Halogen Bond-Assisted Supramolecular Dimerization of Pyridinium-Fused 1,2,4-Selenadiazoles via Four-Center Se2N2 Chalcogen Bonding.

The synthesis and structural characterization of α-haloalkyl-substituted pyridinium-fused 1,2,4-selenadiazoles with various counterions is reported herein, demonstrating a strategy for directed supramolecular dimerization in the solid state. The compounds were obtained through a recently discovered 1,3-dipolar cycloaddition reaction between nitriles and bifunctional 2-pyridylselenyl reagents, and their structures were confirmed by the X-ray crystallography. α-Haloalkyl-substituted pyridinium-fused 1,2,4-selenadiazoles exclusively formed supramolecular dimers via four-center Se···N chalcogen bonding, supported by additional halogen bonding involving α-haloalkyl substituents. The introduction of halogens at the α-position of the substituent R in the selenadiazole core proved effective in promoting supramolecular dimerization, which was unaffected by variation of counterions. Additionally, the impact of cocrystallization with a classical halogen bond donor C6F3I3 on the supramolecular assembly was investigated. Non-covalent interactions were studied using density functional theory calculations and topological analysis of the electron density distribution, which indicated that all ChB, XB and HB interactions are purely non-covalent and attractive in nature. This study underscores the potential of halogen and chalcogen bonding in directing the self-assembly of functional supramolecular materials employing 1,2,4-selenadiazoles derived from recently discovered cycloaddition between nitriles and bifunctional 2-pyridylselenyl reagents.

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Structural basis for dimerization of a paramyxovirus polymerase complex.

The transcription and replication processes of non-segmented, negative-strand RNA viruses (nsNSVs) are catalyzed by a multi-functional polymerase complex composed of the large protein (L) and a cofactor protein, such as phosphoprotein (P). Previous studies have shown that the nsNSV polymerase can adopt a dimeric form, however, the structure of the dimer and its function are poorly understood. Here we determine a 2.7 Å cryo-EM structure of human parainfluenza virus type 3 (hPIV3) L-P complex with the connector domain (CD') of a second L built, while reconstruction of the rest of the second L-P obtains a low-resolution map of the ring-like L core region. This study reveals detailed atomic features of nsNSV polymerase active site and distinct conformation of hPIV3 L with a unique ß-strand latch. Furthermore, we report the structural basis of L-L dimerization, with CD' located at the putative template entry of the adjoining L. Disruption of the L-L interface causes a defect in RNA replication that can be overcome by complementation, demonstrating that L dimerization is necessary for hPIV3 genome replication. These findings provide further insight into how nsNSV polymerases perform their functions, and suggest a new avenue for rational drug design.

Specific features of a scaffolding antibody light chain.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.

Heat flows enrich prebiotic building blocks and enhance their reactivity.

The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality.

BACKGROUND: CTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer-promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif. RESULTS: Previously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein. CONCLUSIONS: Our findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers.

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer's interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

Dimerization of the ß-Hairpin Membrane-Active Cationic Antimicrobial Peptide Capitellacin from Marine Polychaeta: An NMR Structural and Thermodynamic Study.

Capitellacin is the ß-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric ß-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal ß-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the "carpet" mode of membrane activity. An analysis of the known structures of ß-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of ß-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of ß-structural peptides in biological membranes.

G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

Membrane-Driven Dimerization of the Peripheral Membrane Protein KRAS: Implications for Downstream Signaling.

Transient homo-dimerization of the RAS GTPase at the plasma membrane has been shown to promote the mitogen-activated protein kinase (MAPK) signaling pathway essential for cell proliferation and oncogenesis. To date, numerous crystallographic studies have focused on the well-defined GTPase domains of RAS isoforms, which lack the disordered C-terminal membrane anchor, thus providing limited structural insight into membrane-bound RAS molecules. Recently, lipid-bilayer nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses have revealed several distinct structures of the membrane-anchored homodimers of KRAS, an isoform that is most frequently mutated in human cancers. The KRAS dimerization interface is highly plastic and altered by biologically relevant conditions, including oncogenic mutations, the nucleotide states of the protein, and the lipid composition. Notably, PRE-derived structures of KRAS homodimers on the membrane substantially differ in terms of the relative orientation of the protomers at an "α-α" dimer interface comprising two α4-α5 regions. This interface plasticity along with the altered orientations of KRAS on the membrane impact the accessibility of KRAS to downstream effectors and regulatory proteins. Further, nanodisc platforms used to drive KRAS dimerization can be used to screen potential anticancer drugs that target membrane-bound RAS dimers and probe their structural mechanism of action.

PDGFRA K385 mutants in myxoid glioneuronal tumors promote receptor dimerization and oncogenic signaling.

Myxoid glioneuronal tumors (MGNT) are low-grade glioneuronal neoplasms composed of oligodendrocyte-like cells in a mucin-rich stroma. These tumors feature a unique dinucleotide change at codon 385 in the platelet-derived growth factor receptor α (encoded by the PDGFRA gene), resulting in the substitution of lysine 385 into leucine or isoleucine. The functional consequences of these mutations remain largely unexplored. Here, we demonstrated their oncogenic potential in fibroblast and Ba/F3 transformation assays. We showed that the K385I and K385L mutants activate STAT and AKT signaling in the absence of ligand. Co-immunoprecipitations and BRET experiments suggested that the mutations stabilized the active dimeric conformation of the receptor, pointing to a new mechanism of oncogenic PDGF receptor activation. Furthermore, we evaluated the sensitivity of these mutants to three FDA-approved tyrosine kinase inhibitors: imatinib, dasatinib, and avapritinib, which effectively suppressed the constitutive activity of the mutant receptors. Finally, K385 substitution into another hydrophobic amino acid also activated the receptor. Interestingly, K385M was reported in a few cases of brain tumors but not in MGNT. Our results provide valuable insights into the molecular mechanism underlying the activation of PDGFRα by the K385I/L mutations, highlighting their potential as actionable targets in the treatment of myxoid glioneuronal tumors.

Conserved proline residues prevent dimerization and aggregation in the ß-lactamase BlaC.

Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A ß-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the ß-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.

Structural insights reveal interplay between LAG-3 homodimerization, ligand binding, and function.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.

Metabolic remodeling by the PD-L1 inhibitor BMS-202 significantly inhibits cell malignancy in human glioblastoma.

Recently, crystallographic studies have demonstrated that BMS-202, a small-molecule compound characterized by a methoxy-1-pyridine chemical structure, exhibits a high affinity to PD-L1 dimerization. However, its roles and mechanisms in glioblastoma (GBM) remain unclear. The objective of this study is to investigate the antitumor activity of BMS-202 and its underlying mechanisms in GBM using multi-omics and bioinformatics techniques, along with a majority of in vitro and in vivo experiments, including CCK-8 assays, flow cytometry, co-immunoprecipitation, siRNA transfection, PCR, western blotting, cell migration/invasion assays and xenografts therapeutic assays. Our findings indicate that BMS-202 apparently inhibits the proliferation of GBM cells both in vitro and in vivo. Besides, it functionally blocks cell migration and invasion in vitro. Mechanistically, it reduces the expression of PD-L1 on the surface of GBM cells and interrupts the PD-L1-AKT-BCAT1 axis independent of mTOR signaling. Taken together, we conclude that BMS-202 is a promising therapeutic candidate for patients with GBM by remodeling their cell metabolism regimen, thus leading to better survival.

Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes.

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

Homo-BacPROTAC-induced degradation of ClpC1 as a strategy against drug-resistant mycobacteria.

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.