Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

All-Atom Simulations Elucidate the Impact of U2AF2 Cancer-Associated Mutations on Pre-mRNA Recognition.

The U2AF2 splicing factor, made of two tandem RNA recognition motifs (RRMs) joined by a flexible linker, selects the intronic polypyrimidine sequence of premature mRNA, thus ensuring splicing fidelity. Increasing evidence links mutations of key splicing factors, including U2AF2, to a variety of cancers. Nevertheless, the impact of U2AF2 cancer-associated mutations on polypyrimidine recognition remains unclear. Here, we combined extensive (18 µs-long) all-atom molecular dynamics simulations and dynamical network theory analysis (NWA) of U2AF2, in its wild-type form and in the presence of the six most frequent cancer-associated mutations, bound to a poly-U strand. Our results reveal that the selected mutations affect the pre-mRNA binding at two hot spot regions, irrespectively of where these mutants are placed on the distinct U2AF2 domains. Complementarily, NWA traced the existence of cross-communication pathways, connecting each mutation site to these recognition hot spots, whose strength is altered by the mutations. Our outcomes suggest the existence of a structural/dynamical interplay of the two U2AF2's RRMs underlying the recognition of the polypyrimidine tract and reveal that the cancer-associated mutations affect the polypyrimidine selection by altering the RRMs' cooperativity. This mechanism may be shared by other RNA binding proteins hallmarked, like U2AF2, by multidomain architecture and high plasticity.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Allosteric Cross-Talk among Spike's Receptor-Binding Domain Mutations of the SARS-CoV-2 South African Variant Triggers an Effective Hijacking of Human Cell Receptor.

The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike protein's receptor-binding domain (RBD), which, binding to the angiotensin-converting enzyme 2 (ACE2) receptor, mediates viral entry into host cells. Here, all-atom molecular dynamics simulations and dynamical network theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spike's binding affinity toward ACE2, the concomitant N501Y, E484K, and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements and (ii) impairing the binding of antibodies elicited by infected or vaccinated patients. This information unlocks the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, setting the basis for developing the next-generation anti-COVID-19 therapeutics.

Atomic-Level Mechanism of Pre-mRNA Splicing in Health and Disease.

Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.

Is the Rigidity of SARS-CoV-2 Spike Receptor-Binding Motif the Hallmark for Its Enhanced Infectivity? Insights from All-Atom Simulations.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic is setting the global health crisis of our time, causing a devastating societal and economic burden. An idiosyncratic trait of coronaviruses is the presence of spike glycoproteins on the viral envelope, which mediate the virus binding to specific host receptor, enabling its entry into the human cells. In spite of the high sequence identity of SARS-CoV-2 with its closely related SARS-CoV emerged in 2002, the atomic-level determinants underlining the molecular recognition of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor and, thus, the rapid virus spread into human body, remain unresolved. Here, multi-microsecond-long molecular dynamics simulations enabled us to unprecedentedly dissect the key molecular traits liable of the higher affinity/specificity of SARS-CoV-2 toward ACE2 as compared to SARS-CoV. This supplies a minute per-residue contact map underlining its stunningly high infectivity. Harnessing this knowledge is pivotal for urgently developing effective medical countermeasures to face the ongoing global health crisis.

Decrypting the Information Exchange Pathways across the Spliceosome Machinery.

Intron splicing of a nascent mRNA transcript by spliceosome (SPL) is a hallmark of gene regulation in eukaryotes. SPL is a majestic molecular machine composed of an entangled network of proteins and RNAs that meticulously promotes intron splicing through the formation of eight intermediate complexes. Cross-communication among the critical distal proteins of the SPL assembly is pivotal for fast and accurate directing of the compositional and conformational readjustments necessary to achieve high splicing fidelity. Here, molecular dynamics (MD) simulations of an 800 000 atom model of SPL C complex from yeast Saccharomyces cerevisiae and community network analysis enabled us to decrypt the complexity of this huge molecular machine, by identifying the key channels of information transfer across long distances separating key protein components. The reported study represents an unprecedented attempt in dissecting cross-communication pathways within one of the most complex machines of eukaryotic cells, supporting the critical role of Clf1 and Cwc2 splicing cofactors and specific domains of the Prp8 protein as signal conveyors for pre-mRNA maturation. Our findings provide fundamental advances into mechanistic aspects of SPL, providing a conceptual basis for controlling the SPL via small-molecule modulators able to tackle splicing-associated diseases by altering/obstructing information-exchange paths.

All-Atom Simulations Disclose How Cytochrome Reductase Reshapes the Substrate Access/Egress Routes of Its Partner CYP450s.

Cytochromes P450 enzymes (CYP450s) promote the oxidative metabolism of a variety of substrates via the electrons supplied by the cytochrome P450 reductase (CPR) and upon formation of a CPR/CYP450 adduct. In spite of the pivotal regulatory importance of this process, the impact of CPR binding on the functional properties of its partner CYP450 remains elusive. By performing multiple microsecond-long all-atom molecular dynamics simulations of a 520 000-atom model of a CPR/CYP450 adduct embedded in a membrane mimic, we disclose the molecular terms for their interactions, considering the aromatase (HA) enzyme as a proxy of the CYP450 family. Our study strikingly unveils that CPR binding alters HA's functional motions, bolstering a change in the shape and type of the channels traveled by substrates/products during their access/egress to/from the enzyme's active site. Our outcomes unprecedentedly contribute to extricate the many entangled facets of the CYP450 metabolon, redrafting its intricate panorama from an atomic-level perspective.

Exploiting Cryo-EM Structural Information and All-Atom Simulations To Decrypt the Molecular Mechanism of Splicing Modulators.

Splicing modulators (SMs) pladienolides, herboxidienes, and spliceostatins exert their antitumor activity by altering the ability of SF3B1 and PHF5A proteins, components of SF3b splicing factor, to recognize distinct intron branching point sequences, thus finely calibrating constitutive/alternative/aberrant splicing of pre-mRNA. Here, by exploiting structural information obtained from cryo-EM data, and by performing multiple µs-long all-atom simulations of SF3b in apo form and in complex with selected SMs, we disclose how these latter seep into the narrow slit at the SF3B1/PHF5A protein interface. This locks the intrinsic open/closed conformational transitions of SFB1's solenoidal structure into the open state. As a result, SMs prevent the formation of a closed/intron-loaded conformation of the SF3B1 protein by decreasing the internal SF3B1 cross-correlation and reducing SF3B1's functional plasticity. We further compellingly support the proposition that SMs' action exceeds a purely competitive inhibition. Indeed, our simulations also demonstrate that the introduction of recurrent drug resistance/sensitizing mutations in SF3B1 or PHF5A, besides affecting the binding affinity of SMs, likewise influence the functional dynamics of SF3B1. This knowledge clarifies the molecular terms of SF3b modulation by small-molecules, fostering the rational-based discovery of drugs tackling distinct cancer types resulting from dysregulated splicing. This work also supports the coming of age usage of cryo-EM structural data in forthcoming drug-discovery studies.

Disclosing the Impact of Carcinogenic SF3b Mutations on Pre-mRNA Recognition Via All-Atom Simulations.

The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3'-splice-site (3'SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3'SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing.