Investigating the molecular mechanism of h3b‐8800: A splicing modulator inducing preferential lethality in spliceosome‐mutant cancers

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre‐mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B‐8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B‐8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome‐mutant cancer cells remains elusive. Here, microsecond‐long all‐atom simulations addressed the binding/dissociation mechanism of H3B‐8800 to wild type and K700E SF3B1‐containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B‐8800 binding. This supports the hypothesis that the selectivity of H3B‐8800 towards mutant cancer cells is unrelated to its preferential targeting ofK700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B‐8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug‐discovery efforts to specifically target mutant SF3b

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\u2018SS). 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\u2018SS 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 moleculardynamics 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

Molecular basis for functional diversity among microbial Nep1-like proteins

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from \u3bcs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between nontoxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens

Establishing the catalytic and regulatory mechanism of RNA-based machineries

Ribonucleoprotein (RNP)-machineries are comprised of intricate networks of long noncoding RNAs and proteins that allow them to actively participate in transcription, RNA processing, and translation. RNP-machineries thus play vital roles in gene expression and regulation. Recent advances in cryo-EM techniques provided a wealth of near-atomic-level resolution structures setting the basis for understanding how these fascinating multiscale complexes exert their diverse roles. However, these structures represent only isolated snapshots of the plastic and highly dynamic RNP-machineries and are thus insufficient to comprehensively assess their multifaceted mechanisms. In this review, we discuss the role and merit of all-atom simulations in disentangling the mechanism of eukaryotic RNA-based machineries responsible for RNA processing. We showcase how all-atom simulations can capture their large-scale functional movements, trace the signaling pathways that are at the root of their massive conformational remodeling, explain recognition mechanisms of specific RNA sequences, and, lastly, unravel the chemical mechanisms underlying the formation of functional RNA strands. Finally, we review the methodological pitfalls and outline future challenges in modeling key functional aspects of these large molecular engines with all-atom simulations. In addition to providing insights into the most basic processes that govern all forms of life, in-depth mechanistic comprehension of RNP-machineries offers a foundation for developing innovative therapeutic strategies against the variety of human diseases linked to deregulated RNA metabolism. This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics

An Expanded Two-Zn2+-Ion Motif Orchestrates Pre-mRNA Maturation in the 3′-End Processing Endonuclease Machinery

Eukaryotic precursor-messenger RNAs (pre-mRNAs) undergo extensive compositional pre-processing to become mature, protein-coding mRNAs. Among the vital transformations underlying pre-mRNA maturation, the 3′-end processing machinery (3EPM) orchestrates 3′-end pre-mRNA cleavage via a yet elusive catalytic mechanism. Here, all-atom simulations of a 350,000 atom model of 3EPM disclose that its catalytic engine, the CPSF73 endonuclease, cleaves the 3′-end of pre-mRNA via an associative two-Zn2+-ion-aided mechanism, where the metals, besides activating the nucleophile and stabilizing the transition state, as in canonical two-Mg2+-ion catalysis, assist the leaving group's protonation. In spite of the distinctive metal type content, an in depth structural and mechanistic inspection of two-Zn2+-ion- versus two-Mg2+-ion-dependent nucleases unlocks striking similarities between the expanded positive charge of their catalytic motifs, with the metals being assisted by second-shell basic residues/metal ions. This catalytic architecture, hence, emerges as a critical prerequisite for a common and effective mechanism of phosphodiester bond hydrolysis in nuclease enzymes. Ostensibly, our outcomes unveil the salient molecular traits of the 3EPM mechanism, providing tantalizing opportunities in harnessing this emerging drug target to fight the wide variety of human diseases associated with a deregulated pre-mRNA processing

Molecular Basis of SARS-CoV-2 Nsp1-Induced Immune Translational Shutdown as Revealed by All-Atom Simulations

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents the most severe global health crisis in modern human history. One of the major SARS-CoV-2 virulence factors is nonstructural protein 1 (Nsp1), which, outcompeting with the binding of host mRNA to the human ribosome, triggers a translation shutdown of the host immune system. Here, microsecond-long all-atom simulations of the C-terminal portion of the SARS-CoV-2/SARS-CoV Nsp1 in complex with the 40S ribosome disclose that SARS-CoV-2 Nsp1 has evolved from its SARS-CoV ortholog to more effectively hijack the ribosome by undergoing a critical switch of Q/E158 and E/Q159 residues that perfects Nsp1's interactions with the ribosome. Our outcomes offer a basis for understanding the sophisticated mechanisms underlying SARS-CoV-2 diversion and exploitation of human cell components to its deadly purposes

Molecular Basis of SARS-CoV-2 Nsp1-Induced Immune Translational Shutdown as Revealed by All-Atom Simulations

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents the most severe global health crisis in modern human history. One of the major SARS-CoV-2 virulence factors is nonstructural protein 1 (Nsp1), which, outcompeting with the binding of host mRNA to the human ribosome, triggers a translation shutdown of the host immune system. Here, microsecond-long all-atom simulations of the C-terminal portion of the SARS-CoV-2/SARS-CoV Nsp1 in complex with the 40S ribosome disclose that SARS-CoV-2 Nsp1 has evolved from its SARS-CoV ortholog to more effectively hijack the ribosome by undergoing a critical switch of Q/E158 and E/Q159 residues that perfects Nsp1's interactions with the ribosome. Our outcomes offer a basis for understanding the sophisticated mechanisms underlying SARS-CoV-2 diversion and exploitation of human cell components to its deadly purposes

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

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 \u3bcs-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

Investigating the molecular mechanism of h3b‐8800: A splicing modulator inducing preferential lethality in spliceosome‐mutant cancers

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre‐mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B‐8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B‐8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome‐mutant cancer cells remains elusive. Here, microsecond‐long all‐atom simulations addressed the binding/dissociation mechanism of H3B‐8800 to wild type and K700E SF3B1‐containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B‐8800 binding. This supports the hypothesis that the selectivity of H3B‐8800 towards mutant cancer cells is unrelated to its preferential targeting ofK700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B‐8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug‐discovery efforts to specifically target mutant SF3b