Biology subjects

Herrero del Valle, A.

Publications and source records attributed to Herrero del Valle, A..

7 recordsLinked to original sources

bioRxiv2025

Structural basis for Lamassu-based antiviral immunity and its evolution from DNA repair machinery

Bacterial immune systems exhibit remarkable diversity and modularity, as a consequence of the continuous selective pressures imposed by phage predation. Despite recent mechanistic advances, the evolutionary origins of many antiphage immune systems remain elusive, especially for those that encode homologs of the Structural Maintenance of Chromosomes (SMC) superfamily, which are essential for chromosome maintenance and DNA repair across domains of life. Here, we elucidate the structural basis and evolutionary emergence of Lamassu, a bacterial immune system family featuring diverse effectors but a core conserved SMC-like sensor. Using cryo-EM, we determined structures of the Vibrio cholerae Lamassu complex in both apo- and dsDNA-bound states, revealing unexpected stoichiometry and topological architectures. We further demonstrate how Lamassu specifically senses dsDNA in vitro and phage replication origins in vivo, thereby triggering the formation of LmuA tetramers that activate the Cap4 nuclease domain. Our findings reveal that Lamassu evolved via exaptation of the bacterial Rad50-Mre11 DNA repair system to form a compact, modular sensor for viral replication, exemplifying how cellular machinery can be co-opted for novel immune functions.

microbiology↗

bioRxiv2024

Molecular basis of autoimmune disease protection by MDA5 variants

MDA5 recognizes dsRNA from viruses and retroelements. Cooperative filament formation and ATP-dependent proofreading confer MDA5 with the necessary sensitivity and specificity for dsRNA. The gene encoding MDA5 is a hotspot for disease-associated variants. Many MDA5 variants are associated with protection from autoimmune disease, while increasing the risk of infection and chronic inflammation, but how these variants affect MDA5-dependent RNA sensing remains unclear. Here, we determine the consequences of autoimmune-protective MDA5 variants on the molecular structure and activities of MDA5. The rare variants E627* and I923V reduce the cellular interferon response to picornavirus infection and are deficient in filament formation. The I923V variant is ATPase hyperactive, causing premature dissociation from dsRNA. Cryo-EM structures of MDA5 I923V bound to dsRNA at different stages of ATP hydrolysis reveal smaller RNA binding interfaces, leading to excessive proofreading activity. Variants R843H and T946A, which are genetically linked and cause mild phenotypes, have no effect on dsRNA recognition, suggesting an indirect disease mechanism. We conclude that the autoimmune-protective MDA5 variants lead to a loss of MDA5-dependent signaling via multiple distinct mechanisms.

biochemistry↗

bioRxiv2024

An alternative mechanism for activation of innate immune signaling by MDA5

Long double-stranded (ds) RNA in the cytosol acts as a potent inflammatory molecule recognized by the receptor MDA5, triggering the innate immune response. Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly-disassembly dynamic is proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA via single-stranded (ss) RNA loop extrusion, even against a significant opposing force. This compacted state is further stabilized by oligomerization of MDA5s caspase recruitment domain (CARD) and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5 mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.

biophysics↗

bioRxiv2023

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing

Cytosolic long double-stranded RNA (dsRNA), among the most potent proinflammatory signals, is recognized by MDA5. MDA5 binds dsRNA cooperatively, forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNAs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-{beta} responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. This amplifies the RNA footprint expansion that accompanies each round of ATP hydrolysis and allows MDA5 to displace tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling of the MDA5-LGP2 interface suggests that MDA5 interacts with dsRNA stem-bound rather than end-bound LGP2. We conclude that NTPase-dependent binding of LGP2 to internal sites on dsRNA increases the number and signaling output of MDA5-dsRNA complexes. Our work identifies novel molecular mechanisms contributing the selectivity and sensitivity of cytosolic dsRNA sensing. KEY POINTSO_LICooperative ATP hydrolysis in MDA5 filaments confers selectivity for dsRNA and displaces other proteins from RNA C_LIO_LINoncooperative NTP hydrolysis by LGP2 induces binding to internal RNA sites with low selectivity C_LIO_LIRNA stem-bound LGP2 nucleates assembly of MDA5 signaling complexes on a broader set of RNA ligands C_LI

biochemistry↗

bioRxiv2021

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

molecular biology↗

bioRxiv2021

MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

biochemistry↗

bioRxiv2019

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1-3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in {gamma}-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

microbiology↗