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Ghirlando, Rodolfo

Publications and source records attributed to Ghirlando, Rodolfo.

12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Asymmetric loading of TnsE regulates Tn7 targeting of DNA replication structures

Abstract Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a conserved site in the chromosome or replicating DNA structures in conjugal plasmids, ensuring the vertical and horizontal spread of the element. While the pathway targeting the attTn7 site in the bacterial chromosome has been extensively studied, the pathway targeting DNA replication structures remains poorly understood. We have used an integrative structural biology approach to elucidate how the Tn7-encoded protein TnsE recognizes replication sites. Using native mass spectrometry, we found that TnsE forms 1:1 and 2:1 (TnsE:DNA) complexes on 3′-recessed DNA, with gain-of-function TnsE variants favoring the formation of 2:1 complexes. Structural characterization confirms that two TnsE molecules bind to DNA with the C-terminal domain of the protein recognizing duplex DNA, leaving the N-terminal domain to impose DNA substrate specificity and recruit the core transposition machinery. Collectively, our work is consistent with a model where TnsE-mediated target-site selection relies on the formation of an asymmetric TnsE:DNA complex to recruit the Tn7 transposase to DNA replication structures.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (K dimer ) 2–3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ~10 to 30%, relative to that of the wild-type enzyme, without altering K dimer . Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MPro WT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MPro WT -ensitrelvir. In conclusion, chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Structural basis of tRNA recognition by the widespread OB fold

The widespread oligonucleotide/oligosaccharide-binding (OB)-fold recognizes diverse substrates from sugars to nucleic acids and proteins, and plays key roles in genome maintenance, transcription, translation, and tRNA metabolism. OB-containing bacterial Trbp and yeast Arc1p proteins are thought to recognize the tRNA elbow or anticodon regions. Here we report a 2.6 Å co-crystal structure of Aquifex aeolicus Trbp111 bound to tRNA Ile , which reveals that Trbp recognizes tRNAs solely by capturing their 3' ends. Structural, mutational, and biophysical analyses show that the Trbp/EMAPII-like OB fold precisely recognizes the single-stranded structure, 3' terminal location, and specific sequence of the 3' CA dinucleotide — a universal feature of mature tRNAs. Arc1p supplements its OB – tRNA 3' end interaction with additional contacts that involve an adjacent basic region and the tRNA body. This study uncovers a previously unrecognized mode of tRNA recognition by an ancient protein fold, and provides insights into protein-mediated tRNA aminoacylation, folding, localization, trafficking, and piracy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Here, through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 3 10 -helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MPro WT ) from precursor polyproteins. Upon expression, a model precursor of MPro WT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MPro M ) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MPro WT . MPro M and the catalytic domain (MPro 1-199 ) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MPro M and MPro 1-199 , respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor

We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T m ) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [ (-6) MPro H41A and (-6*) MPro H41A , respectively], and its corresponding mature MPro H41A were systematically examined. While the H41A mutation exerts negligible effect on T m and dimer dissociation constant (K dimer ) of MPro H41A , relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in T m and > 85-fold increase in K dimer as compared to MPro H41A . The K d for the binding of the covalent inhibitor GC373 to (-6*) MPro H41A increases ~12-fold, relative to MPro H41A , concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers’ oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Finally, disorder of the terminal residues 1–2 and 302–306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Autoprocessing and oxyanion loop reorganization upon GC373 and nirmatrelvir binding of monomeric SARS-CoV-2 main protease catalytic domain

The monomeric catalytic domain (residues 1–199) of SARS-CoV-2 main protease (MPro 1-199 ) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro 1-199 junction. We report the catalytic activity and the dissociation constants of MPro 1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MPro WT and MPro 1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, in this study, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition

Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.

59 BASIC BIOLOGICAL SCIENCES↗