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Luuk Loeff

@lloeff.bsky.social
1.4K followers 259 following 150 posts

Principal Investigator at Leiden University Medical Center Host-Defence | Structural biology | Single-molecule Biophysics www.loefflab.com

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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Together, our data support a model in which DruH processes replication-associated forked DNA structures to generate 5′ overhangs that trigger activation of the autoinhibited helicase–nuclease DruE, enabling selective DNA degradation during phage defense.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
DruE loads onto the 3′ strand, raising the question of how DruH exonuclease activity promotes DruE activation. Unwinding assays show that DruE initiates on substrates with 5′ overhangs, while blocking the 3′ strand with streptavidin prevents helicase loading and strongly inhibits activity.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
The structure of the holo-complex reveals that the C-terminal domain of DruH directly engages DruE and promotes loading of monomeric DruE onto the 3′ overhang.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Next, we used pulldown assays to identify the preferred DNA substrate of Druantia and determined the structure of the DruE–DruH holo-complex. We find that forked DNA substrates promote assembly of a DNA-bound complex between DruH and monomeric DruE, forming a 1:1 complex.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Next, we determined the structure of DruE, a large dimeric helicase–nuclease. The structure reveals an autoinhibited assembly, while biochemical reconstitution and phage assays show that DruE couples ATP-dependent DNA unwinding to nuclease-mediated DNA degradation both essential for immunity.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Given the predicted helicase activity of druE, we hypothesized that Type III Druantia directly targets phage-derived nucleic acids. Guided by AF3 predictions and biochemical assays, we find that DruH functions as a 3′-5′ exonuclease that processes DNA substrates containing exposed 3′ termini.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
To understand the function of DruH, we solved its structure using cryo-EM. DruH is a multidomain protein containing α-helical N- and C-terminal domains connected by six central Ig-like domains.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
We find that Druantia both DruE and DruH are essential for immunity. Interestingly, infected cells continue to grow during phage challenge, indicating that Type III Druantia does not function through an abortive infection mechanism.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Type III Druantia is a two-component defense system consisting of the signature gene druE, a predicted helicase, and druH a large accessory protein of unknown function. To understand the mechanism underlying Druantia immunity, we focused on the Type III system from Pseudomonas aeruginosa.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Together, our data support a model in which DruH processes replication-associated forked DNA structures to generate 5′ overhangs that trigger activation of the autoinhibited helicase–nuclease DruE, enabling selective DNA degradation during phage defense.
100
Luuk Loeff @lloeff.bsky.social · 14/05/2026
The holo-complex shows that DruE loads onto the 3′ strand. Unwinding assays reveal that DruE initiates unwinding on substrates containing 5′ overhangs, while blocking the 3′ strand near the junction with streptavidin prevents productive loading into the helicase and strongly inhibits activity.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Next, we used pulldown assays to identify the preferred DNA substrate of Druantia and determined the structure of the DruE–DruH holo-complex. We find that forked DNA substrates promote assembly of a DNA-bound complex between DruH and monomeric DruE, forming a 1:1 complex.
110
Luuk Loeff @lloeff.bsky.social · 14/05/2026
Next, we determined the structure of DruE, a large dimeric helicase–nuclease. The structure reveals an autoinhibited assembly, while biochemical reconstitution and phage assays show that DruE couples ATP-dependent DNA unwinding to nuclease-mediated DNA degradation both essential for immunity.
100
Luuk Loeff @lloeff.bsky.social · 14/05/2026
Given the predicted helicase activity of druE, we hypothesized that Type III Druantia directly targets phage-derived nucleic acids. Guided by AF3 predictions and biochemical assays, we find that DruH functions as a 3′-5′ exonuclease that processes DNA substrates containing exposed 3′ termini.
100
Luuk Loeff @lloeff.bsky.social · 14/05/2026
To understand the function of DruH, we solved its structure using cryo-EM. DruH is a multidomain protein containing α-helical N- and C-terminal domains connected by six central Ig-like domains.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
We find that Druantia both DruE and DruH are essential for immunity. Interestingly, infected cells continue to grow during phage challenge, indicating that Type III Druantia does not function through an abortive infection mechanism.
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Luuk Loeff @lloeff.bsky.social · 14/05/2026
Type III Druantia is a two-component defense system consisting of the signature gene druE, a predicted helicase, and druH a large accessory protein of unknown function. To understand the mechanism underlying Druantia immunity, we focused on the Type III system from Pseudomonas aeruginosa.
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Luuk Loeff @lloeff.bsky.social · 06/06/2025
Had a great day at @ebi.embl.org Grenoble for the Next-Gen in Infection Biology micro symposium! Big thanks to @kowaeva.bsky.social for the invite!
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
In sum, we combined single-particle cryo-EM with biochemical experiments to reveal the mechanistic basis for the protective function of PYROXD1. Our results further underscore the notion of co-evolution of PYROXD1 with RTCB as a mechanism to enable the tRNA-LC to function under aerobic conditions.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
The initial step of RTCB-catalyzed RNA ligation involves Archease mediated guanylylation of the RTCB catalytic center. We performed mass spectrometry and show that PYROXD1 selectively interacts with the apo-enzyme form of RTCB, while guanylylated RTCB remains active under oxidative stress.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
Superposition of the RTCB–PYROXD1 complex structure with the crystal structure of NAD(P)H-free PYROXD1 reveals structural rearrangements in PYROXD1 induced by NADH binding and RTCB recruitment.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
The structure of the RTCB-PYROXD1 complex reveals that the PYROXD1 CTD engages with the catalytic center cleft of RTCB, thereby blocking the substrate binding sites and rendering the catalytic center inaccessible. Mutation of the interacting residues resulted in a loss of protection.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
The structure shows clear density of both the nicotinamide and flavin dinucleotides within the catalytic center of PYROXD1 that are required for complex formation.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
To uncover the mechanism of oxidative stress protection by PYROXD1 we used cryo-EM to obtain a structure of the RTCB-PYROXD1 complex.
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Luuk Loeff @lloeff.bsky.social · 11/03/2025
The human tRNA ligase RTCB contains an active site that contains a highly reactive cystine group that is sensitive to oxidative inactivation.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Our data suggests that Shedu specifically targets free DNA ends, providing self vs non-self-recognition. Our findings underline that recognition of pathogen-specific nucleic acid structures is a conserved feature of innate immunity across all domains of life.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Next, we generated gene edited phages to carrying the most frequent mutations (shoutout to @benadler.bsky.social from @doudna-lab.bsky.social). These experiments show that loss UvsW is responsible for the escape phenotype.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Lastly, we isolated phages that escape the Shedu immune system. All the escape mutants carry mutations in UvsW a helicase that is involved in the recombination-dependent DNA replication pathway of T-even phages, a process that relies on the generation of free DNA ends.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
To validate DNA-end targeting in vivo, we transformed cells with linear plasmid DNA fragments carrying short homology arms. Cells expressing SduA were 10x less efficient in assembling the fragments into a functional plasmid compared to the control.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
The end-bound DNA is projected over the active site of protomer SduA.2 and protrudes towards the active site of SduA.4. This positions the phosphate backbone of nucleotide 14 towards the active site, and results in cleavage at a fixed distance from the 5’-end.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
The structure shows that Shedu binds dsDNA ends, using its N-terminal clamp. To be able to fit the DNA in the clamp in the unwound, while base pairing is retained.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
But how does Shedu provide phage defense and how does it discriminate self from non-self? 🤔 After exhausting many options, the answer came from a cryo-EM structure of Shedu bound to a dsDNA substrate.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
DUF4263 adopts a PD(D/E)XK like fold consistent with its predicted nuclease function. The conserved residues that coordinate a magnesium ion point towards its active site. Mutational analysis shows that these residues are essential for both phage defense and DNA cleavage.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Given the putative nuclease function of Shedu, we performed cleavage assays on oligos and observed that SduA is a dsDNA nuclease.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Each protomer is composed of two domains connected by an alpha-helical linker. In absence of the N-term domain, DUF4263 elicits toxicity in cells. This indicates that the N-term domain regulates the activity of DUF4263.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
To understand the defense mechanism of Shedu we purified SduA from E.coli and determined its structure using cryo-EM. The structure shows SduA forms a tetrameric complex through its DUF4263 domain, while the N-terminal domains form clamp like structures on each end.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
We tested several orthologs of SduA for phage defense and could show robust phage defense in E. coli. Infection assays in liquid culture show that Shedu uses a mechanism that is distinct from abortive infection, suggesting that Shedu can distinguish self from non-self.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
Shedu systems are characterized by the presence of a DUF4263 domain. By performing phylogenetic analysis, we found that SduA can be grouped into two clusters (Type I & II) based on their size and position of the signature domain.
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Luuk Loeff @lloeff.bsky.social · 31/12/2024
What a great way to end the year! ✨ Today in @cellpress.bsky.social we report the structure and function of the Shedu anti-phage defense system. tinyurl.com/4crj6dnx A long 🧵...
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Luuk Loeff @lloeff.bsky.social · 12/11/2024
To fill up my empty feed, my first post on #bluesky. Earlier this year we showed how the DdmDE system from pandemic Vibrio cholerae defends against invading plasmids. A great collaboration between the labs of Martin Jinek (UZH) and @mblokesch.bsky.social (EPFL). www.science.org/doi/10.1126/...
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