Abstract EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency has been established as the cause of Kleefstra syndrome 1, the pathogenesis of G9a dysfunction in human disease remains largely unknown. We identified seven de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome 1. In vitro studies reveal that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant exhibit growth retardation, facial/skull dysmorphia and aberrant behavior. Here we report pathogenic EHMT2 variants that likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency via a distinct molecular mechanism, defining an autosomal dominant EHMT2 -related Kleefstra syndrome.

Loading of calcium ions into the numerous carboxy-proximal binding sites in the repeats-in-toxin (RTX) domains drives the cooperative and vectorial folding of RTX β-roll structures involved in cell binding and membrane penetration of RTX cytolysins. Two additional binding sites for calcium ions, coordinated by the side chains of residues D880, D918, and N936, were identified in the structure of the acylated cap of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA). We show that this calcium-binding structure plays a key role in membrane insertion of the toxin. An N936L residue substitution did not impact toxin acylation or CR3 receptor binding but disrupted the calcium-driven folding of the acylated segment and ablated the membrane penetration capacity of the toxin. Similarly, substitution of the corresponding D639 residue of Escherichia coli α-hemolysin abolished its cytolytic capacity. Moreover, disruption of the β-turn structures in the calcium-binding sites of the acylated segment of CyaA (G934L) and α-hemolysin (G637L) strongly impaired the cytotoxic capacities of both toxins. On the contrary, a D880L substitution yielded a CyaA toxin with an enhanced CR3-independent cell penetration and pore-forming capacity. Hydrogen/deuterium exchange probing revealed that the D880L substitution altered the fold of the acylated segment and the interaction of its two acylated β-hairpins. Hence, loading the calcium-binding sites in the acylated segment controls the structure and rules the interaction and the functional cooperation of the two acylated β-hairpins that facilitate penetration of the CyaA polypeptide into the cell membrane.

Human NQO1 is a homodimeric flavoenzyme essential for the redox metabolism of many substances and implicated in major global health challenges such as cancer and Alzheimer's disease. X-ray crystallographic studies have identified several residues within its substrate binding site (including Tyr126 and Tyr128) that may regulate catalytic competent binding of substrates, cofactor redox properties, half-site reactivity, and/or functional inter-active site negative cooperativity. To elucidate the functional role of Tyr126 and Tyr128, we generated point mutants at these positions and assessed their dynamics and kinetic properties. Hydrogen-deuterium exchange coupled to mass spectrometry revealed that non-conservative mutations, particularly at Tyr126, notably disrupted dynamics not only within the substrate binding site but also in structural elements connecting the two active sites of the NQO1 homodimer. Rapid-mixing pre-steady-state kinetics experiments of the reduction of NQO1 by NAD(P)H showed that mutations to Phe caused a mild decrease in hydride transfer (HT) efficiency from the coenzyme to the FAD cofactor. In contrast, mutations to Ala resulted in a significantly greater impact and mutations to Glu nearly abolished HT. Despite these effects, some mutations moderately affected the non-synchronous catalysis between the two alternating active sites, but hardly produced an impact on the selectivity for NADPH versus NADH as hydride donor coenzymes. However, all variants exhibited markedly impaired enzyme turnover, highlighting alterations in the enzyme's substrate specificity toward quinones. The data presented here demonstrate that Tyr126 and Tyr128 optimize both substrate binding geometry as well as overall enzyme conformational dynamics during the asymmetric catalytic cycle of the NQO1 homodimer.

The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl‒/H+ stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl‒ ions to the extracellular side, opening of the inner gate, and water wires that facilitate H+ transport. Surprisingly, these water wires occurred independently of Cl‒ binding, prompting us to reassess the relationship between Cl‒ binding and Cl‒/H+ coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl‒ binding affinity, we conclude that, while Cl‒ binding is necessary for coupling, even weak binding can support Cl‒/H+ coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl‒/H+ exchange mechanism.

Proteins frequently undergo large-scale conformational excursions in their native ensemble. Such structural transitions are particularly critical for enabling access to binding sites when they are buried in the protein interior. Here, we map the conformational landscape of AlbAS, a natural isoform of the transcription factor AlbA from the gut microbe Klebsiella oxytoca, which sequesters the antibiotic albicidin in a solvent-inaccessible binding tunnel. Combining equilibrium, time-resolved experiments, structural mass spectrometry and calorimetry with statistical modeling, we show that AlbAS displays large differences in local and global stability and dynamics, with ∼600-fold difference in unfolding rates across different parts of the structure. Several residues lining the ligand-binding pocket and the inter-sub-domain residues rapidly exchange protons with the solvent in hydrogen-deuterium exchange mass spectrometry experiments, indicative of anisotropic distributions of local stabilities, with the N-terminal subdomain being less stable. The AlbAS conformational landscape is thus quite rugged, encompassing numerous partially structured states in equilibrium, including partial unlocking of the N-terminal subdomain at a time-constant of 6 ms that exposes the binding sites to aid in albicidin binding.

Misfolding diseases that result in loss of function represent a considerable burden for both individuals and society. Primary Hyperoxaluria Type 1 (PH1) is a rare genetic disorder caused by the deficit of alanine:glyoxylate aminotransferase (AGT), which leads to excessive endogenous oxalate formation translating into progressive renal damage due to stones formation. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (leading to enhanced degradation, aggregation and/or mitochondrial mistargeting). The main therapeutic approach is a double kidney and liver transplantation, but treatments based on RNA silencing are currently available, although they normalize urinary oxalate excretion only in about 50 % of patients, calling for the need of alternative options. In this work, we developed and characterized a novel biotechnological approach using six single-domain nanobodies (NB-AGT-1 to -6) as potential therapeutics counteracting PH1 misfolding. We show that NB-AGTs are very stable proteins and bind pathogenic and non-pathogenic variants of AGT with high affinities (Kd values from low nM to low pM). Structural studies indicate that NB-AGTs bind to different protein epitopes thus being selective for different variants. Preliminary experiments in a cellular model of PH1 show that internalization of engineered NB-AGT-3 enhances the specific activity of disease-associated variants. Overall, our results provide the basis to consider NBs as a promising approach for PH1 as premise for their application to other loss-of-function misfolding diseases.

Retinoic acid receptors (RARs) are ligand-dependent transcription factors essential for various biological processes, including embryogenesis, differentiation, and apoptosis. RARs function as heterodimers with retinoid X receptors (RXRs) and regulate gene expression via retinoic acid response elements (RAREs). Their transcriptional activity is modulated by coregulators, with corepressors maintaining repression in the absence of ligand and coactivators enabling transcription upon ligand binding. Structural studies reveal that DNA binding induces conformational changes affecting coregulator interactions. However, the precise structural organization of RAR/RXR-coregulator complexes and the allosteric influence of DNA on receptor function remain incompletely understood. Our study presents an integrative analysis of the RAR/RXR heterodimer bound to four distinct and relevant RAREs (DR0, DR1, DR5, and IR0) in complex with either a corepressor (NCoR) or a coactivator (TIF-2) nuclear receptor interaction domain. By combining small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we revealed that the heterodimer adopts distinct conformations depending on the DNA sequence, influencing interdomain distances and receptor interactions. Additionally, we uncovered the dynamic interplay between ligand, DNA, and coregulator binding. This study provides new insights into the structural features of coregulator proteins and highlights the allosteric influence of RAREs on receptor function.

EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency was established as the cause of Kleefstra syndrome twenty years ago, the pathogenesis of G9a dysfunction in human disease remains largely unknown. Here, we report clinical and molecular correlates of six de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome. In vitro studies revealed that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant ( Ehmt2 c.3385_3396del ) exhibited growth retardation, facial/skull dysmorphia and aberrant behavior. EHMT2 variants described here likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency causing Kleefstra syndrome via a distinct molecular mechanism. ### Competing Interest Statement B.S. is a shareholder in Episign, Inc. The other authors declare no conflicts of interest Ministry of Education, Youth and Sports of the Czech Republic, LX22NPO5107, LM2023067, LM2023042 Charles University, https://ror.org/024d6js02, UNCE/MED/007 Ministerio de Ciencia, Innovación y Universidades, https://ror.org/05r0vyz12, PID2021-128087OB-I00 Genome Canada, https://ror.org/029s29983, Genomics Applications Partnership Program Grant (OGI-188) European Union, https://ror.org/019w4f821, Marie Skłodowska-Curie grant agreement No 101003406, ERA fellowship (2D-TOPMASS, grant 101090276), CZ.02.1.01/0.0/0.0/18_046/0015974, CZ.02.01.01/00/23_020/0008540, 305444 Ministry of Health of the Czech Republic, NU23-07-00281, NW24-04-00067 Fundacion Inocente, FII2024-125

Human cis-prenyltransferase (hcis-PT) synthesizes long-chain isoprenoids essential for N-linked protein glycosylation. This heteromeric complex comprises the catalytic subunit DHDDS and the regulatory Nogo-B receptor (NgBR). Although NgBR dramatically enhances DHDDS activity, the molecular basis for this allosteric regulation remains unclear. Here, we combined crystallography, hydrogen-deuterium exchange mass spectrometry (HDX-MS), molecular dynamics simulations, and network analysis to uncover the structural dynamics and communication pathways within hcis-PT. By solving the apo structure of hcis-PT, we reveal only a localized flexibility at the active site and the NgBR C-terminus. However, HDX-MS demonstrated widespread substrate-induced stabilization, particularly at the NgBR βD-βE loop, highlighting it as an allosteric hub. Functional mutagenesis scanning identified NgBRS249 as critical for enzymatic activity, independent of structural perturbations. Network analysis of MD simulations pinpointed this residue as a central node in inter-subunit communication, with perturbations disrupting downstream allosteric pathways, altering enzymatic activity. Our findings reveal a dynamic regulatory network centered at the inter-subunit interface, wherein specific NgBR residues modulate DHDDS activity through allosteric signaling. This work elucidates a conserved mechanism of subunit coordination in long-chain cis-prenyltransferases and suggests avenues for therapeutic targeting of hcis-PT-related disorders.

Proteolysis is a crucial step in both bottom-up and structural proteomics workflows, directly influencing peptide identification and sequence coverage in mass spectrometry-based analyses. While classical proteomics typically relies on highly specific enzymes with well-defined cleavage patterns, structural MS approaches such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) often employ nonspecific or semispecific proteases, producing complex peptide mixtures that require more detailed digestion analysis. To address these needs and streamline the entire process, we developed DigDig, a standalone, Java-based software tool for evaluating and comparing proteolytic digestion across diverse experimental conditions. DigDig processes output files from common search engines and provides customizable visualizations of key digestion metrics, including sequence coverage, reproducibility, peptide redundancy, cleavage site preferences, and peptide length distributions. A distinguishing feature is its ability to detect and report repetitive peptide sequences, which are frequently missed by standard tools. We demonstrate its capabilities using data sets from both specific and nonspecific digestions, highlighting its utility in digestion quality control, protease characterization, and method development, particularly in HDX-MS workflows. DigDig is freely available at https://peterslab.org/DigDig/.

Misfolding diseases that result in loss of function represent a considerable burden for both individuals and society. Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by mutations in the alanine:glyoxylate aminotransferase 1 (AGT) enzyme. The underlying molecular mechanisms causing PH1 are associated with protein misfolding (enhanced aggregation and mitochondrial mistargeting). The main therapeutic approach to increase patients’ lifespan and quality of life is a double kidney and liver transplantation. Alternative treatments such as gene and enzyme replacement and pharmacological chaperones are currently being introduced, but other alternatives are necessary. In this work, we developed and characterized a novel biotechnological approach using six single-domain nanobodies (NB-AGT-1 to -6) as potential therapeutics for PH1 misfolding. We show that NB-AGTs are very stable proteins and bind to pathogenic and non-pathogenic variants of AGT with extreme affinities (with Kd values from low nM to low pM). Structural studies showed that NB-AGTs bind to different epitopes of AGT with selectivity for different AGT variants. Experiments in cellular PH1 models showed that internalization of engineered NB-AGT-3 enhanced the specific activity of disease-associated variants. Overall, we show that NBs are a novel and promising approach to treat PH1 and other loss-of-function misfolding diseases. ### Competing Interest Statement The authors have declared no competing interest.

Protein prenylation plays a critical role in regulating the cellular localization of small GTPases and is essential for multiple myeloma (MM) pathology. Geranylgeranyl diphosphate synthase (GGPPS), producing a key prenylation moiety, exists in a dimeric or hexameric form, depending on the species. However, the functional significance of this oligomerization remains unclear. Using crystallography, mass spectrometry, and fluorescence spectroscopy, we show that the GGPPSR235C mutant-found in the widely studied MM cell line RPMI-8226-exhibits weakened inter-dimer interactions, reduced hexamer stability, and increased apparent substrate affinity and product release kinetics. These effects are even more pronounced in a dimeric mutant, GGPPSY246D, demonstrating that interdimer interactions within the hexamer help stabilize a lid region over the active site, thereby stabilizing product binding in an inhibitory conformation. Together, these findings reveal that hexamerization regulates GGPPS activity through product inhibition and underscore the importance of cell line selection and characterization in drug discovery efforts.

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant, whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown.

The human cis-prenyltransferase complex (hcis-PT) plays a major role in protein N-glycosylation by synthesizing the precursor for the glycan carrier dolichol-phosphate. Expressed in all human cells, hcis-PT is a heterotetramer consisting of two heterodimers formed between the catalytic subunit dehydrodolichyl diphosphate synthase (DHDDS) and the regulatory subunit Nogo-B receptor (NgBR). Although NgBR has no endogenous cis-prenyltransferase activity, it accelerates the activity of DHDDS ∼400-fold.

Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1‐causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen‐deuterium exchange (HDX) to characterize the wild‐type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5‐phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

Voltage-gated proton channels (HV1) harbor four transmembrane helices and are devoid of a canonical ion-conductive pore, rendering them non-conventional voltage-gated cation channels. Their identification in unicellular species, such as the marine coccolithophore Emiliania huxleyi (EhHV1), has unraveled an intrinsic structural and functional diversity within this channel family. Indeed, sequence analysis of EhHV1 predicts a distinctive extracellular domain (ECD), which is absent from mammalian HV channels and enriched in histidine residues. Moreover, key histidine residues responsible for zinc-dependent regulation of human HV1 are not conserved in EhHV1. Here, we determined the contribution of the ECD of EhHV1 to zinc-mediated channel inhibition and voltage-dependent channel activity. Following detergent-mediated solubilization and purification of EhHV1 overexpressed in insect cells, size-exclusion chromatography-coupled multiangle light scattering and crosslinking analyses revealed that the purified (>99%) EhHV1 maintains a dimeric stoichiometry as their mammalian orthologs. Furthermore, the purified EhHV1 retains functionality as determined by a proton flux assay following proteoliposome reconstitution. To resolve the structural role of the ECD in EhHV1, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses. Intriguingly, incubation of EhHV1 with zinc resulted in significantly reduced deuterium uptake in the ECD, indicating its involvement in zinc binding. Notably, we also observed reduced deuterium exchange around R282 of the transmembrane S4 helix (R3), consistent with the notion that zinc binding prevents its intermittent exposure to the intracellular side of the membrane, thereby reducing channel opening probability. Moreover, voltage-clamp analyses showed that while the deletion of the ECD in EhHV1 renders the channel non-functional, mutating all ten ECD histidine residues (H163-206A) abolished zinc-mediated channel inhibition. Thus, we hypothesize that the EhHV1 ECD exhibits complex zinc-binding properties and serves as an allosteric site involved in the mobility of the S4 segment.