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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 Zn 2+ 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 Zn 2+ 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.

α-Carboxyketose synthases, including 3-deoxy-d- arabino heptulosonate 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.

3-Deoxy-d- arabino heptulosonate-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, K i = 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 IC 50 = 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.

To revitalize the antibiotic pipeline, it is critical to identify and validate new antimicrobial targets 1 . In Mycobacteria tuberculosis and Francisella tularensis, biotin biosynthesis is a key fitness determinant during infection 2-5 , 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 models 6-9 . 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.

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 K i of 36 μM, followed by effectively irreversible conversion to an E*·I complex, with a k 2 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.

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.

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.

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.

Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilO Δ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous β-strand in the second αββ motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.

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, Mn 2+ , binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with K i = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, t R , 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 k cat value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent K M,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.

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 β-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 β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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.

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilEΔ1-28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilXNm and PilVNm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilXNm and PilVNm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A number of Gram-negative pathogens utilize type III secretion systems (T3SSs) to inject bacterial effector proteins into the host. An important component of T3SSs is a conserved ATPase that captures chaperone-effector complexes and energizes their dissociation to facilitate effector translocation. To date, there has been limited work characterizing the chaperone-T3SS ATPase interaction despite it being a fundamental aspect of T3SS function. In this study, we present the 2.1 Å resolution crystal structure of the Salmonella enterica SPI-2-encoded ATPase, SsaN. Our structure revealed a local and functionally important novel feature in helix 10 that we used to define the interaction domain relevant to chaperone binding. We modeled the interaction between the multicargo chaperone, SrcA, and SsaN and validated this model using mutagenesis to identify the residues on both the chaperone and ATPase that mediate the interaction. Finally, we quantified the benefit of this molecular interaction on bacterial fitness in vivo using chromosomal exchange of wild-type ssaN with mutants that retain ATPase activity but no longer capture the chaperone. Our findings provide insight into chaperone recognition by T3SS ATPases and demonstrate the importance of the chaperone-T3SS ATPase interaction for the pathogenesis of Salmonella., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair., (Copyright © 2013 Wiley Periodicals, Inc.)

The study of biological transporters can be hampered by a dearth of methodology for tracking their activity within cells. Here, we present a means of monitoring the function of transport machinery within bacteria, exploiting a genetically encoded riboswitch-based sensor to detect the accumulation of the substrate in the cytoplasm. This method was used to investigate the model ABC transporter BtuC2D2F, which permits vitamin B12 uptake in Escherichia coli. We exploited the wealth of structural data available for this transporter to probe the functional and mechanistic importance of key residues of the substrate binding protein BtuF that are predicted to support its interaction with its substrate or with the BtuC channel-forming subunits. Our results reveal molecular interaction requirements for substrate binding proteins and demonstrate the utility of riboswitch-based sensors in the study of biological transport., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ability of Deinococcus radiodurans to recover from extensive DNA damage is due in part to its ability to efficiently repair its genome, even following severe fragmentation by hundreds of double-strand breaks. The single-strand annealing pathway plays an important role early during the recovery process, making use of a protein, DdrB, shown to greatly stimulate ssDNA annealing. Here, we report the structure of DdrB bound to ssDNA to 2.3 Å. Pentameric DdrB was found to assemble into higher-order structures that coat ssDNA. To gain further mechanistic insight into the protein's function, a number of point mutants were generated altering both DNA binding and higher order oligomerization. This work not only identifies higher-order DdrB associations but also suggests the presence of an extended DNA binding surface running along the 'top' surface of a DdrB pentamer and continuing down between two individual subunits of the ring structure. Together this work sheds new insight into possible mechanisms for DdrB function in which higher-order assemblies of DdrB pentamers assist in the pairing of complementary ssDNA using an extended DNA binding surface.

Background: Heparin cofactor II (HCII) is a circulating protease inhibitor, one which contains an N-terminal acidic extension (HCII 1-75) unique within the serpin superfamily. Deletion of HCII 1-75 greatly reduces the ability of glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, and abrogates HCII binding to thrombin exosite 1. While a minor portion of HCII 1-75 can be visualized in a crystallized HCII-thrombin S195A complex, the role of the rest of the extension is not well understood and the affinity of the HCII 1-75 interaction has not been quantitatively characterized. To address these issues, we expressed HCII 1-75 as a small, N-terminally hexahistidine-tagged polypeptide in E. coli., Results: Immobilized purified HCII 1-75 bound active α-thrombin and active-site inhibited FPR-ck- or S195A-thrombin, but not exosite-1-disrupted γT-thrombin, in microtiter plate assays. Biotinylated HCII 1-75 immobilized on streptavidin chips bound α-thrombin and FPR-ck-thrombin with similar KD values of 330-340 nM. HCII 1-75 competed thrombin binding to chip-immobilized HCII 1-75 more effectively than HCII 54-75 but less effectively than the C-terminal dodecapeptide of hirudin (mean Ki values of 2.6, 8.5, and 0.29 μM, respectively). This superiority over HCII 54-75 was also demonstrated in plasma clotting assays and in competing the heparin-catalysed inhibition of thrombin by plasma-derived HCII; HCII 1-53 had no effect in either assay. Molecular modelling of HCII 1-75 correctly predicted those portions of the acidic extension that had been previously visualized in crystal structures, and suggested that an α-helix found between residues 26 and 36 stabilizes one found between residues 61-67. The latter region has been previously shown by deletion mutagenesis and crystallography to play a crucial role in the binding of HCII to thrombin exosite 1., Conclusions: Assuming that the KD value for HCII 1-75 of 330-340 nM faithfully predicts that of this region in intact HCII, and that 1-75 binding to exosite 1 is GAG-dependent, our results support a model in which thrombin first binds to GAGs, followed by HCII addition to the ternary complex and release of HCII 1-75 for exosite 1 binding and serpin mechanism inhibition. They further suggest that, in isolated or transferred form, the entire HCII 1-75 region is required to ensure maximal binding of thrombin exosite 1.

We report here the optimization of an HldE kinase inhibitor to low nanomolar potency, which resulted in the identification of the first reported compounds active on selected E. coli strains. One of the most interesting candidates, compound 86, was shown to inhibit specifically bacterial LPS heptosylation on efflux pump deleted E. coli strains. This compound did not interfere with E. coli bacterial growth (MIC > 32 μg/mL) but sensitized this pathogen to hydrophobic antibiotics like macrolides normally inactive on Gram-negative bacteria. In addition, 86 could sensitize E. coli to serum complement killing. These results demonstrate that HldE kinase is a suitable target for drug discovery. They also pave the way toward novel possibilities of treating or preventing bloodstream infections caused by pathogenic Gram negative bacteria by inhibiting specific virulence factors.

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5 Å resolution X-ray structure shows that a single region from the AHNAK C terminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30 nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Type III secretion (T3S) is an essential virulence factor used by gram-negative pathogenic bacteria to deliver effector proteins into the host cell to establish and maintain an intracellular infection. Chlamydia is known to use T3S to facilitate invasion of host cells but many proteins in the system remain uncharacterized. The C. trachomatis protein CT584 has previously been implicated in T3S. Thus, we analyzed the CT584 ortholog in C. pneumoniae (Cpn0803) and found that it associates with known T3S proteins including the needle-filament protein (CdsF), the ATPase (CdsN), and the C-ring protein (CdsQ). Using membrane lipid strips, Cpn0803 interacted with phosphatidic acid and phosphatidylinositol, suggesting that Cpn0803 may associate with host cells. Crystallographic analysis revealed a unique structure of Cpn0803 with a hydrophobic pocket buried within the dimerization interface that may be important for binding small molecules. Also, the binding domains on Cpn0803 for CdsN, CdsQ, and CdsF were identified using Pepscan epitope mapping. Collectively, these data suggest that Cpn0803 plays a role in T3S.

XRCC4 and XLF are key proteins in the repair of DNA double-strand breaks through nonhomologous end-joining. Together, they form a complex that stimulates the ligation of double-strand breaks. Owing to the suggested filamentous nature of this complex, structural studies via X-ray crystallography have proven difficult. Multiple truncations of the XLF and XRCC4 proteins were cocrystallized, but yielded low-resolution diffraction (~20 Å). However, a combination of microseeding, dehydration and heavy metals improved the diffraction of XRCC4(Δ157)-XLF(Δ224) crystals to 3.9 Å resolution. Although molecular replacement alone was unable to produce a solution, when combined with the anomalous signal from tantalum bromide clusters initial phasing was successfully obtained., (© 2011 International Union of Crystallography. All rights reserved.)

Background: PH domains represent one of the most common domains in the human proteome. These domains are recognized as important mediators of protein-phosphoinositide and protein-protein interactions. Phosphoinositides are lipid components of the membrane that function as signaling molecules by targeting proteins to their sites of action. Phosphoinositide based signaling pathways govern a diverse range of important cellular processes including membrane remodeling, differentiation, proliferation and survival. Myo-Inositol phosphates are soluble signaling molecules that are structurally similar to the head groups of phosphoinositides. These molecules have been proposed to function, at least in part, by regulating PH domain-phosphoinositide interactions. Given the structural similarity of inositol phosphates we were interested in examining the specificity of PH domains towards the family of myo-inositol pentakisphosphate isomers., Results: In work reported here we demonstrate that the C-terminal PH domain of pleckstrin possesses the specificity required to discriminate between different myo-inositol pentakisphosphate isomers. The structural basis for this specificity was determined using high-resolution crystal structures. Moreover, we show that while the PH domain of Grp1 does not possess this high degree of specificity, the PH domain of protein kinase B does., Conclusions: These results demonstrate that some PH domains possess enough specificity to discriminate between myo-inositol pentakisphosphate isomers allowing for these molecules to differentially regulate interactions with phosphoinositides. Furthermore, this work contributes to the growing body of evidence supporting myo-inositol phosphates as regulators of important PH domain-phosphoinositide interactions. Finally, in addition to expanding our knowledge of cellular signaling, these results provide a basis for developing tools to probe biological pathways.

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 μM), with the latter two values being in close agreement with literature values.

Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 A structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 A revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS.

MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

Background: The Actinobacteria phylum represents one of the largest and most diverse groups of bacteria, encompassing many important and well-characterized organisms including Streptomyces, Bifidobacterium, Corynebacterium and Mycobacterium. Members of this phylum are remarkably diverse in terms of life cycle, morphology, physiology and ecology. Recent comparative genomic analysis of 19 actinobacterial species determined that only 5 genes of unknown function uniquely define this large phylum 1. The cellular functions of these actinobacteria-specific proteins (ASP) are not known., Results: Here we report the first characterization of one of the 5 actinobacteria-specific proteins, ASP1 (Gene ID: SCO1997) from Streptomyces coelicolor. The X-ray crystal structure of ASP1 was determined at 2.2 A. The overall structure of ASP1 retains a similar fold to the large NP-1 family of nucleoside phosphorylase enzymes; however, the function is not related. Further comparative analysis revealed two regions expected to be important for protein function: a central, divalent metal ion binding pore, and a highly conserved elbow shaped helical region at the C-terminus. Sequence analyses revealed that ASP1 is paralogous to another actinobacteria-specific protein ASP2 (SCO1662 from S. coelicolor) and that both proteins likely carry out similar function., Conclusion: Our structural data in combination with sequence analysis supports the idea that two of the 5 actinobacteria-specific proteins, ASP1 and ASP2, mediate similar function. This function is predicted to be novel since the structures of these proteins do not match any known protein with or without known function. Our results suggest that this function could involve divalent metal ion binding/transport.

Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism.

The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.

DNA double-strand breaks represent one of the most severe forms of DNA damage in mammalian cells. One pathway for repairing these breaks occurs via nonhomologous end-joining (NHEJ) and depends on XRCC4, LigaseIV, and Cernunnos, also called XLF. Although XLF stimulates XRCC4/LigaseIV to ligate mismatched and noncohesive DNA ends, the mechanistic basis for this function remains unclear. Here we report the structure of a partially functional 224 residue N-terminal fragment of human XLF. Despite only weak sequence similarity, XLF(1-170) shares structural homology with XRCC4(1-159). However, unlike the highly extended 130 A helical domain observed in XRCC4, XLF adopts a more compact, folded helical C-terminal region involving two turns and a twist, wrapping back to the structurally conserved N terminus. Mutational analysis of XLF and XRCC4 reveals a potential interaction interface, suggesting a mechanism for how XLF stimulates the ligation of mismatched ends.

Background: Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains., Results: In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 muM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 A. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate., Conclusion: The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules.

The biosynthesis of methionine is an attractive antibiotic target given its importance in protein and DNA metabolism and its absence in mammals. We have performed a high-throughput screen of the methionine biosynthesis enzyme cystathionine beta-lyase (CBL) against a library of 50 000 small molecules and have identified several compounds that inhibit CBL enzyme activity in vitro. These hit molecules were of two classes: those that blocked CBL activity with mixed steady-state inhibition and those that covalently interacted with the enzyme at the active site pyridoxal phosphate cofactor with slow-binding inhibition kinetics. We determined the crystal structure of one of the slow-binding inhibitors in complex with CBL and used this structure as a guide in the synthesis of a small, focused library of analogues, some of which had improved enzyme inhibition properties. These studies provide the first lead molecules for antimicrobial agents that target cystathionine beta-lyase in methionine biosynthesis.

Dihydrofolate reductase (DHFR) is a vital metabolic enzyme and thus a clinically prominent target in the design of antimetabolites. In this work, we identify 1,4-bis-{[N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (compound 1) as the correct structure of the previously reported DHFR inhibitor 1,4-bis-{(iminothioureidomethyl)aminomethyl}-3,6-dimethyl-benzene (compound 2). The fact that compound 1 has an uncharacteristic structure for DHFR inhibitors, and an affinity (KI of 11.5 nM) comparable to potent inhibitors such as methotrexate and trimethoprim, made this inhibitor of interest for further analysis. We have conducted a characterization of the primary interactions of compound 1 and DHFR using a combination of X-ray structure and SAR analysis. The crystal structure of E. coli DHFR in complex with compound 1 and NADPH reveals that one portion of this inhibitor exploits a unique binding surface, the M20 loop. The importance of this interface was further confirmed by SAR analysis and additional structural characterization.

Pleckstrin is an important intracellular protein involved in the phosphoinositide-signalling pathways of platelet activation. This protein contains both N- and C-terminal pleckstrin-homology (PH) domains (N-PH and C-PH). The crystal structure of C-PH was solved by molecular replacement and refined at 2.1 Angstroms resolution. Two molecules were observed within the asymmetric unit and it is proposed that the resulting dimer interface could contribute to the previously observed oligomerization of pleckstrin in resting platelets. Structural comparisons between the phosphoinositide-binding loops of the C-PH crystal structure and the PH domains of DAPP1 and TAPP1, the N-terminal PH domain of pleckstrin and a recently described solution structure of C-PH are presented and discussed.

The XRCC4 protein is of critical importance for the repair of broken chromosomal DNA by non-homologous end joining (NHEJ). The absence of XRCC4 abolishes chromosomal NHEJ almost completely. One reason for this severe phenotype is that XRCC4 binds and modulates the stability and activity of the NHEJ-specific ligase, DNA ligase IV. XRCC4 in solution is in equilibrium between the dimeric and tetrameric forms. Previous structural studies have shown that the interface between dimers is located in the same region as that implicated in DNA ligase IV interaction. With the use of equilibrium sedimentation analysis, we show here that only the XRCC4 dimer can associate with DNA ligase IV, forming a monodisperse complex of 2:1 stoichiometry in solution. In addition, physical analysis of XRCC4/DNA ligase IV complex formation, combined with mutational analysis of XRCC4, indicates that tetramerization and DNA ligase IV binding are mutually exclusive. We propose that the putative function of the XRCC4 tetramer is distinct from its DNA ligase IV-associated function.

We have used the recently determined crystal structures of Escherichia coli (E. coli) MutS, MutL and MutH to guide construction of 47 amino-acid substitutions in these proteins and analyzed their behavior in mismatch repair and recombination in vitro and in vivo. We find that the active site of the MutH endonuclease is composed of regions from two separate structural domains and that the C-terminal 5 residues of MutH influence both DNA binding and cleavage. We also find that the non-specific DNA-binding activity of MutL is required for mismatch repair and probably functions after strand cleavage by MutH. Alteration of residues in either the mismatch recognition domain, the ATPase active site, or the domain interfaces linking the two activities can diminish the differential binding of MutS to homoduplex versus heteroduplex and results in the loss of mismatch-specific MutH activation. Finally, every mutation that abolishes mismatch repair is deficient in blocking homeologous recombination, suggesting that mismatch repair and prevention of homeologous recombination use the same MutS-MutL complexes for signaling in E. coli.

Human MutLalpha, a heterodimer of hMLH1 and hPMS2, is essential for DNA mismatch repair. Inactivation of the hmlh1 or hpms2 genes by mutation or epigenesis causes genomic instability and a predisposition to hereditary non-polyposis cancer. We report here the X-ray crystal structures of the conserved N-terminal 40 kDa fragment of hPMS2, NhPMS2, and its complexes with ATPgammaS and ADP at 1.95, 2.7 and 2.7 A resolution, respectively. The NhPMS2 structures closely resemble the ATPase fragment of Escherichia coli MutL, which coordinates protein-protein interactions in mismatch repair by undergoing structural transformation upon binding of ATP. Unlike the E.coli MutL, whose ATPase activity requires protein dimerization, the monomeric form of NhPMS2 is active both in ATP hydrolysis and DNA binding. NhPMS2 is the first example of a GHL ATPase active as a monomer, suggesting that its activity may be modulated by hMLH1 in MutLalpha, and vice versa. The potential heterodimer interface revealed by crystallography provides a mutagenesis target for functional studies of MutLalpha.

The MutS protein initiates DNA mismatch repair by recognizing mispaired and unpaired bases embedded in duplex DNA and activating endo- and exonucleases to remove the mismatch. Members of the MutS family also possess a conserved ATPase activity that belongs to the ATP binding cassette (ABC) superfamily. Here we report the crystal structure of a ternary complex of MutS-DNA-ADP and assays of initiation of mismatch repair in conjunction with perturbation of the composite ATPase active site by mutagenesis. These studies indicate that MutS has to bind both ATP and the mismatch DNA simultaneously in order to activate the other mismatch repair proteins. We propose that the MutS ATPase activity plays a proofreading role in DNA mismatch repair, verification of mismatch recognition, and authorization of repair.

XRCC4 is essential for carrying out non-homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4-defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 A resolution. Xrcc4 protein forms a strikingly elongated dumb-bell-like tetramer. Each of the N-terminal globular head domains consists of a beta-sandwich and a potentially DNA-binding helix- turn-helix motif. The C-terminal stalk comprising a single alpha-helix >120 A in length is partly incorporated into a four-helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV. The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in 'divalent metal ion-dependent' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.