tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).

Enzymes known as PylRS offer the remarkable ability to expand the natural genetic code of a living cell with unnatural amino acids. Currently, over 200 unnatural amino acids can be genetically encoded with the help of PylRS and its partner tRNAPyl, enabling us to endow proteins with novel properties, or regulate protein activity using light or inducible cross-linking. One intriguing feature of PylRS enzymes is their ability to avoid cross-reactivity when two PylRS homologs from different organisms-such as those from the archaea Methanosarcina mazei and Methanomethylophilus alvus-are co-expressed in a single cell. This makes it possible to simultaneously encode two unnatural amino acids in a single protein. This study illuminates the elusive mechanism of PylRS specificity by using cryo-electron microscopy, biochemistry and molecular simulations. The interaction of PylRS from M. alvus with its tRNAPyl is best described as two pieces of a jigsaw puzzle; in which PylRS recognizes the unique shape of its cognate tRNA instead of specific nucleotides in the tRNA sequence like other tRNA-binding enzymes. This finding may streamline the rational design of tools for simultaneous genetic incorporation of multiple unnatural amino acids, thereby facilitating the development of valuable proteins for research, medicine, and biotechnology.

Identifying the molecular basis of Laminin N-terminal domain Ca2+ binding using a hybrid approach.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2.

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2'-5'-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2'-5'-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Correction: Dramatic and concerted conformational changes enable rhodocetin to block α2ß1 integrin selectively.

[This corrects the article DOI: 10.1371/journal.pbio.2001492.].

Dramatic and concerted conformational changes enable rhodocetin to block α2ß1 integrin selectively.

The collagen binding integrin α2ß1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αß and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2ß1 integrin. Besides the release of the nonbinding RCαß tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the "closed" conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2ß1 integrin.

Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5'-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC.

The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes, but they do not predominate in detergent-solubilized VDAC samples. In this study, a cholesterol analog, cholesteryl-hemisuccinate (CHS), was shown to induce the formation of detergent-soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation, and size-exclusion chromatography small angle x-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 µM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.

Modulating antibody-dependent cellular cytotoxicity of epidermal growth factor receptor-specific heavy-chain antibodies through hinge engineering.

Human IgG1 and IgG3 antibodies (Abs) can mediate Ab-dependent cellular cytotoxicity (ADCC), and engineering of the Ab Fc (point mutation; defucosylation) has been shown to affect ADCC by modulating affinity for FcRγIIIa. In the absence of a CH 1 domain, many camelid heavy-chain Abs (HCAbs) naturally bear very long and flexible hinge regions connecting their VH H and CH 2 domains. To better understand the influence of hinge length and structure on HCAb ADCC, we produced a series of hinge-engineered epidermal growth factor receptor (EGFR)-specific chimeric camelid VH H-human Fc Abs and characterized their affinities for recombinant EGFR and FcRγIIIa, their binding to EGFR-positive tumor cells, and their ability to elicit ADCC. In the case of one chimeric HCAb (EG2-hFc), we found that variants bearing longer hinges (IgG3 or camelid hinge regions) showed dramatically improved ADCC in comparison with a variant bearing the human IgG1 hinge, in similar fashion to a variant with reduced CH 2 fucosylation. Conversely, an EG2-hFc variant bearing a truncated human IgG1 upper hinge region failed to elicit ADCC. However, there was no consistent association between hinge length and ADCC for four similarly engineered chimeric HCAbs directed against distinct EGFR epitopes. These findings demonstrate that the ADCC of some HCAbs can be modulated simply by varying the length of the Ab hinge. Although this effect appears to be heavily epitope-dependent, this strategy may be useful to consider during the design of VH H-based therapeutic Abs for cancer.

Human DDX21 binds and unwinds RNA guanine quadruplexes.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

Reductive power of the archaea right-handed coiled coil nanotube (RHCC-NT) and incorporation of mercury clusters inside protein cages.

Coiled coils are well described as powerful oligomerization motifs and exhibit a large diversity of functions, including gene regulation, cell division, membrane fusion and drug extrusion. The archaea S-layer originated right-handed coiled coil -RHCC-NT- is characterized by extreme stability and is free of cysteine and histidine moieties. In the current study, we have followed a multidisciplinary approach to investigate the capacity of RHCC-NT to bind a variety of ionic complex metal ions. At the outside of the RHCC-NT, one mercury ion forms an electrostatic interaction with the S-methyl moiety of the single methionine residue present in each coil. We demonstrate that RHCC-NT is reducing and incorporating metallic mercury in the large-sized interior cavities which are lined up along the tetrameric channel.

Interaction studies of a protein and carbohydrate system using an integrated approach: a case study of the miniagrin-heparin system.

The major challenges in biophysical characterization of human protein-carbohydrate interactions are obtaining monodispersed preparations of human proteins that are often post-translationally modified and lack of detection of carbohydrates by traditional detection systems. Light scattering (dynamic and static) techniques offer detection of biomolecules and their complexes based on their size and shape, and do not rely on chromophore groups (such as aromatic amino acid sidechains). In this study, we utilized dynamic light scattering, analytical ultracentrifugation and small-angle X-ray scattering techniques to investigate the solution properties of a complex resulting from the interaction between a 15 kDa heparin preparation and miniagrin, a miniaturized version of agrin. Results from dynamic light scattering, sedimentation equilibrium, and sedimentation velocity experiments signify the formation of a monodisperse complex with 1:1 stoichiometry, and low-resolution structures derived from the small-angle X-ray scattering measurements implicate an extended conformation for a side-by-side miniagrin‒heparin complex.

Validation of a simultaneous method for determining polycyclic aromatic compounds and alkylated isomers in biota.

RATIONALE: There is a need for a validated method to improve detection limits and simultaneously quantify polycyclic aromatic compounds (PACs, both parent and alkylated homologues) in biota by gas chromatography/tandem mass spectrometry because of their environmental significance. The validation of the method was performed in accordance to the Eurachem Guide to Quality in Analytical Chemistry. METHODS: Gas chromatography coupled with a triple quadrupole mass spectrometer used in multiple reaction monitoring (MRM) mode was used for detection and quantification. Retention time windows and selective MRM ion transitions were optimized for a suite of PACs. The developed method was validated by comparing our measurements made on a reference material of freeze-dried mussel tissue (Mytilus edulis) with the certified values. RESULTS: Linearity was observed between 10-1000 pg/µL (PAHs) and 2-500 pg/µL (alkyl-PACs including S-based PACs). The overall mean (±SD) for the limits of detection of 43 PACs studied were 0.305 ± 0.276 and 2.69 ± 1.10 ng/g, respectively. For the 14 certified target analytes, the percent relative error ranged from 1.3 to 33%. With the exception of benzo(a)pyrene, the between-day and within-day repeatability for all target analytes was lower than 15% RSD. CONCLUSIONS: This is the first report of a fully validated method to simultaneously quantify PACs in biota performed in an ISO accredited laboratory.

Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork.

High mobility group AT-hook 2 (HMGA2) protein is composed of three AT-hook domains. HMGA2 expresses at high levels in both embryonic stem cells and cancer cells, where it interacts with and stabilizes replication forks (RFs), resulting in elevated cell proliferation rates. In this study, we demonstrated that HMGA2 knockdown reduces cell proliferation. To understand the features required for interaction between HMGA2 and RFs, we studied the solution structure of HMGA2, free and in complex with RFs, using an integrated host of biophysical techniques. Circular dichroism and NMR experiments confirmed the disordered state of unbound HMGA2. Dynamic light scattering and sedimentation velocity experiments demonstrated that HMGA2 and RF are monodisperse in solution, and form an equimolar complex. Small-angle x-ray scattering studies revealed that HMGA2 binds in a side-by-side orientation to RF where 3 AT-hooks act as a clamp to wrap around a distorted RF. Thus, our data provide insights into how HMGA2 interacts with stalled RFs and the function of the process.

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3'-Tail of the Long Non-coding RNA BC200 (BCYRN1).

RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3'-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3'-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.

Archaea S-layer nanotube from a "black smoker" in complex with cyclo-octasulfur (S8 ) rings.

Elemental sulfur exists primarily as an S80 ring and serves as terminal electron acceptor for a variety of sulfur-fermenting bacteria. Hyperthermophilic archaea from black smoker vents are an exciting research tool to advance our knowledge of sulfur respiration under extreme conditions. Here, we use a hybrid method approach to demonstrate that the proteinaceous cavities of the S-layer nanotube of the hyperthermophilic archaeon Staphylothermus marinus act as a storage reservoir for cyclo-octasulfur S8. Fully atomistic molecular dynamics (MD) simulations were performed and the method of multiconfigurational thermodynamic integration was employed to compute the absolute free energy for transferring a ring of elemental sulfur S8 from an aqueous bath into the largest hydrophobic cavity of a fragment of archaeal tetrabrachion. Comparisons with earlier MD studies of the free energy of hydration as a function of water occupancy in the same cavity of archaeal tetrabrachion show that the sulfur ring is energetically favored over water.

Impact of G-quadruplex loop conformation in the PITX1 mRNA on protein and small molecule interaction.

Intramolecular G-quadruplexes (G4s) are G-rich nucleic acid structures that fold back on themselves via interrupting loops to create stacked planar G-tetrads, in which four guanine bases associate via Hoogsteen hydrogen bonding. The G4 structure is further stabilized by monovalent cations centered between the stacked tetrads. The G-tetrad face on the top and bottom planes of G4s are often the site of interaction with proteins and small molecules. To investigate the potential impact of interrupting loops on both G4 structure and interaction with proteins/small molecules, we characterized a specific G4 from the 3'-UTR of PITX1 mRNA that contains loops of 6 nucleotides using biophysical approaches. We then introduced mutations to specific loops to determine the impact on G4 structure and the ability to interact with both proteins and a G4-specific ligand. Our results suggest that mutation of a specific loop both affects the global G4 structure and impacts the ability to interact with a G4 binding protein and small molecule ligand.