In eukaryotes, genomic DNA is packaged into hierarchical higher-order chromatin structures. Regulation of this organization is essential for the faithful maintenance and controlled expression of genetic information. Assembly of the nucleosome─the fundamental unit of chromatin─depends on a tightly coordinated network of histone chaperones that escort histones from their initial synthesis through multiple intermediates to their final deposition into nucleosomes. This review provides an overview of the stepwise journey of histones and the regulatory mechanisms that govern this process. We also highlight major outstanding questions and discuss future directions in the field.

The rapid emergence of multidrug-resistant (MDR) bacteria threatens the effectiveness of existing antibiotics and has created an urgent need for agents with novel mechanisms of action. Calcium-dependent antibiotics (CDAs), a structurally diverse group of nonribosomal cyclic lipopeptides, have gained attention for their potent activity against Gram-positive pathogens, including strains resistant to conventional therapies. Their hallmark feature is Ca2+-mediated activation: the calcium ion binding to the lipopeptide enables selective interactions with bacterial lipids or cell-wall precursors. This review examines the relationship between CDA structure, Ca2+ coordination, mechanistic diversity, and biological activity. Together, these features position CDAs as promising templates for next-generation therapeutics to combat the escalating MDR crisis.

Protein dynamics─the fluctuating nature of protein structure once described by Gregorio Weber as "kicking and screaming"─is understood to be an intrinsic feature of proteins and their function. Yet it is often difficult to pin down exactly how those dynamics assist function. Allosteric regulation is a widespread protein function that was once seen to operate solely through conformational change. Over the last two decades, a series of experimental studies has shown that thermally activated, rapid-time scale dynamics can underlie allosteric ligand binding cooperativity, even in the absence of conformational change. This concept is known as "dynamic allostery", in which localized dynamics represent conformational entropy that can effectively serve as a set of thermodynamic "nano-levers". Here, we review these studies and their collective finding: that changes in the amplitudes of picosecond-nanosecond timescale side-chain dynamics can exert a large entropic driving force in protein binding events. The studies require NMR relaxation measurements of methyl "order parameters" (O2axis). We focus on the recent example from Sgt2, a chaperone in yeast's guided tail-anchoring protein pathway. Sgt2 harbors an intrinsically disordered C-terminal tail that allosterically enhances side-chain dynamics in other domains, which in turn abrogates binding to partner Get4/5. Motivated by this example, order parameters are explained in simple terms and discussed empirically to raise confidence in them as meaningful reporters of local motion. Specific studies are highlighted to show that different proteins utilize distinct dynamic strategies for allosteric coupling. Finally, the surprising role of disordered tails in controlling dynamic allostery is discussed.

Lysyl oxidases (LOXs) initiate the posttranslational cross-linking of collagen by oxidizing lysine to allysine residues, a process crucial for the mechanical properties of the extracellular matrix. However, dysregulated LOX levels─particularly those of the LOXL2 isoform─have been implicated in numerous fibrotic diseases and cancers. Accordingly, considerable effort has been devoted to understanding the biological role of LOXL2. Despite this interest, little is known about how the type, structure, and neighboring groups of the amine influence LOXL2 activity. Here, we determined Michaelis-Menten kinetics for a panel of lysine-based model substrates to assess the structural determinants of LOXL2-catalyzed oxidation. We show that LOXL2 oxidizes exclusively primary, α-unbranched amines. In addition, our studies revealed that an additional positively charged group enhances LOXL2 activity.

Asparaginyl ligases are powerful tools for peptide and protein engineering due to their ability to efficiently catalyze a variety of site-specific transpeptidation reactions. Although engineering efforts have enhanced the transpeptidation efficiency of several enzymes, attempts to modify their substrate specificity have been more limited. In a recent study, we produced the first asparaginyl ligase with engineered P2' substrate specificity by mutating Tyr188 to Ala in OaAEP1. Here, we report the engineering of two additional asparaginyl ligases from different plant families, VyPAL2 and butelase 1. We show that mutating the corresponding Tyr residue located in the S2' pocket of these enzymes also expands their substrate scope, enabling the mutant enzymes to process substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling that their parent enzymes process poorly. These findings further establish the role of the conserved S2' Tyr residue as a general determinant of substrate specificity for asparaginyl ligases and provide a path toward more extensive engineering efforts.

Efficient enzymatic depolymerization of recalcitrant polysaccharides such as chitin and cellulose relies on processive glycoside hydrolases (GHs), whose efficiency can be enhanced through cooperation with lytic polysaccharide monooxygenases (LPMOs). For processive GHs, retained binding during successive catalytic events aids depolymerization of crystalline substrates but comes at the expense of slow dissociation (low koff) that limits turnover. Here, we engineered a series of mutants of the processive exochitobiohydrolase SmChiB from Serratia marcescens, in which a major determinant of substrate affinity and processivity, Trp220, was replaced by Tyr, Phe, His, Gln, or Ala. Functional analysis showed that these mutants had stepwise reductions in substrate affinity and processivity, with the latter being a signature of an increased koff. Molecular dynamics simulations confirmed that Trp220 plays an important role in substrate binding. The less processive SmChiB variants, and in particular W220Y, were able to reach wild-type-like performance only at high substrate concentrations. Importantly, the use of LPMOs to decrystallize the substrate and thereby increase its effective concentration, enhanced the performance of the less processive mutants to a much greater extent than for wild-type SmChiB. In some reaction setups, the combination of W220Y with an LPMO yielded twice as much soluble product compared to the wild-type enzyme under identical conditions. Thus, when combined with LPMOs, less processive GHs become more favorable because they are intrinsically more efficient catalysts when acting on noncrystalline substrates. These findings shed light on how the interplay between GHs and LPMOs can be optimized for efficient enzymatic conversion of recalcitrant polysaccharides.

Antibiotics have revolutionized human health by significantly reducing morbidity and mortality associated with bacterial infections. Antibiotics exert bactericidal or bacteriostatic effects through inhibition of cell wall synthesis and disruption of cell membrane integrity, inhibition of protein, nucleic acid synthesis, and other metabolic pathways. Despite their remarkable success since the mid-20th century, antimicrobial resistance (AMR) has emerged as a major global health concern, undermining current treatments and complicating infection management. Key drivers of AMR include the overuse and misuse of antibiotics in clinical settings as well as bacterial adaptations such as genetic mutations and horizontal gene transfer. Mechanistically, these changes can lead to enzymatic inactivation of antibiotics, modification of drug targets, changes in permeability, and active efflux of antimicrobial agents. As resistance rises, antibiotic discovery and development have lagged, creating an urgent need for novel therapeutic strategies and chemical scaffolds. This review examines the antibiotic mechanisms and antibiotic evasion strategies, highlighting genetic and omics approaches used to identify high-priority targets for future drug discovery.

Several bacterial pathogens secrete multidomain enzymes known as lytic polysaccharide monooxygenases (LPMOs) that are important for virulence. One example is the Vibrio cholerae virulence factor GbpA (VcGbpA), in which an N-terminal LPMO domain is followed by two domains of unknown function called GbpA2 and GbpA3, and a C-terminal chitin-binding domain called CBM73. In-depth functional characterization of full-length and truncated variants of VcGbpA and a homologue from V. campbellii (previously V. harveyi, VhGbpA) showed that the catalytic LPMO domains of these proteins exhibit properties similar to natural single-domain LPMOs with established roles in chitin degradation. Interestingly, binding to chitin and efficient degradation of this substrate were affected by the presence of the GbpA2 and GbpA3 domains. Combined with structural predictions and analyses of sequence conservation, our data show that GbpA3 has evolved to interact with the reduced catalytic copper site in the LPMO domain to prevent off-pathway reactions in the absence of substrate. Substrate binding by CBM73 weakens this interaction, enabling the activation of the LPMO only when substrate is present. These observations shed new light into the functionality of these multidomain LPMOs and uncover a novel mechanism for regulating LPMO activity.

Repeat expansions in the genome are associated with numerousgeneticdiseases. The instability of repeat sequences is driven in part byslipped-out structures, such as hairpins. APOBEC3A (A3A), a cytosinedeaminase, preferentially targets single-stranded DNA, including repeatregions capable of forming such secondary structures. In this study,we investigated how small molecules that selectively bind C–Cmismatches in CCG hairpin repeats modulate A3A-mediated deamination.Using model oligonucleotides containing (CCG)9 repeatsand mismatch-binding ligands (MBLs: AmND and AmBzND), we show thatthese ligands selectively stabilize the stem regions of hairpin structures,suppressing deamination in the stem and directing A3A activity tothe loop regions. The inhibitory effect was dose-dependent, and deaminationoccurred preferentially at loop cytosines. These findings demonstratethat hairpin stabilization with small molecules can modulate A3A site-selectivitywith implications for understanding repeat instability and its therapeuticcontrol. Furthermore, this approach may serve as a basis for developingchemical tools to manipulate repeat-associated genome functions.

The transcription of the HIV-1 long-terminal repeat (LTR), driven by the viral transactivator Tat, represents a distinct and druggable step in HIV-1 replication. Targeting Tat-mediated transcription is a promising antiretroviral strategy due to its mechanistic distinction from host cell transcription, although specificity remains a challenge due to potential off-target effects. To identify selective inhibitors, a two-step screening approach was employed. A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was first used to identify compounds that disrupt Tat-trans-activation response (TAR) RNA interactions, yielding 655 initial hits, including five 2-phenyl acrylate derivatives with >50% inhibition. A secondary library of 194 structurally related compounds was then screened using a dual-luciferase reporter assay to evaluate transcriptional inhibition and cytotoxicity. From this, 46 compounds met selection criteria (>50% F-Luc inhibition, > 90% R-Luc activity, > 90% viral inhibition, and >70% cell viability). Among them, methyl (E)-2-(2-((4-(decyloxy)phenoxy)methyl)phenyl)-3-methoxyacrylate showed the highest potency, with IC50 and EC50 values of 1.44 and 0.83 μM, respectively. Time-of-addition (TOA) assays indicated inhibition of the Tat-dependent transcription phase. Surface plasmon resonance analysis revealed binding to the Tat peptide but not to TAR RNA, suggesting the binding target for 019854-B06. Immunoblotting and coimmunoprecipitation showed that 019854-B06 neither promotes Tat degradation nor disrupts the Tat/CycT1 complex, supporting a Tat-centric, nondegradative mechanism. Therefore, this study identifies a novel 2-phenyl acrylate-based inhibitor of Tat-mediated HIV-1 transcription through an integrated biophysical and functional screening strategy.