Belén Chaves-Arquero

In drug discovery, traditional automated library synthesis has typically involved single-step synthetic procedures targeting a single vector of interest. However, achieving greater structural diversity requires exploring multistep and multivectorial approaches. These methodologies enable the preparation of compounds with varying structures in a single experiment. Here, we present a novel method for multistep library synthesis in continuous flow. This approach offers unique opportunities, such as exploring linkers between two defined vectors or rapidly mapping synergistic structure-activity relationship (SAR) by concurrently exploring multiple vectors. Our method incorporates up to eight different synthetic methodologies, including established chemistries, metal-catalysed transformations, and modern metallaphotoredox couplings. This broad range of synthetic methodologies ensures a high level of diversity in the compounds generated, providing a powerful tool to accelerate exploration of the chemical space in drug discovery programs.

A substantial part of living cells activity involves transcription regulation. The RNA polymerases responsible for this job need to know ‘where/when' to start and stop in the genome, answers that may change throughout life and upon external stimuli. In Saccharomyces cerevisiae, RNA Pol II transcription termination can follow two different routes: the poly(A)-dependent one used for most of the mRNAs and the Nrd1/Nab3/Sen1 (NNS) pathway for non-coding RNAs (ncRNA). The NNS targets include snoRNAs and cryptic unstable transcripts (CUTs) generated by pervasive transcription. This review recapitulates the state of the art in structural biology and biophysics of the Nrd1, Nab3 and Sen1 components of the NNS complex, with special attention to their domain structures and interactions with peptide and RNA motifs, and their heterodimerization. This structural information is put into the context of the NNS termination mechanism together with possible prospects for evolution in the field.

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4⁺ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1–21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + β secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

Heterodimerization of RNA binding proteins Nrd1 and Nab3 is essential to communicate the RNA recognition in the nascent transcript with the Nrd1 recognition of the Ser₅-phosphorylated Rbp1 C-terminal domain in RNA polymerase II. The structure of a Nrd1–Nab3 chimera reveals the basis of heterodimerization, filling a missing gap in knowledge of this system. The free form of the Nrd1 interaction domain of Nab3 (NRID) forms a multi-state three-helix bundle that is clamped in a single conformation upon complex formation with the Nab3 interaction domain of Nrd1 (NAID). The latter domain forms two long helices that wrap around NRID, resulting in an extensive protein–protein interface that would explain the highly favorable free energy of heterodimerization. Mutagenesis of some conserved hydrophobic residues involved in the heterodimerization leads to temperature-sensitive phenotypes, revealing the importance of this interaction in yeast cell fitness. The Nrd1–Nab3 structure resembles the previously reported Rna14/Rna15 heterodimer structure, which is part of the poly(A)-dependent termination pathway, suggesting that both machineries use similar structural solutions despite they share little sequence homology and are potentially evolutionary divergent.

TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein. We show that TUT4(7) ZnF2 contains two distinct RNA binding surfaces that are used in the interaction with different RNA nucleobases in different targets, i.e that this small domain encodes diversity in TUT4(7) selectivity and molecular function. Interestingly and unlike other well-characterized CCHC ZnFs, ZnF2 is not physically coupled to the flanking ZnF3 and acts independently in miRNA recognition, while the remaining CCHC ZnF of TUT4(7), ZnF1, has lost its intrinsic RNA binding capability. Together, our data suggest that the ZnFs of TUT4(7) are independent units for RNA and, possibly, protein-protein interactions that underlay the protein’s functional flexibility and are likely to play an important role in building its interaction network.

Summary: Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the intrinsically disordered region of eIF4G1, eIF4G1 _1-249 , and its interactions with Pub1 and Pab1. The conformational ensemble of eIF4G1 _1-249 shows an α-helix within the BOX3 conserved element and a dynamic network of fuzzy π-π and π-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. In contrast, the Pub1 RRM3 domain interacts with the RNA1-1 and BOX1 regions of eIF4G1. Mixtures of Pub1, Pab1 and eIF4G1 form micrometer-size protein condensates that require the presence of the eIF4G1 BOX1 element. These homotypic interactions suggest a double key mechanism of eIF4G1 regulation, important for understanding the architecture of stress granule cores.

To investigate the structural impact of phosphorylation on the human histone H1.0 C‐terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T¹¹⁸‐H1.0 (T¹¹⁸PKK motif) and T¹⁴⁰‐H1.0 (T¹⁴⁰PVK motif). Both model peptides are mainly disordered in aqueous solution in their non‐phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T¹¹⁸‐H1.0 and pT¹¹⁸‐H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104–115 and a short α/3₁₀ helix (residues 119–123), that are almost perpendicular in T¹¹⁸‐H1.0 but have a poorly defined orientation in pT¹¹⁸‐H1.0. Peptides T¹⁴⁰‐H1.0 and pT¹⁴⁰‐H1.0 form very similar α helices between residues 141–147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side‐chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT¹¹⁸‐H1.0 and pT¹⁴⁰‐H1.0 has pK_a values below the intrinsic values, which can be explained by non‐specific charge–charge interactions with nearby Lys. The non‐polar Val in the TPVK motif accounts for the pT¹⁴⁰ pK_a being closer to the intrinsic pK_a value than the pT¹¹⁸ pK_a. Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short‐lived intrinsically disordered proteins and domains.

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The C-terminal domain of histone H1.0 (C-H1.0) is involved in DNA binding and is a main determinant of the chromatin condensing properties of histone H1.0. Phosphorylation at the (S/T)-P-X-(K/R) motifs affects DNA binding and is crucial for regulation of C-H1.0 function. Since C-H1.0 is an intrinsically disordered domain, solution NMR is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of C-H1.0. However, its very repetitive, low-amino acid-diverse and Pro-rich sequence, together with the low signal dispersion observed at the ¹H–¹⁵N HSQC spectra of both non- and tri-phosphorylated C-H1.0 preclude the use of standard ¹H-detected assignment strategies. We have achieved an essentially complete assignment of the heavy backbone atoms (¹⁵N, ¹³C′ and ¹³C_α), as well as ¹H^N and ¹³C_β nuclei, of non- and tri-phosphorylated C-H1.0 by applying a novel ¹³C-detected CON-based strategy. No C-H1.0 region with a clear secondary structure tendency was detected by chemical shift analyses, confirming at residue level that C-H1.0 is disordered in aqueous solution. Phosphorylation only affected the chemical shifts of phosphorylated Thr’s, and their adjacent residues. Heteronuclear {¹H}–¹⁵N NOEs were also essentially equal in the non- and tri-phosphorylated states. Hence, structural tendencies and dynamic properties of C-H1.0 free in aqueous solution are unmodified by phosphorylation. We propose that the assignment strategy used for C-H1.0, which is based on the acquisition of only a few 3D spectra, is an excellent choice for short-lived intrinsically disordered proteins with repetitive sequences.