The MCU and MCUb amino-terminal domains tightly interact: mechanisms for low conductance assembly of the mitochondrial calcium uniporter complex.

The mitochondrial calcium (Ca2+) uniporter (MCU) complex is regulated via integration of the MCU dominant negative beta subunit (MCUb), a low conductance paralog of the main MCU pore forming protein. The MCU amino (N)-terminal domain (NTD) also modulates channel function through cation binding to the MCU regulating acidic patch (MRAP). MCU and MCUb have high sequence similarities, yet the structural and functional roles of MCUb-NTD remain unknown. Here, we report that MCUb-NTD exhibits α-helix/ß-sheet structure with a high thermal stability, dependent on protein concentration. Remarkably, MCU- and MCUb-NTDs heteromerically interact with ∼nM affinity, increasing secondary structure and stability and structurally perturbing MRAP. Further, we demonstrate MCU and MCUb co-localization is suppressed upon NTD deletion concomitant with increased mitochondrial Ca2+ uptake. Collectively, our data show that MCU:MCUb NTD tight interactions are promoted by enhanced regular structure and stability, augmenting MCU:MCUb co-localization, lowering mitochondrial Ca2+ uptake and implicating an MRAP-sensing mechanism.

Mitigation of TDP-43 toxic phenotype by an RGNEF fragment in amyotrophic lateral sclerosis models.

Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction. Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers. Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.

Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate and harbors a Zn2+ ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives. High-resolution crystal structures of GmhA complexed to two N-formyl hydroxamate inhibitors confirmed the binding interactions to a central Zn2+ ion coordination site. Some of these compounds were found to be nanomolar inhibitors of GmhA. While devoid of HepG2 cytotoxicity and antibacterial activity of their own, they demonstrated in vitro lipopolysaccharide heptosylation inhibition in Enterobacteriaceae as well as the potentiation of erythromycin and rifampicin in a wild-type Escherichia coli strain. These inhibitors pave the way for a novel treatment of Gram-negative infections.

Role of Half-of-Sites Reactivity and Inter-Subunit Communications in DAHP Synthase Catalysis and Regulation.

α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.

Perceptions of using infographics for scientific communication on social media for COVID-19 topics: a survey study.

Navigating for accurate information, especially health- and science-related content, on social media has been challenging during the COVID-19 pandemic. Although infographics are a popular medium for simplifying text-based information into visual components, their usefulness during a global health crisis has not been explored. The study aims to explore the perceptions of infographics in conveying scientific information related to COVID-19 on social media. Following a social media campaign that published COVID-19 related infographics from May to August 2020, a cross-sectional survey was administered to social media users, primarily students from Western University. Several questions asked respondents to make comparisons with written articles when reporting their perceptions of infographics. Seventy-three percent of students from 361 responses belonged to health-related academic backgrounds. Seventy-two percent felt more likely to share infographics than written articles on social media due to the visual appeal. Nearly 90% felt it was easier to navigate through complicated science and that more scientists should use infographics on social media. Educational background did not influence the perceived usefulness of infographics in understanding scientific information. Infographics are perceived favourably in conveying scientific information about COVID-19 on social media. Findings from this study can inform communication strategies during a pandemic and, more broadly, global crises.

A novel fluorescent reporter sensitive to serine mis-incorporation.

High-fidelity translation was considered a requirement for living cells. The frozen accident theory suggested that any deviation from the standard genetic code should result in the production of so much mis-made and non-functional proteins that cells cannot remain viable. Studies in bacterial, yeast, and mammalian cells show that significant levels of mistranslation (1-10% per codon) can be tolerated or even beneficial under conditions of oxidative stress. Single tRNA mutants, which occur naturally in the human population, can lead to amino acid mis-incorporation at a codon or set of codons. The rate or level of mistranslation can be difficult or impossible to measure in live cells. We developed a novel red fluorescent protein reporter that is sensitive to serine (Ser) mis-incorporation at proline (Pro) codons. The mCherry Ser151Pro mutant is efficiently produced in Escherichia coli but non-fluorescent. We demonstrated in cells and with purified mCherry protein that the fluorescence of mCherry Ser151Pro is rescued by two different tRNASer gene variants that were mutated to contain the Pro (UGG) anticodon. Ser mis-incorporation was confirmed by mass spectrometry. Remarkably, E. coli tolerated mistranslation rates of ~10% per codon with negligible reduction in growth rate. Conformational sampling simulations revealed that the Ser151Pro mutant leads to significant changes in the conformational freedom of the chromophore precursor, which is indicative of a defect in chromophore maturation. Together our data suggest that the mCherry Ser151 mutants may be used to report Ser mis-incorporation at multiple other codons, further expanding the ability to measure mistranslation in living cells.

An Inhibitor-in-Pieces Approach to DAHP Synthase Inhibition: Potent Enzyme and Bacterial Growth Inhibition.

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate biosynthetic pathway and is an antimicrobial target. We used an inhibitor-in-pieces approach, based on the previously reported inhibitor DAHP oxime, to screen inhibitor fragments in the presence and absence of glycerol 3-phosphate to occupy the distal end of the active site. This led to DAHP hydrazone, the most potent inhibitor to date, Ki = 10 ± 1 nM. Three trifluoropyruvate (TFP)-based inhibitor fragments were efficient inhibitors with ligand efficiencies of up to 0.7 kcal mol-1/atom compared with 0.2 kcal mol-1/atom for a typical good inhibitor. The crystal structures showed the TFP-based inhibitors binding upside down in the active site relative to DAHP oxime, providing new avenues for inhibitor development. The ethyl esters of TFP oxime and TFP semicarbazone prevented E. coli growth in culture with IC50 = 0.21 ± 0.01 and 0.77 ± 0.08 mg mL-1, respectively. Overexpressing DAHP synthase relieved growth inhibition, demonstrating that DAHP synthase was the target. Growth inhibition occurred in media containing aromatic amino acids, suggesting that growth inhibition was due to depletion of some other product(s) of the shikimate pathway, possibly folate.

Identification of Bioactive SNM1A Inhibitors.

SNM1A is a nuclease required to repair DNA interstrand cross-links (ICLs) caused by some anticancer compounds, including cisplatin. Unlike other nucleases involved in ICL repair, SNM1A is not needed to restore other forms of DNA damage. As such, SNM1A is an attractive target for selectively increasing the efficacy of ICL-based chemotherapy. Using a fluorescence-based exonuclease assay, we screened a bioactive library of compounds for inhibition of SNM1A. Of the 52 compounds initially identified as hits, 22 compounds showed dose-response inhibition of SNM1A. An orthogonal gel-based assay further confirmed nine small molecules as SNM1A nuclease activity inhibitors with IC50 values in the mid-nanomolar to low micromolar range. Finally, three compounds showed no toxicity at concentrations able to significantly potentiate the cytotoxicity of cisplatin. These compounds represent potential leads for further optimization to sensitize cells toward chemotherapeutic agents inducing ICL damage.

Broad and Differential Animal Angiotensin-Converting Enzyme 2 Receptor Usage by SARS-CoV-2.

The COVID-19 pandemic has caused an unprecedented global public health and economic crisis. The origin and emergence of its causal agent, SARS-CoV-2, in the human population remains mysterious, although bat and pangolin were proposed to be the natural reservoirs. Strikingly, unlike the SARS-CoV-2-like coronaviruses (CoVs) identified in bats and pangolins, SARS-CoV-2 harbors a polybasic furin cleavage site in its spike (S) glycoprotein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as its receptor to infect cells. Receptor recognition by the S protein is the major determinant of host range, tissue tropism, and pathogenesis of coronaviruses. In an effort to search for the potential intermediate or amplifying animal hosts of SARS-CoV-2, we examined receptor activity of ACE2 from 14 mammal species and found that ACE2s from multiple species can support the infectious entry of lentiviral particles pseudotyped with the wild-type or furin cleavage site-deficient S protein of SARS-CoV-2. ACE2 of human/rhesus monkey and rat/mouse exhibited the highest and lowest receptor activities, respectively. Among the remaining species, ACE2s from rabbit and pangolin strongly bound to the S1 subunit of SARS-CoV-2 S protein and efficiently supported the pseudotyped virus infection. These findings have important implications for understanding potential natural reservoirs, zoonotic transmission, human-to-animal transmission, and use of animal models.IMPORTANCE SARS-CoV-2 uses human ACE2 as a primary receptor for host cell entry. Viral entry mediated by the interaction of ACE2 with spike protein largely determines host range and is the major constraint to interspecies transmission. We examined the receptor activity of 14 ACE2 orthologs and found that wild-type and mutant SARS-CoV-2 lacking the furin cleavage site in S protein could utilize ACE2 from a broad range of animal species to enter host cells. These results have important implications in the natural hosts, interspecies transmission, animal models, and molecular basis of receptor binding for SARS-CoV-2.

Structural Insights Into PfARO and Characterization of its Interaction With PfAIP.

Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins.

Mimicking the human environment in mice reveals that inhibiting biotin biosynthesis is effective against antibiotic-resistant pathogens.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets1. In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection2-5, making it a high-priority target. However, biotin biosynthesis has been overlooked for priority pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. This can be attributed to the lack of attenuation observed for biotin biosynthesis genes during transposon mutagenesis studies in mouse infection models6-9. Previous studies did not consider the 40-fold higher concentration of biotin in mouse plasma compared to human plasma. Here, we leveraged the unique affinity of streptavidin to develop a mouse infection model with human levels of biotin. Our model suggests that biotin biosynthesis is essential during infection with A. baumannii, K. pneumoniae and P. aeruginosa. Encouragingly, we establish the capacity of our model to uncover in vivo activity for the biotin biosynthesis inhibitor MAC13772. Our model addresses the disconnect in biotin levels between humans and mice, and explains the failure of potent biotin biosynthesis inhibitors in standard mouse infection models.

Modifying a covarying protein-DNA interaction changes substrate preference of a site-specific endonuclease.

Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein-DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

NeuNAc Oxime: A Slow-Binding and Effectively Irreversible Inhibitor of the Sialic Acid Synthase NeuB.

NeuB is a bacterial sialic acid synthase used by neuroinvasive bacteria to synthesize N-acetylneuraminate (NeuNAc), helping them to evade the host immune system. NeuNAc oxime is a potent slow-binding NeuB inhibitor. It dissociated too slowly to be detected experimentally, with initial estimates of its residence time in the active site being >47 days. This is longer than the lifetime of a typical bacterial cell, meaning that inhibition is effectively irreversible. Inhibition data fitted well to a model that included a pre-equilibration step with a Ki of 36 µM, followed by effectively irreversible conversion to an E*·I complex, with a k2 of 5.6 × 10-5 s-1. Thus, the inhibitor can subvert ligand release and achieve extraordinary residence times in spite of a relatively modest initial dissociation constant. The crystal structure showed the oxime functional group occupying the phosphate-binding site normally occupied by the substrate PEP and the tetrahedral intermediate. There was an ≈10% residual rate at high inhibitor concentrations regardless of how long NeuB and NeuNAc oxime were preincubated together. However, complete inhibition was achieved by incubating NeuNAc oxime with the actively catalyzing enzyme. This requirement for the enzyme to be actively turning over for the inhibitor to bind to the second subunit demonstrated an important role for intersubunit communication in the inhibitory mechanism.

Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage.

Repair of two major forms of DNA damage, single strand breaks and base modifications, are dependent on XRCC1. XRCC1 orchestrates these repair processes by temporally and spatially coordinating interactions between several other repair proteins. Here we show that XRCC1 contains a central DNA binding domain (CDB, residues 219-415) encompassing its first BRCT domain. In contrast to the N-terminal domain of XRCC1, which has been reported to mediate damage sensing in vitro, we demonstrate that the DNA binding module identified here lacks binding specificity towards DNA containing nicks or gaps. Alanine substitution of residues within the CDB of XRCC1 disrupt DNA binding in vitro and lead to a significant reduction in XRCC1 retention at DNA damage sites without affecting initial recruitment. Interestingly, reduced retention at sites of DNA damage is associated with an increased rate of repair. These findings suggest that DNA binding activity of XRCC1 plays a significant role in retention at sites of damage and the rate at which damage is repaired.

Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases.

LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.

Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink.

DNA interstrand crosslinks (ICLs) covalently join opposing strands, blocking both replication and transcription, therefore making ICL-inducing compounds highly toxic and ideal anti-cancer agents. While incisions surrounding the ICL are required to remove damaged DNA, it is currently unclear which endonucleases are needed for this key event. SNM1A has been shown to play an important function in human ICL repair, however its suggested role has been limited to exonuclease activity and not strand incision. Here we show that SNM1A has endonuclease activity, having the ability to cleave DNA structures that arise during the initiation of ICL repair. In particular, this endonuclease activity cleaves single-stranded DNA. Given that unpaired DNA regions occur 5' to an ICL, these findings suggest SNM1A may act as either an endonuclease and/or exonuclease during ICL repair. This finding is significant as it expands the potential role of SNM1A in ICL repair.

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic.

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn2+, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with Ki = 1.5 ± 0.4 µM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, tR, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The kcat value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent KM,E4P value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.

Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations.

Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a ß-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptake rates. Collectively, our data reveal that the ß-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

UNLABELLED: FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCE: FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB.

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.