F. Ribeiro ◽  
Vanesa Amarelle ◽  
Liliane F. C. Ribeiro ◽  
María-Eugenia Guazzaroni
Keyword(s):  
Protein Engineering ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Protein Function ◽  
Cellular Response ◽  
Protein Domain ◽  
Signal Molecule ◽  
Synthetic Protein ◽  
Periplasmic Binding Proteins ◽  
Domain Insertion

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

scholarly journals Biophysical and functional characterization of Norrin signaling through Frizzled4

2018 ◽  
Vol 115 (35) ◽  
pp. 8787-8792 ◽  
Author(s):  
Injin Bang ◽  
Hee Ryung Kim ◽  
Andrew H. Beaven ◽  
Jinuk Kim ◽  
Seung-Bum Ko ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wnt Signaling ◽  
Conformational Changes ◽  
Cellular Response ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Intracellular Loop ◽  
Ligand Interaction ◽  
Rich Domain ◽  
Linker Domain

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin–dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD–Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin–dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.

scholarly journals Single-molecule observation of ligand binding and conformational changes in FeuA

2019 ◽  
Author(s):  
Marijn de Boer ◽  
Giorgos Gouridis ◽  
Yusran Abdillah Muthahari ◽  
Thorben Cordes
Keyword(s):  
Data Analysis ◽  
Ligand Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Analysis Procedure ◽  
Closed Conformation ◽  
Data Analysis Procedure ◽  
Ligand Interactions ◽  
Substrate Binding Protein

ABSTRACTThe specific binding of ligands by proteins and the coupling of this process to conformational changes are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10000-fold and temporally stabilize the formed conformation 250-fold.SIGNIFICANCE STATEMENTLigand binding and the coupling of this process to conformational changes in proteins are fundamental to their function. We developed a single-molecule approach that allows the simultaneous observation of ligand binding and conformational changes in the substrate-binding protein FeuA. This allows to directly observe the ligand binding process, ligand-driven conformational changes as well as rare short-lived conformational transitions that are uncoupled from the ligand. These findings provide insight into the fundamental relation between ligand-protein interactions and conformational changes. Our findings are, however, not only of interest to understand protein function, but the developed data analysis procedure allows the determination of (relative) distance changes in single-molecule FRET experiments, for situations in which donor and acceptor fluorophore are influenced by quenching processes.

scholarly journals Structural Basis for 2′-5′-Oligoadenylate Binding and Enzyme Activity of a Viral RNase L Antagonist

2015 ◽  
Vol 89 (13) ◽  
pp. 6633-6645 ◽  
Author(s):  
Kristen M. Ogden ◽  
Liya Hu ◽  
Babal K. Jha ◽  
Banumathi Sankaran ◽  
Susan R. Weiss ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Cellular Response ◽  
Substrate Binding ◽  
Core Structure ◽  
Structural Basis ◽  
Rnase L ◽  
Binding Mechanism ◽  
Oligoadenylate Synthetase ◽  
Terminal Domain

ABSTRACTSynthesis of 2′-5′-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HΦ(S/T)Φ (where Φ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave β-sheet that contains the catalytic cleft, but it lacks two α-helical regions and two β-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A.IMPORTANCEThe C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2′-5′-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two α-helical regions and β-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity

1990 ◽  
Vol 10 (10) ◽  
pp. 5496-5501
Author(s):  
N Giese ◽  
W J LaRochelle ◽  
M May-Siroff ◽  
K C Robbins ◽  
S A Aaronson
Keyword(s):  
Monoclonal Antibody ◽  
Growth Factor ◽  
Conformational Changes ◽  
Protein Domain ◽  
Platelet Derived Growth Factor ◽  
Receptor Interaction ◽  
Pdgf Receptor ◽  
Amino Acid Residues ◽  
Disulfide Linkages ◽  
Transforming Activity

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

Sign in / Sign up

Export Citation Format

Share Document