Tandem Domains Research Articles

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose Nicotinamide adenine dinucleotide (NAD+) regeneration as the function of this enzyme and name it Metabolosome Nicotinamide Adenine Dinucleotide Hydrogen (NADH) dehydrogenase (MNdh). Its partner shell protein BMC-TSE (tandem domain BMC shell protein of the single layer type for electron transfer) assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, Electron Paramagnetic Resonance spectroscopy, protein voltammetry, and structural modeling verified with X-ray footprinting. This finding represents a paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

A distinct co-expressed sulfurtransferase extends the physiological role of mercaptopropionate dioxygenase in Pseudomonas aeruginosa PAO1

Oxidation and assimilation of persulfides in bacteria is often catalyzed by a persulfide dioxygenase and sulfurtransferase in consecutive reactions. Enzymes responsible for the oxidation of persulfides have not been clearly defined in Pseudomonas aeruginosa PAO1. The characterized mercaptopropionate dioxygenase (MDO) in P. aeruginosa PAO1 has been proposed to catalyze the oxidation of 3-mercaptopropionate. However, the physiological role of MDO is uncertain given the expression of a sulfurtransferase (ST) enzyme on the same operon as the thiol dioxygenase. The st gene had a co-occurrence frequency with mdo of 0.94 demonstrating the co-expression and physiological link of the two genes. There are four tandem rhodanese domains in the ST enzyme with two of the domains containing potential catalytic Cys residues (Cys191 and Cys435) capable of forming a persulfide. Only Cys435 was accessible in thiol quantification assays, and results from H/D-X MS analyses further established the accessibility of the domain containing Cys435. Both thiosulfate and mercaptopyruvate served as sulfur donors to the ST enzyme, with Cys435 forming the persulfide intermediate. Kinetic investigations of MDO suggested the enzyme had a broader substrate specificity than previously identified, oxidizing both mercaptopropionate and mercaptopyruvate thiol and persulfide substrates. The results obtained from these investigations provide insight into the overall mechanism and physiological role of the mdo operon in sulfide oxidation and assimilation.

Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains.

Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function). Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs. Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs. Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome.

Immuno- and expression analysis of Ehrlichia canis immunoreactive proteins.

Ehrlichia canis is the primary etiologic agent of canine monocytic ehrlichiosis, a serious and sometimes fatal hemorrhagic disease of dogs. Diagnosis of E. canis infection is often retrospectively confirmed by serologic detection of antibodies by immunofluorescent microscopy. Our laboratory previously identified numerous major immunoreactive proteins with species-specific linear antibody epitopes that are useful for immunodiagnosis of CME. More recently, we have defined the entire antibody-reactive immunome of E. canis, substantially increasing the number of major immunoreactive proteins known to exist. In this study, we analyzed and compared seven recently identified antibody reactive E. canis proteins with established diagnostic antigens including tandem repeat proteins TRP19, TRP36 and TRP140 and observed comparable immunoreactivity. Many of these proteins were conserved in different E. canis strains. Multiple linear antibody epitopes were mapped in a highly conserved TRP (Ecaj_0126), including within the tandem repeat domain. Temporal antibody responses were examined, and multiple proteins reacted with antibodies in sera as early as 21 days post experimental infection. Host-specific expression of the proteins was examined which revealed that some proteins exhibited higher expression in mammalian cells, while others in tick cells. This study has identified new immunodiagnostic candidates that exhibit different host expression patterns, information which may be useful for developing ultrasensitive immunodiagnostics and effective vaccines for CME.

Backbone 1H, 15N, and 13C resonance assignments of the FF1 domain from P190A RhoGAP in 5 and 8M urea.

The Rho GTPase (Ras homolog GTPases) system is a crucial signal transducer that regulates various cellular processes, including cell cycle and migration, genetic transcription, and apoptosis. In this study, we investigated the unfolded state of the first FF domain (FF1) of P190A RhoGAP, which features four tandem FF domains. For signal transduction, FF1 is phosphorylated at tyrosine 308 (Y308), which is buried in the hydrophobic core and is inaccessible to kinases in the folded domain. It was proposed, therefore, that the phosphorylation occurs in a transiently populated unfolded state of FF1. To probe the folding pathway of the RhoGAP FF1 domain, here we have performed a nearly complete backbone resonance assignments of a putative partially unfolded state of FF1 in 5M urea and its fully unfolded state in 8M urea.

The first report of prion protein gene sequences in Dybowski’s frog and the American bullfrog: high amyloid propensity of the frog prion protein

Prion diseases are fatal infectious neurodegenerative diseases caused by the proteinase K-sensitive form of prion protein (PrPSc). The exact origin of prion seeding and the transition factor of PrPSc has not been elucidated. The main hosts of prion diseases are herbivores, so the feces and corpses of Amphibians can seed PrPSc through ecosystems. The frog is an excellent candidate for transmission studies for this reason, but genetic analyses of the prion protein gene (PRNP) in the context of prion-related characteristics of frog species are lacking. We amplified frog PRNP gene sequences in Dybowski’s frog and the American bullfrog by polymerase chain reaction (PCR) and amplicon sequencing. In addition, we carried out multiple sequencing alignments and annotated major PrP components including signal peptide, tandem repeat domain, and PrPC-PrPSc interaction region of frog PrPs by bioinformatics tools. We predicted secondary and tertiary structures and amyloid propensities of frog PrPs using AlphaFold2 and AMYCO, respectively. We obtained DNA sequences of the PRNP gene in Dybowski’s frog and the American bullfrog, as well as a partially conserved palindromic sequence (PrPC-PrPSc interaction region) and absence of tandem repeat region of PrP in seven frog species. We analyzed protein structure of among these frog species and found that the high Himalaya frog has high aggregation propensity and the western clawed frog does not have the N-terminal signal peptide. To the best of our knowledge, this was the first comparative genetic study regarding prion-related features of frog species.

Structural mechanisms of SLF1 interactions with Histone H4 and RAD18 at the stalled replication fork.

DNA damage that obstructs the replication machinery poses a significant threat to genome stability. Replication-coupled repair mechanisms safeguard stalled replication forks by coordinating proteins involved in the DNA damage response (DDR) and replication. SLF1 (SMC5-SMC6 complex localization factor 1) is crucial for facilitating the recruitment of the SMC5/6 complex to damage sites through interactions with SLF2, RAD18,and nucleosomes. However, the structural mechanisms of SLF1's interactions are unclear. In this study, we determined the crystal structure of SLF1's ankyrin repeat domain bound to an unmethylated histone H4 tail, illustrating how SLF1 reads nascent nucleosomes. Using structure-based mutagenesis, we confirmed a phosphorylation-dependent interaction necessary for a stable complex between SLF1's tandem BRCA1 C-Terminal domain (tBRCT) and the phosphorylated C-terminal region (S442 and S444) of RAD18. We validated a functional role of conserved phosphate-binding residues inSLF1, and hydrophobic residues in RAD18 that areadjacent to phosphorylation sites, both of whichcontribute to the strong interaction. Interestingly, we discovered a DNA-binding property of this RAD18-binding interface, providing an additional domain of SLF1 to enhance binding to nucleosomes. Our results provide critical structural insights into SLF1's interactions with post-replicative chromatin and phosphorylation-dependent DDR signalling, enhancing our understanding of SMC5/6 recruitment and/or activity during replication-coupled DNA repair.

The Pvc15 ATPase selectively associates effector proteins with the Photorhabdus virulence cassette.

The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.

Protein-Sequence-Based Search of Nonreceptor ITAM-Like Regions to Identify Cytosolic Syk-Recruiting Proteins.

The recruitment of the protein spleen tyrosine kinase (Syk) to membrane-bound immune receptors is an essential step in initiating an immune response mediated through the activated receptors. Syk recognizes intracellular phosphorylated regions of membrane receptors known as immunoreceptor tyrosine-based activation motifs (ITAMs) defined by a sequence with two tyrosine (Y) amino acids separated by a certain spacing of six to eight residues: YXX(I/L)X6-8YXX(I/L). Syk with doubly phosphorylated ITAM is high-affinity and negatively regulated when Syk itself becomes phosphorylated. While the role of Syk in immune signaling is well characterized, recent information affords new functionality to Syk related to cytoplasmic processes, including the clearance of stress granules and P-bodies, both formed by liquid-liquid phase separation. Little to nothing is known about the molecular interactions involving Syk in these cytoplasmic processes. Given the essential role of receptor ITAMs in recruiting and localizing Syk for immune signaling, we explore here the possibility of a similar localization mechanism occurring for cytoplasmic processes by searching sequences of proteins related to Syk cytoplasmic function for regions that resemble receptor ITAMs. Protein sequence databases were generated from a Syk-dependent phosphoproteome and from genes related to P-bodies. A search of these databases for ITAM-like sequences yielded 102 unique hits, and 33 of these were synthesized and tested experimentally for binding to Syk tandem SH2 domains. The equilibrium dissociation constants were 0.1-50 μM for 28 peptides, and binding was negatively regulated by phosphorylation for many peptides. These results identify cytoplasmic proteins with potential for regulating the localization of Syk in a phosphorylation-dependent manner to nonmembrane cellular regions.

Structure and Dynamics of the CCCH-Type Tandem Zinc Finger Domain of POS-1 and Implications for RNA Binding Specificity.

CCCH-type tandem zinc finger (TZF) motifs are found in many RNA-binding proteins involved in regulating mRNA stability, translation, and splicing. In Caenorhabditis elegans, several RNA-binding proteins that regulate embryonic development and cell fate determination contain CCCH TZF domains, including POS-1. Previous biochemical studies have shown that despite high levels of sequence conservation, POS-1 recognizes a broader set of RNA sequences compared to the human homologue tristetraprolin. However, the molecular basis of these differences remains unknown. In this study, we refined the consensus RNA sequence and determined the differing binding specificities of the two zinc fingers of POS-1. We also determined the solution structure and characterized the internal dynamics of the TZF domain of POS-1. From the structure, we identified unique features that define the RNA binding specificity of POS-1. We also observed that the TZF domain of POS-1 is in equilibrium between interconverting conformations. Transitions between these conformations require internal motions involving many residues with correlated dynamics in each ZF. We propose that the correlated dynamics are necessary to allow allosteric communication between the nucleotide-binding pockets observed in the N-terminal ZF. Our study shows that both the structure and conformational plasticity of POS-1 are important in ensuring recognition of its RNA binding targets.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind CaV2 calcium channel C-termini in vitro

PDZ domain mediated interactions with voltage-gated calcium (CaV) channel C-termini play important roles in localizing membrane Ca2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and CaV2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to CaV2 chanels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and CaV2 channels from cnidarians and placozoans interact in vitro, and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis. Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly bound the divergent C-terminal ligands of cnidarian and placozoan CaV2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore CaV2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates bilaterian animals, and that evolutionary changes in CaV2 channel C-terminal sequences resulted in altered binding modalities with Mint.

Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood. Syt1 has additional roles in docking dense core vesicles (DCV) and synaptic vesicles (SV) to the plasma membrane (PM) and in regulating fusion pore dynamics. Thus, Syt1 perturbations could affect release through vesicle docking, fusion triggering, fusion pore regulation, or a combination of these. Here, using a human neuroendocrine cell line, we show that neutralization of highly conserved polybasic patches in either C2 domain of Syt1 impairs both DCV docking and efficient release of serotonin from DCVs. Interestingly, the same mutations resulted in larger fusion pores and faster release of serotonin during individual fusion events. Thus, Syt1's roles in vesicle docking, fusion triggering, and fusion pore control may be functionally related.

Solution NMR backbone assignment of the N-terminal tandem Zα1-Zα2 domains of Z-DNA binding protein 1.

The detection of nucleic acids that are present in atypical conformations is a crucial trigger of the innate immune response. Human Z-DNA binding protein 1 (ZBP1) is a pattern recognition receptor that harbors two Zα domains that recognize Z-DNA and Z-RNA. ZBP1 detects this alternate nucleic acid conformation as foreign, and upon stabilization of these substrates, it triggers activation of an immune response. Here, we present the backbone chemical shift assignment of a construct encompassing the Zα1 and Zα2 domains as well as the interconnecting linker of ZBP1. These assignments can be directly transferred to the isolated Zα1 and Zα2 domains, thereby demonstrating that these domains maintain virtually identical structures in the tandem context.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind Ca V 2 calcium channel C-termini in vitro .

PDZ domain mediated interactions with voltage-gated calcium (Ca V ) channel C-termini play important roles in localizing membrane Ca 2+ signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca V 2.2 in mammals. In this study, we show through various in silico analyses that Mint is an animal-specific gene with a highly divergent N-terminus but a strongly conserved C-terminus comprised of a phosphotyrosine binding domain, two tandem PDZ domains (PDZ-1 and PDZ-2), and a C-terminal auto-inhibitory element that binds and inhibits PDZ-1. In addition to Ca V 2 channels, most genes that interact with Mint are also deeply conserved including amyloid precursor proteins, presenilins, neurexin, and CASK and Veli which form a tripartite complex with Mint in bilaterians. Through yeast and bacterial 2-hybrid experiments, we show that Mint and Ca V 2 channels from cnidarians and placozoans interact in vitro , and in situ hybridization revealed co-expression in dissociated neurons from the cnidarian Nematostella vectensis . Unexpectedly, the Mint orthologue from the ctenophore Hormiphora californiensis strongly binds the divergent C-terminal ligands of cnidarian and placozoan Ca V 2 channels, despite neither the ctenophore Mint, nor the placozoan and cnidarian orthologues, binding the ctenophore Ca V 2 channel C-terminus. Altogether, our analyses suggest that the capacity of Mint to bind CaV2 channels predates pre-bilaterian animals, and that evolutionary changes in Ca V 2 channel C-terminal sequences resulted in altered binding modalities with Mint.

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding.

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

H3K14ac facilitates the reinstallation of constitutive heterochromatin in Drosophila early embryos by engaging Eggless/SetDB1

Constitutive heterochromatin, a fundamental feature of eukaryotic nucleus essential for transposon silencing and genome stability, is rebuilt on various types of repetitive DNA in the zygotic genome during early embryogenesis. However, the molecular program underlying this process remains poorly understood. Here, we show that histone H3 lysine 14 acetylation (H3K14ac) is engaged in the reinstallation of constitutive heterochromatin in Drosophila early embryos. H3K14ac partially colocalizes with H3 lysine 9 trimethylation (H3K9me3) and its methyltransferase Eggless/SetDB1 around the mid-blastula transition. Concealing H3K14ac by either antibody injection or maternal knockdown of Gcn5 diminishes Eggless/SetDB1 nuclear foci and reduces the deposition of H3K9me3. Structural analysis reveals that Eggless/SetDB1 recognizes H3K14ac via its tandem Tudor domains, and disrupting the binding interface causes defects in Eggless/SetDB1 distribution and derepression of a subset of transposons. Therefore, H3K14ac, a histone modification normally associated with active transcription, is a crucial component of the early embryonic machinery that introduces constitutive heterochromatic features to the newly formed zygotic genome.

Structure, biosynthesis and regulation of the T1 antigen, a phase-variable surface polysaccharide conserved in many Salmonella serovars

The bacterial genus Salmonella includes diverse isolates with multiple variations in the structure of the main polysaccharide component (O antigen) of membrane lipopolysaccharides. In addition, some isolates produce a transient (T) antigen, such as the T1 polysaccharide identified in the 1960s in an isolate of Salmonella enterica Paratyphi B. The structure and biosynthesis of the T1 antigen have remained enigmatic. Here, we use biophysical, biochemical and genetic methods to show that the T1 antigen is a complex linear glycan containing tandem homopolymeric domains of galactofuranose and ribofuranose, linked to lipid A–core, like a typical O antigen. T1 is a phase-variable antigen, regulated by recombinational inversion of the promoter upstream of the T1 genetic locus through a mechanism not observed for other bacterial O antigens. The T1 locus is conserved across many Salmonella isolates, but is mutated or absent in most typhoidal serovars and in serovar Enteritidis.

Conserved cysteines prevent C-mannosylation of mucin Cys domains.

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucinCysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

Thermodynamic coupling of the tandem RRM domains of hnRNP A1 underlie its pleiotropic RNA binding functions.

The functional properties of RNA binding proteins (RBPs) require allosteric regulation through interdomain communication. Despite the importance of allostery to biological regulation, only a few studies have been conducted to describe the biophysical nature by which interdomain communication manifests in RBPs. Here, we show for hnRNP A1 that interdomain communication is vital for the unique stability of its amino-terminal domain, which consists of two RNA recognition motifs (RRMs). These RRMs exhibit drastically different stability under pressure. RRM2 unfolds as an individual domain but remains stable when appended to RRM1. Variants that disrupt interdomain communication between the tandem RRMs show a significant decrease in stability. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered carboxyl-terminal domain to engage in protein-protein interactions and influenced the protein's RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.