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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 505 records · Page 28
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Broad betacoronavirus neutralization by a stem helix–specific human antibody

Targeting a range of betacoranaviruses In the past 20 years, three highly pathogenic β-coronaviruses have crossed from animals to humans, including the most recent: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A spike protein that decorates these viruses has an S1 domain that binds host cell receptors and an S2 domain that fuses the viral and cell membranes to allow cell entry. The S1 domain is the target of many neutralizing antibodies but is more genetically variable than S2, and antibodies can exert selective pressure, leading to resistant variants. Pinto et al . identified five monoclonal antibodies that interact with a helix in the S2 domain. The most broadly neutralizing antibody inhibited all β-coronavirus subgenera and reduced viral burden in hamsters infected with SARS-CoV-2. —VV

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cyanobacteriochromes: A Rainbow of Photoreceptors

Widespread phytochrome photoreceptors use photoisomerization of linear tetrapyrrole (bilin) chromophores to measure the ratio of red to far-red light. Cyanobacteria also contain distantly related cyanobacteriochrome (CBCR) proteins that share the bilin-binding GAF domain of phytochromes but sense other colors of light. CBCR photocycles are extremely diverse, ranging from the near-UV to the near-IR. Photoisomerization of the bilin triggers photoconversion of the CBCR input, thereby modulating the biochemical signaling state of output domains such as histidine kinase bidomains that can interface with cellular signal transduction pathways. CBCRs thus can regulate several aspects of cyanobacterial photobiology, including phototaxis, metabolism of cyclic nucleotide second messengers, and optimization of the cyanobacterial light-harvesting apparatus. This review examines spectral tuning, photoconversion, and photobiology of CBCRs and recent developments in understanding their evolution and in applying them in synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A genomic analysis reveals the diversity of cellulosome displaying bacteria

Introduction Several species of cellulolytic bacteria display cellulosomes, massive multi-cellulase containing complexes that degrade lignocellulosic plant biomass (LCB). A greater understanding of cellulosome structure and enzyme content could facilitate the development of new microbial-based methods to produce renewable chemicals and materials. Methods To identify novel cellulosome-displaying microbes we searched 305,693 sequenced bacterial genomes for genes encoding cellulosome proteins; dockerin-fused glycohydrolases (DocGHs) and cohesin domain containing scaffoldins. Results and discussion This analysis identified 33 bacterial species with the genomic capacity to produce cellulosomes, including 10 species not previously reported to produce these complexes, such asAcetivibrio mesophilus. Cellulosome-producing bacteria primarily originate from theAcetivibrio, Ruminococcus, Ruminiclostridium, andClostridiumgenera. A rigorous analysis of their enzyme, scaffoldin, dockerin, and cohesin content reveals phylogenetically conserved features. Based on the presence of a high number of genes encoding both scaffoldins and dockerin-fused GHs, the cellulosomes inAcetivibrioandRuminococcusbacteria possess complex architectures that are populated with a large number of distinct LCB degrading GH enzymes. Their complex cellulosomes are distinguishable by their mechanism of attachment to the cell wall, the structures of their primary scaffoldins, and by how they are transcriptionally regulated. In contrast, bacteria in theRuminiclostridiumandClostridiumgenera produce ‘simple’ cellulosomes that are constructed from only a few types of scaffoldins that based on their distinct complement of GH enzymes are predicted to exhibit high and low cellulolytic activity, respectively. Collectively, the results of this study reveal conserved and divergent architectural features in bacterial cellulosomes that could be useful in guiding ongoing efforts to harness their cellulolytic activities for bio-based chemical and materials production.

Microbiology↗
DOE OSTI2024

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists

Polycystin-1 (PC1) is the protein product of thePKD1gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Life Sciences & Biomedicine - Other Topics↗
NASA NTRS2001

Familial orthostatic tachycardia due to norepinephrine transporter deficiency

Orthostatic intolerance (OI) or postural tachycardia syndrome (POTS) is a syndrome primarily affecting young females, and is characterized by lightheadedness, palpitations, fatigue, altered mentation, and syncope primarily occurring with upright posture and being relieved by lying down. There is typically tachycardia and raised plasma norepinephrine levels on upright posture, but little or no orthostatic hypotension. The pathophysiology of OI is believed to be very heterogeneous. Most studies of the syndrome have focused on abnormalities in norepinephrine release. Here the hypothesis that abnormal norepinephrine transporter (NET) function might contribute to the pathophysiology in some patients with OI was tested. In a proband with significant orthostatic symptoms and tachycardia, disproportionately elevated plasma norepinephrine with standing, impaired systemic, and local clearance of infused tritiated norepinephrine, impaired tyramine responsiveness, and a dissociation between stimulated plasma norepinephrine and DHPG elevation were found. Studies of NET gene structure in the proband revealed a coding mutation that converts a highly conserved transmembrane domain Ala residue to Pro. Analysis of the protein produced by the mutant cDNA in transfected cells demonstrated greater than 98% reduction in activity relative to normal. NE, DHPG/NE, and heart rate correlated with the mutant allele in this family. CONCLUSION: These results represent the first identification of a specific genetic defect in OI and the first disease linked to a coding alteration in a Na+/Cl(-)-dependent neurotransmitter transporter. Identification of this mechanism may facilitate our understanding of genetic causes of OI and lead to the development of more effective therapeutic modalities.

Review, Tutorial↗
NASA NTRS2003

An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

NASA Discipline Plant Biology↗
DOE OSTI2020

ADEPT: a domain independent sequence alignment strategy for gpu architectures

Bioinformatic workflows frequently make use of automated genome assembly and protein clustering tools. At the core of most of these tools, a significant portion of execution time is spent in determining optimal local alignment between two sequences. This task is performed with the Smith-Waterman algorithm, which is a dynamic programming based method. With the advent of modern sequencing technologies and increasing size of both genome and protein databases, a need for faster Smith-Waterman implementations has emerged. Multiple SIMD strategies for the Smith-Waterman algorithm are available for CPUs. However, with the move of HPC facilities towards accelerator based architectures, a need for an efficient GPU accelerated strategy has emerged. Existing GPU based strategies have either been optimized for a specific type of characters (Nucleotides or Amino Acids) or for only a handful of application use-cases. In this paper, we present ADEPT, a new sequence alignment strategy for GPU architectures that is domain independent, supporting alignment of sequences from both genomes and proteins. Our proposed strategy uses GPU specific optimizations that do not rely on the nature of sequence. We demonstrate the feasibility of this strategy by implementing the Smith-Waterman algorithm and comparing it to similar CPU strategies as well as the fastest known GPU methods for each domain. ADEPT’s driver enables it to scale across multiple GPUs and allows easy integration into software pipelines which utilize large scale computational systems. We have shown that the ADEPT based Smith-Waterman algorithm demonstrates a peak performance of 360 GCUPS and 497 GCUPs for protein based and DNA based datasets respectively on a single GPU node (8 GPUs) of the Cori Supercomputer. Overall ADEPT shows 10x faster performance in a node-to-node comparison against a corresponding SIMD CPU implementation. ADEPT demonstrates a performance that is either comparable or better than existing GPU strategies. We demonstrated the efficacy of ADEPT in supporting existing bionformatics software pipelines by integrating ADEPT in MetaHipMer a high-performance denovo metagenome assembler and PASTIS a high-performance protein similarity graph construction pipeline. Our results show 10% and 30% boost of performance in MetaHipMer and PASTIS respectively.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Discovery and Development of a Small-Molecule Inhibitor Targeting the GAS41 YEATS Domain in Nonsmall Cell Lung Cancer

Abstract GAS41 is frequently overexpressed in Non-Small Cell Lung Cancer (NSCLC). GAS41 contains a YEATS domain, which recognizes acetylated lysine residues on histones to recruit protein complexes and facilitate transcription. Suppression of GAS41 in NSCLC models inhibits cellular proliferation and markedly reduces tumor growth in mouse xenografts, justifying the development of small-molecule inhibitors. We have employed structure-based design and medicinal chemistry optimization to discover DLG-41, a submicromolar inhibitor binding to the GAS41 YEATS domain. DLG-41 potently disrupts the association of GAS41 YEATS with chromatin in mammalian cells and inhibits the proliferation of NSCLC cell lines with submicromolar potency without significantly affecting normal lung fibroblasts. DLG-41 induces more effective growth inhibition in A549 versus GAS41-knockout cells, demonstrating on-target activity. DLG-41 treatment upregulates the CDKN1A gene and downregulates pathways associated with lung cancer cell identity, tumor migration, and invasion. DLG-41 is a promising chemical probe for targeting GAS41 protein in NSCLC models and has potential for future development.

Listunov, Dymytrii [University of Michigan , , , ,↗
DOE OSTI2021

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Virology↗
DOE OSTI2024

Structure of a tripartite protein complex that targets toxins to the type VII secretion system

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE–PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

[NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism. The biosynthesis of the NiFe(CN) 2 CO cofactor involves a suite of maturation proteins, including the HypA and HypB nickel metallochaperones. Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis . Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored. Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens. The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity. Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB. The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPase-mediated nickel delivery pathway. The HypA-HypB 2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone. Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis . Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

[NiFe] hydrogenase↗
DOE OSTI2022

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Netrin-like domain of sFRP4, a Wnt antagonist inhibits stemness, metastatic and invasive properties by specifically blocking MMP-2 in cancer stem cells from human glioma cell line U87MG

Rapid proliferation, high stemness potential, high invasiveness and apoptotic evasion are the distinctive hallmarks of glioma malignancy. The dysregulation of the Wnt/β-catenin pathway is the key factor regulating glioma malignancy. Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), which has a prominent pro-apoptotic role in glioma stem cells, has two functional domains, the netrin-like domain (NLD), and cysteine-rich domain (CRD) both of which contribute to apoptotic properties of the whole protein. However, there are no reports elucidating the specific effects of individual domains of sFRP4 in inhibiting the invasive properties of glioma. This study explores the efficacy of the domains of sFRP4 in inhibiting the key hallmarks of glioblastoma such as invasion, metastasis, and stemness. We overexpressed sFRP4 and its domains in the glioblastoma cell line, U87MG cells and observed that both CRD and NLD domains played prominent roles in attenuating cancer stem cell properties. Significantly, we could demonstrate for the first time that both NLD and CRD domains negatively impacted the key driver of metastasis and migration, the matrix metalloproteinase-2 (MMP-2). Mechanistically, compared to CRD, NLD domain suppressed MMP-2 mediated invasion more effectively in glioma cells as observed in matrigel invasion assay and a function-blocking antibody assay. Fluorescent matrix degradation assay further revealed that NLD reduces matrix degradation. NLD also significantly disrupted fibronectin assembly and decreased cell adhesion in another glioma cell line LN229. In conclusion, the NLD peptide of sFRP4 could be a potent short peptide therapeutic candidate for targeting MMP-2-mediated invasion in the highly malignant glioblastoma multiforme.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis

Abstract Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular‐like “rosette” state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide‐binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high‐resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG‐3 PDZ domains from M. brevicollis . Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG‐3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity.

Gao, Melody↗
DOE OSTI2024

Multi-domain automated patterning of DNA-functionalized hydrogels

DNA-functionalized hydrogels are capable of sensing oligonucleotides, proteins, and small molecules, and specific DNA sequences sensed in the hydrogels’ environment can induce changes in these hydrogels’ shape and fluorescence. Fabricating DNA-functionalized hydrogel architectures with multiple domains could make it possible to sense multiple molecules and undergo more complicated macroscopic changes, such as changing fluorescence or changing the shapes of regions of the hydrogel architecture. However, automatically fabricating multi-domain DNA-functionalized hydrogel architectures, capable of enabling the construction of hydrogel architectures with tens to hundreds of different domains, presents a significant challenge. We describe a platform for fabricating multi-domain DNA-functionalized hydrogels automatically at the micron scale, where reaction and diffusion processes can be coupled to program material behavior. Using this platform, the hydrogels’ material properties, such as shape and fluorescence, can be programmed, and the fabricated hydrogels can sense their environment. DNA-functionalized hydrogel architectures with domain sizes as small as 10 microns and with up to 4 different types of domains can be automatically fabricated using ink volumes as low as 50 μL. We also demonstrate that hydrogels fabricated using this platform exhibit responses similar to those of DNA-functionalized hydrogels fabricated using other methods by demonstrating that DNA sequences can hybridize within them and that they can undergo DNA sequence-induced shape change.

Rubanov, Moshe (ORCID:0000000180507407)↗
DOE OSTI2023

Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS–PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS–PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein–protein interactions supports a predicted molecular basis for enzyme–carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.

59 BASIC BIOLOGICAL SCIENCES↗