Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics.

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.

Deciphering the principles of the RNA editing code via large-scale systematic probing.

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.

Structures of the Cmr-ß Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies.

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the "reproducibility crisis," demonstrating the need for rigorous, platform-appropriate validation.

Rescue of expression and function of long QT syndrome-causing mutant hERG channels by enhancing channel stability in the plasma membrane.

The human ether-g-go-go-related gene (hERG) encodes the Kv11.1 (or hERG) channel that conducts the rapidly activating delayed rectifier potassium current (IKr). Naturally occurring mutations in hERG impair the channel function and cause long QT syndrome type 2 (LQT2). Many missense hERG mutations lead to a lack of channel expression on the cell surface, representing a major mechanism for the loss-of-function of mutant channels. While it is generally thought that a trafficking defect underlies the lack of channel expression on the cell surface, in the present study, we demonstrate that the trafficking defective mutant hERG G601S can reach the plasma membrane but is unstable and quickly degrades, which is akin to Wild Type (WT) hERG channels under low K+ conditions. We previously showed that Serine (S) residue at 624 in the innermost position of the selectivity filter of hERG is involved in hERG membrane stability such that substitution of Serine 624 with Threonine (S624T) enhances hERG stability and renders hERG insensitive to low K+ culture. Here, we report that the intragenic addition of S624T substitution to trafficking defective hERG mutants G601S, N470D and P596R led to a complete rescue of the function of these otherwise loss-of-function mutant channels to a level similar to the WT channel, representing the most effective rescue means for the function of mutant hERG channels. These findings not only provide novel insights into hERG mutation-mediated channel dysfunction, but also point to the critical role of S624 in hERG stability on the plasma membrane.

G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Forty Years of Inferential Methods in the Journals of the Society for Molecular Biology and Evolution.

We are launching a series to celebrate the 40th anniversary of the first issue of Molecular Biology and Evolution. In 2024, we will publish virtual issues containing selected papers published in the Society for Molecular Biology and Evolution journals, Molecular Biology and Evolution and Genome Biology and Evolution. Each virtual issue will be accompanied by a perspective that highlights the historic and contemporary contributions of our journals to a specific topic in molecular evolution. This perspective, the first in the series, presents an account of the broad array of methods that have been published in the Society for Molecular Biology and Evolution journals, including methods to infer phylogenies, to test hypotheses in a phylogenetic framework, and to infer population genetic processes. We also mention many of the software implementations that make methods tractable for empiricists. In short, the Society for Molecular Biology and Evolution community has much to celebrate after four decades of publishing high-quality science including numerous important inferential methods.

The Tree Reconstruction Game: Phylogenetic Reconstruction Using Reinforcement Learning.

The computational search for the maximum-likelihood phylogenetic tree is an NP-hard problem. As such, current tree search algorithms might result in a tree that is the local optima, not the global one. Here, we introduce a paradigm shift for predicting the maximum-likelihood tree, by approximating long-term gains of likelihood rather than maximizing likelihood gain at each step of the search. Our proposed approach harnesses the power of reinforcement learning to learn an optimal search strategy, aiming at the global optimum of the search space. We show that when analyzing empirical data containing dozens of sequences, the log-likelihood improvement from the starting tree obtained by the reinforcement learning-based agent was 0.969 or higher compared to that achieved by current state-of-the-art techniques. Notably, this performance is attained without the need to perform costly likelihood optimizations apart from the training process, thus potentially allowing for an exponential increase in runtime. We exemplify this for data sets containing 15 sequences of length 18,000 bp and demonstrate that the reinforcement learning-based method is roughly three times faster than the state-of-the-art software. This study illustrates the potential of reinforcement learning in addressing the challenges of phylogenetic tree reconstruction.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.