RESUMO
In various autoimmune diseases, dysfunctional TREX1 (Three prime Repair Exonuclease 1) leads to accumulation of endogenous single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and DNA/RNA hybrids in the cytoplasm and triggers immune activation through the cGAS-STING pathway. Although inhibition of TREX1 could be a useful strategy for cancer immunotherapy, profiling cellular functions in terms of its potential substrates is a key step. Particularly important is the functionality of processing DNA/RNA hybrids and RNA substrates. The exonuclease activity measurements conducted here establish that TREX1 can digest both ssRNA and DNA/RNA hybrids but not dsRNA. The newly solved structures of TREX1-RNA product and TREX1-nucleotide complexes show that 2'-OH does not impose steric hindrance or specific interactions for the recognition of RNA. Through all-atom molecular dynamics simulations, we illustrate that the 2'-OH-mediated intra-chain hydrogen bonding in RNA would affect the binding with TREX1 and thereby reduce the exonuclease activity. This notion of higher conformational rigidity in RNA leading TREX1 to exhibit weaker catalytic cleavage is further validated by the binding affinity measurements with various synthetic DNA-RNA junctions. The results of this work thus provide new insights into the mechanism by which TREX1 processes RNA and DNA/RNA hybrids and contribute to the molecular-level understanding of the complex cellular functions of TREX1 as an exonuclease.
Assuntos
DNA , RNA , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , Exodesoxirribonucleases/metabolismo , Fosfoproteínas/metabolismo , RNA/genética , Animais , CamundongosRESUMO
Capreomycidine (Cap) is a nonproteinogenic amino acid and building block of nonribosomal peptide (NRP) natural products. We report the formation and activation of Cap in capreomycin biosynthesis. CmnC and CmnD catalyzed hydroxylation and cyclization, respectively, of l-Arg to form l-Cap. l-Cap is then adenylated by CmnG-A before being incorporated into the nonribosomal peptide. The co-crystal structures of CmnG-A with l-Cap and adenosine nucleotides provide insights into the specificity and engineering opportunities of this unique adenylation domain.
Assuntos
Aminoácidos , Peptídeo Sintases , Peptídeo Sintases/metabolismo , Capreomicina , Especificidade por Substrato , Peptídeos/químicaRESUMO
The three prime repair exonuclease 2 (TREX2) is an essential 3'-to-5' exonuclease that functions in cell proliferation, genome integrity and skin homeostasis maintenance. The abnormal expression level of TREX2 can result in broken chromosome, increased susceptibility to skin carcinogenesis and Psoriasis. However, the molecular mechanisms of how TREX2 binds and processes its natural substrates, dsDNA or chromosomal DNA, to maintain genome stability remain unclear. In this study, we present four new crystal structures: apo-TREX2, TREX2 in complex with two different dsDNA substrates, and TREX2 in complex with a processed dsDNA product. Analysis of the structures reveals that TREX2 stacks with the 5'-terminal of dsDNA by a Leu20-Pro21-Asn22 cluster for precisely trimming the 3'-overhang. In addition, TREX2 specifically interacts with the non-scissile strand of dsDNA by an α-helix-loop region. The unique interaction patterns of the TREX2-dsDNA complex highlight the requirement of long double-stranded region for TREX2 binding and provide evidence of the functional role of TREX2 in processing chromosomal DNA. Moreover, the non-processive property of TREX2 is elucidated by the structure of TREX2-product complex. Our work discloses the first structural basis of the molecular interactions between TREX2 and its substrates and unravels the mechanistic actions of TREX2.
Assuntos
Apoproteínas/química , DNA/química , Exodesoxirribonucleases/química , Subunidades Proteicas/química , Motivos de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Spatial hindrance-based pro-antibodies (pro-Abs) are engineered antibodies to reduce monoclonal antibodies' (mAbs) on-target toxicity using universal designed blocking segments that mask mAb antigen-binding sites through spatial hindrance. By linking through protease substrates and linkers, these blocking segments can be removed site-specifically. Although many types of blocking segments have been developed, such as coiled-coil and hinge-based Ab locks, the molecular structure of the pro-Ab, particularly the region showing how the blocking fragment blocks the mAb, has not been elucidated by X-ray crystallography or cryo-EM. To achieve maximal effect, a pro-Ab must have high antigen-blocking and protease-restoring efficiencies, but the unclear structure limits its further optimization. Here, we utilized molecular dynamics (MD) simulations to study the dynamic structures of a hinge-based Ab lock pro-Ab, pro-Nivolumab, and validated the simulated structures with small- and wide-angle X-ray scattering (SWAXS). The MD results were closely consistent with SWAXS data (χ2 best-fit = 1.845, χ2 allMD = 3.080). The further analysis shows a pronounced flexibility of the Ab lock (root-mean-square deviation = 10.90 Å), yet it still masks the important antigen-binding residues by 57.3%-88.4%, explaining its 250-folded antigen-blocking efficiency. The introduced protease accessible surface area method affirmed better protease efficiency for light chain (33.03 Å2) over heavy chain (5.06 Å2), which aligns with the experiments. Overall, we developed MD-SWAXS validation method to study the dynamics of flexible blocking segments and introduced methodologies to estimate their antigen-blocking and protease-restoring efficiencies, which would potentially be advancing the clinical applications of any spatial hindrance-based pro-Ab.
Assuntos
Anticorpos Monoclonais , Simulação de Dinâmica Molecular , Espalhamento a Baixo Ângulo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Difração de Raios X , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Antígenos/química , Antígenos/imunologia , Humanos , Conformação Proteica , Cristalografia por Raios XRESUMO
L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products, including capreomycin, viomycin, zwittermicin, staphyloferrin and dapdiamide. A previous study reported that CmnB and CmnK are two enzymes that are involved in the formation of L-Dap in the biosynthesis of capreomycin. CmnB catalyzes the condensation reaction of O-phospho-L-serine and L-glutamic acid to generate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid, which subsequently undergoes oxidative hydrolysis via CmnK to generate the product L-Dap. Here, the crystal structure of CmnB in complex with the reaction intermediate PLP-α-aminoacrylate is reported at 2.2â Å resolution. Notably, CmnB is the second known example of a PLP-dependent enzyme that forms a monomeric structure in crystal packing. The crystal structure of CmnB also provides insights into the catalytic mechanism of the enzyme and supports the biosynthetic pathway of L-Dap reported in previous studies.
Assuntos
Aminoácidos , Capreomicina , Cristalografia por Raios X , beta-Alanina , Ácido Glutâmico/metabolismoRESUMO
Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistance genes (ARGs). These ARGs are hidden in various environmental bacteria and may become a latent crisis for antibiotic therapy via horizontal gene transfer. In this study, we focus on a resistance gene cph, which encodes a phosphotransferase (Cph) that confers resistance to the antituberculosis drug capreomycin (CMN). Sequence Similarity Network (SSN) analysis classified 353 Cph homologues into five major clusters, where the proteins in cluster I were found in a broad range of actinobacteria. We examine the function and antibiotics targeted by three putative resistance proteins in cluster I via biochemical and protein structural analysis. Our findings reveal that these three proteins in cluster I confer resistance to CMN, highlighting an important aspect of CMN resistance within this gene family. This study contributes towards understanding the sequence-structure-function relationships of the phosphorylation resistance genes that confer resistance to CMN.
Assuntos
Antibacterianos , Capreomicina , Capreomicina/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Bactérias/genética , Genes Bacterianos , Imunidade InataRESUMO
Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.
Assuntos
Actinobacteria/enzimologia , Antibióticos Antituberculose/metabolismo , Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/metabolismo , Capreomicina/metabolismo , Capreomicina/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Actinobacteria/efeitos dos fármacos , Actinobacteria/metabolismo , Antibióticos Antituberculose/química , Proteínas de Bactérias/genética , Capreomicina/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Conformação ProteicaRESUMO
CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form ß-hydroxy l-Arg (CmnC and VioC) or ß,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnCL136Q,S138G,D249Y switches the substrate stereoselectivity from l-Arg to d-Arg with â¼6% relative activity. The crystal structure of CmnCL136Q,S138G,D249Y in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.