Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Structural polymorphism driven by a register shift in a CGAG-rich region found in the promoter of the neurodevelopmental regulator AUTS2 gene.

The AUTS2 gene has been shown to influence brain development by controlling the number of neurons, promoting the growth of axons and dendrites and regulating neuronal migration. The expression of two isoforms of AUTS2 protein is precisely regulated and misregulation of their expression has been correlated with neurodevelopmental delay and autism spectrum disorder. A CGAG-rich region, which includes a putative protein binding site (PPBS), d(AGCGAAAGCACGAA), was found in the promoter region of AUTS2 gene. We show that oligonucleotides from this region adopt thermally stable non-canonical hairpin structures stabilized by G:C and sheared G:A base pairs arranged in a repeating structural motif we termed CGAG block. These motifs are formed consecutively, in a way that exploits a shift in register throughout the whole CGAG repeat to maximize the number of consecutive G:C and G:A base pairs. The differences in CGAG repeat shifting affect the structure of the loop region, where PPBS residues are predominantly located, specifically the loop length, types of base pairs and the pattern of base-base stacking. Finally, we propose a previously unexplored mechanism, by which different folds in the CGAG-rich region could cause a switch in expression between the full-length and C-terminal isoforms of AUTS2.

Intercalation of a Heterocyclic Ligand between Quartets in a G-Rich Tetrahelical Structure.

YES G-rich oligonucleotide VK2 folds into an AGCGA-quadruplex tetrahelical structure distinct and significantly different from G-quadruplexes, even though it contains four G3 tracts. Herein, a bis-quinolinium ligand 360A with high affinity for G-quadruplex structures and selective telomerase inhibition is shown to strongly bind to VK2. Upon binding, 360A does not induce a conformational switch from VK2 to an expected G-quadruplex. In contrast, NMR structural study revealed formation of a well-defined VK2-360A complex with a 1:1 binding stoichiometry, in which 360A intercalates between GAGA- and GCGC-quartets in the central cavity of VK2. This is the first high-resolution structure of a G-quadruplex ligand intercalating into a G-rich tetrahelical fold. This unique mode of ligand binding into tetrahelical DNA architecture offers insights into the stabilization of an AGCGA-quadruplex by a heterocyclic ligand and provides guidelines for rational design of novel VK2 binding molecules with selectivity for different DNA secondary structures.

Use of paramagnetic systems to speed-up NMR data acquisition and for structural and dynamic studies.

NMR spectroscopy is a powerful experimental technique to study biological systems at the atomic resolution. However, its intrinsic low sensitivity results in long acquisition times that in extreme cases lasts for days (or even weeks) often exceeding the lifetime of the sample under investigation. Different paramagnetic agents have been used in an effort to decrease the spin-lattice (T1) relaxation times of the studied nuclei, which are the main cause for long acquisition times necessary for signal averaging to enhance the signal-to-noise ratio of NMR spectra. Consequently, most of the experimental time is "wasted" in waiting for the magnetization to recover between successive scans. In this review, we discuss how to set up an optimal paramagnetic relaxation enhancement (PRE) system to effectively reduce the T1 relaxation times avoiding significant broadening of NMR signals. Additionally, we describe how PRE-agents can be used to provide structural and dynamic information and can even be used to follow the intermediates of chemical reactions and to speed-up data acquisition. We also describe the unique challenges and benefits associated with the application of PRE to solid-state NMR spectroscopy, explaining how the use of PREs is more complex for membrane mimetic systems as PREs can also be exploited to change the alignment of oriented membrane systems. Functionalization of membrane mimetics, such as bicelles, can provide a controlled region of paramagnetic effect that has the potential, together with the desired alignment, to provide crucial biologically relevant structural information. And finally, we discuss how paramagnetic metals can be utilized to further increase the dynamic nuclear polarization (DNP) effects and how to preserve the enhancements when dissolution DNP is implemented.

Energetic Basis of AGCGA-Rich DNA Folding into a Tetrahelical Structure.

It was recently discovered that, besides well-known G-quadruplexes and i-motifs, DNA may adopt another type of noncanonical structure called AGCGA-quadruplexes. Here, the folding of the VK2 fragment from the regulatory region of the PLEKHG3 gene is studied and, for the first time, the energetic contributions that stabilize this unique fold are described. Similarly to the B-DNA, it is stabilized by hydrophobic desolvation and, in contrast to G-quadruplexes, also by specific binding of water molecules. Compared to B-DNA, VK2 folding is enthalpically less favorable due to poorer base-stacking interactions, resulting in substantial conformational flexibility. This entropically favorable conformational "breathing" stabilizes the AGCGA-quadruplexes. In conclusion, AGCGA-quadruplexes have a distinguishing thermodynamic fingerprint and the corresponding driving forces enabling their folding are consistent with the observed structural features.

Metal-Chelated Polymer Nanodiscs for NMR Studies.

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1 ) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene-co-maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu2+ -chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid-nanodisc components. The chelated nanodiscs also decrease the proton T1 values for a water-soluble DNA G-quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed-up data acquisition from lipid bilayer samples and also to provide structural information from water-soluble biomolecules.

High-Resolution Structure of RNA G-Quadruplex Containing Unique Structural Motifs Originating from the 5'-UTR of Human Tyrosine Kinase 2 (TYK2).

Tyrosine kinase 2 (TYK2) is a member of the JAK family of nonreceptor-associated tyrosine kinases together with highly homologous JAK1, JAK2, and JAK3 paralogues. Overexpression of TYK2 is associated with several inflammatory diseases, including severe complications during the COVID-19 infection. Since the downregulation of JAK paralogues could lead to serious health consequences or even death, it is critical to avoid it when designing drugs to suppress TYK2. To achieve the required specificity only for TYK2, researchers have recently selectively targeted TYK2 mRNA by developing antisense oligonucleotides. In this work, we expand the target space of TYK2 mRNA by showing that the mRNA adopts tetra-helical noncanonical structures called G-quadruplexes. We identified a TYKwt RNA oligonucleotide from the 5'-UTR of TYK2 mRNA, which adopts multiple different parallel G-quadruplexes that exist at equilibrium. Using NMR spectroscopy, we showed that some of the G-quadruplexes adopt unique structural motifs, mainly due to the formation of a stable GA bulge. Using guanine to uridine substitutions, we prepared the oligonucleotide TYK3_U6, which serves as an excellent model for the bulged G-quadruplexes formed by the TYKwt oligonucleotide. NMR structural analysis, including data on the residual coupling constants (RDC) of the loop regions, unveiled that the studied three-quartet parallel G-quadruplex contains many unusual structural features such as a G(U)A bulge, a guanine residue in the syn conformation, A and U residues stacked on the top G-quartet, and a well-defined adenine from a three-residue long propeller loop oriented in the groove, all of which could be valuable targets for future drug design.

Protein G-quadruplex interactions and their effects on phase transitions and protein aggregation.

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-ß Generates Toxic Hetero-oligomers.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and ß-amyloid (Aß) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aß(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aß(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aß(M1-42) heteromers comprised of unstructured Aß(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aß(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aß(M1-42) is electrostatically driven and induces unfolding of Aß(M1-42). Neurotoxicity profiling of Aß(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aß(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Tetrahelical structural family adopted by AGCGA-rich regulatory DNA regions.

Here we describe AGCGA-quadruplexes, an unexpected addition to the well-known tetrahelical families, G-quadruplexes and i-motifs, that have been a focus of intense research due to their potential biological impact in G- and C-rich DNA regions, respectively. High-resolution structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy demonstrate that AGCGA-quadruplexes comprise four 5'-AGCGA-3' tracts and are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets, respectively. Residues in the core of the structure are connected with edge-type loops. Sequences of alternating 5'-AGCGA-3' and 5'-GGG-3' repeats could be expected to form G-quadruplexes, but are shown herein to form AGCGA-quadruplexes instead. Unique structural features of AGCGA-quadruplexes together with lower sensitivity to cation and pH variation imply their potential biological relevance in regulatory regions of genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development.

A tetrahelical DNA fold adopted by tandem repeats of alternating GGG and GCG tracts.

DNA can form diverse higher-order structures, whose details are greatly dependent on nucleotide sequence. G-rich sequences containing four or more repeats of three guanines are expected to form G-quadruplexes. Here we show that DNA sequences with GGGAGCG repeats found in the regulatory region of the PLEKHG3 gene are capable of forming tetrahelical DNA structures that are distinct from G-quadruplexes. The d(GGGAGCGAGGGAGCG) sequence, VK1, forms a dimer. Two VK1 sequences connected by an adenine residue, VK2, fold into a monomer, which shares identical structural characteristics with the VK1 fold. Their four-stranded architectures are stabilized by four G-C, four G-A and six G-G base pairs. No G-quartets or Hoogsteen-type hydrogen-bonded guanine residues are present and the overall topology is conserved in the presence of Li(+), Na(+), K(+) and NH4(+) ions. Unique structural features include two edgewise loops on each side of the structure stabilized by three G-G base pairs in N1-carbonyl symmetric geometry.