I04-Macromolecular Crystallography
|
Abhishek
Sirohiwal
,
Juliane
John
,
Yury
Kutin
,
Rohit
Kumar
,
Federico
Baserga
,
Vivek
Srinivas
,
Hugo
Lebrette
,
Maximilian C.
Pöverlein
,
Ana P.
Gamiz-Hernandez
,
Joachim
Heberle
,
Müge
Kasanmascheff
,
Martin
Högbom
,
Ville R. I.
Kaila
Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.
|
May 2026
|
|
I04-Macromolecular Crystallography
|
Philipp
Münick
,
Dimitrios-Ilias
Balourdas
,
Julianne S.
Funk
,
Büşra
Yüksel
,
Danai
Mavridi
,
Justin
Heftel
,
Birgit
Dreier
,
Jonas V.
Schaefer
,
Birgit
Schäfer
,
Stefan
Knapp
,
Tümay
Telatar
,
Baki
Akgül
,
Andreas
Plückthun
,
Thorsten
Stiewe
,
Andreas C.
Joerger
,
Volker
Dötsch
Abstract: The tumor suppressor p53 is the most frequently mutated protein in tumors and a target for drug development. More than 2000 cancer-associated p53 missense mutations have been reported, most of them located in the DNA-binding domain (DBD). Due to the low intrinsic thermostability of the latter, they often lead to unfolding at physiological temperature. Stabilizing the DBD with small molecules has been shown to be effective in reactivating the cavity-creating cancer mutant Y220C. Unfortunately, the majority of p53 mutants seem to lack druggable binding pockets for small molecules. Here we show that a designed ankyrin repeat protein (DARPin) that binds to the p53 DBD stabilizes temperature-sensitive (TS) p53 cancer mutants, thereby compensating for mutation-induced loss of stability. We determined high-resolution crystal structures of multiple DARPin–mutant p53 complexes, providing mechanistic insights into this mode of stabilization. Reporter gene assays across a comprehensive panel of cancer-associated mutants revealed reactivation of the majority of TS mutants, whereas DNA-contact mutants and those with local misfolding of the DNA-binding surface remained inactive, as expected. We demonstrate that this reactivation induces the transcription of canonical p53 target genes and elicits antiproliferative effects in cancer cell lines. A combination of this DARPin with an mRNA/lipid nanoparticle-based transfection approach may have the potential to reactivate most TS p53 mutants and resensitize cancer cells to chemotherapy.
|
May 2026
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[24948, 32736]
Open Access
Abstract: Iron is an essential element that can be growth-limiting in microbial communities, particularly those present within host organisms. To acquire iron, many bacteria secrete siderophores, secondary metabolites that chelate ferric iron. These iron chelates can be transported back into the cell via TonB-dependent transporters in the outer membrane, followed by intracellular liberation of the iron. Pathogenic Escherichia coli and Salmonella produce siderophores during gut infection. In response to iron starvation, the human gut symbiont Bacteroides thetaiotaomicron upregulates an iron piracy system, XusABC, which steals iron-bound siderophores from the invading pathogens. Here, we investigated the molecular details of xenosiderophore uptake across the outer membrane by the XusAB complex. Our crystal and cryogenic electron microscopy structures explain how the XusB lipoprotein recognizes iron-bound xenosiderophores and passes them on to the XusA TonB-dependent transporter. Moreover, we show that Xus homologues can transport a variety of siderophores with different iron-chelating functional groups.
|
May 2026
|
|
Krios I-Titan Krios I at Diamond
|
Merijn B. L.
Vriends
,
Elisha
Moran
,
Martín
Calvelo
,
Thomas
Hansen
,
Isabelle B.
Pickles
,
Xincheng
Xin
,
Marieke
Biezeno
,
Zachary W. B.
Armstrong
,
Maria J.
Ferraz
,
Lei
Li
,
Alice
Lilley
,
Ruth
Harvey
,
Dmitri V.
Filippov
,
Qinghua
Liao
,
Sybrin P.
Schröder
,
Gijsbert A.
Van Der Marel
,
Marta
Artola
,
Johannes M. F. G.
Aerts
,
James N.
Blaza
,
Jeroen D. C.
Codée
,
Carme
Rovira
,
Herman S.
Overkleeft
,
Gideon J.
Davies
Diamond Proposal Number(s):
[28576]
Open Access
Abstract: Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an N-acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a 4H5 half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.
|
Mar 2026
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[36363]
Open Access
Abstract: The human long noncoding RNA (lncRNA) RMRP, initially identified as part of the RNase MRP complex, is linked to various human diseases. However, its structural flexibility and broader cellular roles are not well understood. Here, we offer a comprehensive analysis of RMRP’s structure in solution, its interactions with human proteins, and its mitochondrial functions. Using small-angle X-ray scattering (SAXS), we show that RMRP adopts different Mg2+-dependent shapes, shifting from an extended Y-shaped form to a more compact one as Mg2+ levels increase. We identified and characterized interactions between RMRP and the DEAD-box RNA helicases DDX5 and DDX3X, with DDX5 binding strongly and exhibiting ATP-dependent helicase activity on RMRP, while DDX3X mainly acts as an expression regulator. Both helicases are crucial for the proper mitochondrial localization of RMRP, working within a complex regulatory network. Functionally, reducing RMRP levels disrupts mitochondrial stability, leading to membrane depolarization and an increase in reactive oxygen species, without affecting cell growth. Mechanistically, RMRP specifically controls nuclear-encoded mitochondrial proteins involved in cristae structure (DNAJC11) and respiratory chain function (NDUFS8). Our results position RMRP as a structurally adaptable lncRNA that collaborates with RNA helicases to preserve mitochondrial health through specific gene regulation. These insights provide perspectives on RMRP’s biology and the molecular mechanisms underlying RMRP-related disorders, which could inform future therapies for conditions resulting from RMRP dysfunction.
|
Feb 2026
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[15916, 21426]
Open Access
Abstract: Most prokaryotes divide using filaments of the tubulin-like FtsZ protein, while some archaea employ instead ESCRT-III-like proteins and their filaments for cell division and cytokinesis. The alternative archaeal system comprises Cdv proteins and is thought to bear some resemblance to ESCRT-III-based membrane remodeling in other domains of life, including eukaryotes, especially during abscission. Here, we present biochemical, crystallographic, and cryo-EM studies of the Sulfolobus Cdv machinery. CdvA, an early non-ESCRT component, adopts a PRC‐domain/coiled-coil fold and polymerizes into long double-stranded helical filaments, mainly via hydrophobic interfaces. Monomeric CdvB adopts the canonical ESCRT-III fold in both a closed and a distinct “semiopen” conformation. Soluble CdvB2 filaments are composed of subunits in the closed state, appearing to transition to the open, active state only when polymerized on membranes. Short N-terminal amphipathic helices in all CdvB paralogues, B, B1, and B2, mediate membrane binding and are required for liposome recruitment in vitro. We provide a molecular overview of archaeal ESCRT-III-based cytokinesis machinery, the definitive demonstration that CdvB proteins are bona fide ESCRT-III homologues, and reveal the molecular basis for membrane engagement. Thus, we illuminate conserved principles of ESCRT-mediated membrane remodeling and extend them to an anciently diverged archaeal lineage.
|
Jan 2026
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[37115]
Open Access
Abstract: α7 nicotinic receptors are neurotransmitter-gated ion channels involved in neurological and inflammatory diseases. Ligands acting on its neurotransmitter binding site and on the channel domain of α7 have been extensively developed, yielding a wide range of orthosteric effectors and allosteric positive modulators. Here, we present the functional and structural characterization of two camelid antibody fragments, or nanobodies, F1 and E6, that inhibit α7 activity by acting as negative allosteric modulators, an underrepresented class of ligands. Cryo-EM structures of the nanobodies in complex with α7 show that both nanobodies form a pentameric bundle at the apex of the receptor, each nanobody interacting through a conserved set of residues at α7 subunit interfaces. Electrophysiological experiments suggest that E6 inhibits the activity of α7 by stabilizing its resting conformation, and that internanobodies interactions are key to its activity. Those two nanobodies expand the toolbox for human α7 modulation, opening new possibilities for its pharmacological control with far reaching potentialities in clinics.
|
Jan 2026
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[26835]
Abstract: Pyruvate kinase (PK) is a crucial glycolytic protein involved in vital cellular processes ranging from cell proliferation to immune responses. The activity and functions of PK are tightly regulated by diverse mechanisms, including posttranslational Nϵ-lysine acetylation. Although previous studies have explored the impact of acetylation on selected lysine residues within the M2 isoform of PK (PKM2), a more comprehensive selection of acetylation sites and their respective effects on both PKM2 and the highly homologous PKM1 isoform is lacking. Here, we describe the structural, functional, and regulatory effects of site-specific acetylation on an expanded set of conserved lysines in PKM2 and selected lysines in PKM1. To study homogeneously acetylated proteins, we genetically encoded the incorporation of acetylated lysine into PKM variants expressed in bacteria and cultured mammalian cells. Our integrated biochemical, structural, and computational approach revealed K115 acetylation as an inhibitory modification in both PKM1 and PKM2 that stabilizes a closed active site conformation of the proteins. We also show that, in contrast to K115 acetylation, previously reported acetylation of K305 inhibits PKM2 but has no effect on the activity and oligomerization of PKM1. These findings propose the existence of both uniform and isoform-specific regulatory mechanisms of PKM, mediated by acetylation.
|
Dec 2025
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[36390]
Open Access
Abstract: RAD51AP1 is an emergent key factor in homologous recombination (HR), the major pathway for accurate repair of DNA double-strand breaks, and in alternative lengthening of telomeres (ALT). Depletion of RAD51AP1 diminishes HR and overexpression is common in cancer, where it is associated with malignancy. Here, we show that RAD51AP1 has a hitherto unknown role in modulating the RAD51 recombinase, the central player in HR. Through a combination of biochemistry and structural biology, we reveal that RAD51AP1 possesses at least three RAD51-binding sites that facilitate its binding across two adjacent RAD51 molecules. We uncover a previously unidentified RAD51-binding mode that stabilizes the RAD51 N-terminal domain and protomer interface in the filaments. We uncover a previously undescribed role for RAD51AP1 in stabilizing RAD51-ssDNA filaments and promoting strand exchange. Our structural data provide the molecular basis for how RAD51AP1 binding induces conformational changes that promote RAD51 DNA association and oligomerization, therefore promoting filament nucleation, stabilization, and strand exchange. Further, we resolved structures of RAD51-ssDNA filaments in the presence of Mg2+-ATP and upon hydrolysis to Mg2+-ADP, revealing that RAD51 filaments expand upon ATP hydrolysis and explaining how ADP reduces RAD51–DNA binding. Our findings reveal RAD51AP1 as a versatile RAD51 modulator and RAD51 filament remodeler and shed previously unidentified insights into the modulation of HR, which is critical for the maintenance of genome stability.
|
Dec 2025
|
|
I06-Nanoscience (XPEEM)
|
Gregg
Wildenberg
,
Kevin M.
Boergens
,
Lola
Lambert
,
Ruiyu
Li
,
Allison
Craig
,
Michael K. L.
Man
,
Amin
Moradi
,
Janek
Rieger
,
Hengli
Duan
,
Sarnjeet S.
Dhesi
,
Gabriel
Karras
,
Francesco
Maccherozzi
,
Keshav
Dani
,
Rudolf
Tromp
,
Sense Jan
Van Der Molen
,
Sarah B.
King
,
Narayanan
Kasthuri
Diamond Proposal Number(s):
[40333]
Open Access
Abstract: Photoemission electron microscopy (PEEM) offers a potential third modality for large-volume connectomics alongside transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We image osmium stained, ultrathin brain sections on gold coated silicon at synaptic resolution using commercial PEEMs. At coarser resolution, we demonstrate that ultraviolet laser illumination enables gigavoxel-per-second acquisition rates without thermal damage. PEEM combines TEM-like parallel detection with SEM-compatible solid supports into a potentially scalable and cost-effective approach for large-volume connectomes.
|
Nov 2025
|
|
