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Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought., Author summary Spore germination is essential for many spore-forming pathogens to initiate infection. In order for spores to germinate, they must degrade a thick, protective layer of cell wall known as the cortex. The enzymes that digest this layer selectively recognize the spore-specific cell wall modification, muramic-∂-lactam (MAL). MAL is made in part through the activity of the CwlD amidase, which is found in all spore-forming bacteria. While Bacillus subtilis CwlD appears to have amidase activity on its own, Clostridioides difficile CwlD activity depends on its binding partner, the GerS lipoprotein. To understand why C. difficile CwlD requires GerS, we determined the X-ray crystal structure of the CwlD:GerS complex and discovered that GerS binds to a site distant from CwlD’s active site. We also found that GerS stabilizes CwlD binding to its co-factor, Zn2+, indicating that GerS allosterically activates CwlD amidase. Notably, regulation at the level of Zn2+ binding has not previously been described for bacterial amidases, and GerS is the first protein to be shown to allosterically activate an amidase. Since binding partners of bacterial amidases were only first discovered 15 years ago, our results suggest that diverse mechanisms remain to be discovered for these critical enzymes.

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. Spore germination depends on the degradation of the protective spore peptidoglycan layer known as the spore cortex. Cortex degradation is mediated by enzymes that recognize the spore-specific peptidoglycan modification, muramic-∂-lactam (MAL). In C. difficile, MAL synthesis depends on the activity of the CwlD amidase and the GerS lipoprotein, which directly binds CwlD. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind zinc stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD binding to zinc, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of zinc co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

SUMMARYOxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generate sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual “open” conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a “closed” conformation when bound to its substrate. Combined crystallographic and solution scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of the enzyme. Additionally, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.

The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential bio-marker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Site-directed mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman’s assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.

DNA polymerase β (Pol β) is essential for maintaining genomic integrity. During short-patch base excision repair (BER), Pol β incorporates a nucleotide into a single-gapped DNA substrate. Pol β may also function in long-patch BER, where the DNA substrate consists of larger gap sizes or 5′-modified downstream DNA. We have recently shown that Pol β fills small gaps in DNA during microhomology-mediated end-joining as part of a process that increases genomic diversity. Our previous results with single-nucleotide gapped DNA show that Pol β undergoes two pre-catalytic conformational changes upon binding to the correct nucleotide substrate. Here we use FRET to investigate nucleotide incorporation of Pol β with various DNA substrates. The results show that increasing the gap size influences the fingers closing step by increasing its reverse rate. However, the 5′-phosphate group has a more significant effect. The absence of the 5′-phosphate decreases the DNA binding affinity of Pol β and results in a conformationally more open binary complex. Moreover, upon addition of the correct nucleotide in the absence of 5′-phosphate, a slow fingers closing step is observed. Interestingly, either increasing the gap size or removing the 5′-phosphate group results in loss of the noncovalent step. Together, these results suggest that the character of the DNA substrate impacts the nature and rates of pre-catalytic conformational changes of Pol β. Our results also indicate that conformational changes are important for the fidelity of DNA synthesis by Pol β.

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase β (Pol β) functions in base excision repair to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. To probe the mechanism that may underlie E288K's loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT because of an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure.

With the formidable growth in the volume of genetic information, it has become essential to identify and characterize mutations in macromolecules not only to predict contributions to disease processes but also to guide the design of therapeutic strategies. While mutations of certain residues have a predictable phenotype based on their chemical nature and known structural position, many types of mutations evade prediction based on current information. Described in this work are the crystal structures of two cancer variants located in the palm domain of DNA polymerase β (pol β), S229L and G231D, whose biological phenotype was not readily linked to a predictable structural implication. Structural results demonstrate that the mutations elicit their effect through subtle influences on secondary interactions with a residue neighboring the active site. Residues 229 and 231 are 7.5 and 12.5 Å, respectively, from the nearest active site residue, with a β-strand between them. A residue on this intervening strand, M236, appears to transmit fine structural perturbations to the catalytic metal-coordinating residue D256, affecting its conformational stability.

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor., Author summary The major nosocomial pathogen Clostridioides difficile depends on spore germination to initiate infection. Interestingly, C. difficile’s germinant sensing mechanism differs markedly from other spore-forming bacteria, since it uses bile acids to induce germination and lacks the transmembrane germinant receptors conserved in almost all spore-forming organisms. Instead, C. difficile is thought to use CspC, a soluble pseudoprotease, to sense these unique bile acid germinants. To gain insight into how a pseudoprotease senses germinant and propagates this signal, we solved the crystal structure of C. difficile CspC. Guided by this structure, we identified mutations that alter the sensitivity of C. difficile spores to not only bile acid germinant but also to amino acid and calcium co-germinants. Taken together, our study implicates CspC in either directly or indirectly sensing these diverse small molecules and thus raises new questions regarding how C. difficile spores physically detect bile acid germinants and co-germinants.

Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ HAT complex and contains a unique combination of domains typically found in chromatin-associated factors, which include plant homeodomain (PHD) fingers, a bromodomain and a proline-tryptophan-tryptophan-proline (PWWP) domain. Bromodomains are conserved structural motifs generally known to recognize acetylated histones, and the BRPF1 bromodomain preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We solved the X-ray crystal structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptides. Site-directed mutagenesis on residues in the BRPF1 bromodomain-binding pocket was carried out to investigate the contribution of specific amino acids on ligand binding. Our results provide critical insights into the molecular mechanism of ligand binding by the BRPF1 bromodomain, and reveal that ordered water molecules are an essential component driving ligand recognition.

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.

The original signature of the transferrin (TF) family of proteins was the ability to bind ferric iron with high affinity in the cleft of each of two homologous lobes. However, in recent years, new family members that do not bind iron have been discovered. One new member is the inhibitor of carbonic anhydrase (ICA), which as its name indicates, binds to and strongly inhibits certain isoforms of carbonic anhydrase. Recently, mouse ICA has been expressed as a recombinant protein in a mammalian cell system. Here, we describe the 2.4 A structure of mouse ICA from a pseudomerohedral twinned crystal. As predicted, the structure is bilobal, comprised of two α-β domains per lobe typical of the other family members. As with all but insect TFs, the structure includes the unusual reverse γ-turn in each lobe. The structure is consistent with the fact that introduction of two mutations in the N-lobe of murine ICA (mICA) (W124R and S188Y) allowed it to bind iron with high affinity. Unexpectedly, both lobes of the mICA were found in the closed conformation usually associated with presence of iron in the cleft, and making the structure most similar to diferric pig TF. Two new ICA family members (guinea pig and horse) were identified from genomic sequences and used in evolutionary comparisons. Additionally, a comparison of selection pressure (dN/dS) on functional residues reveals some interesting insights into the evolution of the TF family including that the N-lobe of lactoferrin may be in the process of eliminating its iron binding function.

Most high M(r) thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys(1)-Cys(2)) which forms an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys(2) position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from Drosophila melanogaster and Caenorhabditis elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pK(a) as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pK(a) of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.

Eukaryotic Rad51 recombinase plays a central role in maintaining genome stability by promoting homology-directed repair of DNA double-strand breaks and stalled replication forks. Rad51 forms presyn...

The first high-resolution crystal structure of spiroiminodihydantoin (dSp1) was obtained in the context of the DNA polymerase β active site and reveals two areas of significance. First, the structure verifies the recently determined S configuration at the spirocyclic carbon. Second, the distortion of the DNA duplex is similar to that of the single-oxidation product 8-oxoguanine. For both oxidized lesions, adaptation of the syn conformation results in similar backbone distortions in the DNA duplex. The resulting conformation positions the dSp1 A-ring as the base-pairing face whereas the B-ring of dSp1 protrudes into the major groove.

Spores are the major transmissive form of the nosocomial pathogen Clostridium difficile, a leading cause of healthcare-associated diarrhea worldwide. Successful transmission of C. difficile requires that its hardy, resistant spores germinate into vegetative cells in the gastrointestinal tract. A critical step during this process is the degradation of the spore cortex, a thick layer of peptidoglycan surrounding the spore core. In Clostridium sp., cortex degradation depends on the proteolytic activation of the cortex hydrolase, SleC. Previous studies have implicated Csps as being necessary for SleC cleavage during germination; however, their mechanism of action has remained poorly characterized. In this study, we demonstrate that CspB is a subtilisin-like serine protease whose activity is essential for efficient SleC cleavage and C. difficile spore germination. By solving the first crystal structure of a Csp family member, CspB, to 1.6 Å, we identify key structural domains within CspB. In contrast with all previously solved structures of prokaryotic subtilases, the CspB prodomain remains tightly bound to the wildtype subtilase domain and sterically occludes a catalytically competent active site. The structure, combined with biochemical and genetic analyses, reveals that Csp proteases contain a unique jellyroll domain insertion critical for stabilizing the protease in vitro and in C. difficile. Collectively, our study provides the first molecular insight into CspB activity and function. These studies may inform the development of inhibitors that can prevent clostridial spore germination and thus disease transmission., Author Summary Clostridium difficile is the leading cause of health-care associated diarrhea worldwide. C. difficile infections begin when its spores transform into vegetative cells during a process called germination. In Clostridium sp., germination requires that the spore cortex, a thick, protective layer, be removed by the cortex hydrolase SleC. While previous studies have shown that SleC activity depends on a subtilisin-like protease, CspB, the mechanisms regulating CspB function have not been characterized. In this study, we solved the first crystal structure of the Csp family of proteases and identified its key functional regions. We determined that CspB carries a unique jellyroll domain required for stabilizing the protein both in vitro and in C. difficile and a prodomain required for proper folding of the protease. Unlike all other prokaryotic subtilisin-like proteases, the prodomain remains bound to CspB and inhibits its protease activity until the germination signal is sensed. Our study provides new insight into how germination is regulated in C. difficile and may inform the development of inhibitors that can prevent germination and thus C. difficile transmission.

Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe N hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

Thioredoxin reductase (TR) from Drosophila melanogaster (DmTR) is a member of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases and catalyzes the reduction of the redox-active disulfide bond of thioredoxin. DmTR is notable for having high catalytic activity without the presence of a selenocysteine (Sec) residue (which is essential for the mammalian thioredoxin reductases). We report here the X-ray crystal structure of DmTR at 2.4 A resolution (Rwork = 19.8%, Rfree = 24.7%) in which the enzyme was truncated to remove the C-terminal tripeptide sequence Cys-Cys-Ser. We also demonstrate that tetrapeptides equivalent to the oxidized C-terminal active sites of both mouse mitochondrial TR (mTR3) and DmTR are substrates for the truncated forms of both enzymes. This truncated enzyme/peptide substrate system examines the kinetics of the ring-opening step that occurs during the enzymatic cycle of TR. The ring-opening step is 300-500-fold slower when Sec is replaced with Cys in mTR3 when using this system. Conversely, when Cys is replaced with Sec in DmTR, the rate of ring opening is only moderately increased (5-36-fold). Structures of these tetrapeptides were oriented in the active site of both enzymes using oxidized glutathione bound to GR as a template. DmTR has a more open tetrapeptide binding pocket than the mouse enzyme and accommodates the peptide Ser-Cys-Cys-Ser(ox) in a cis conformation that allows for the protonation of the leaving-group Cys by His464', which helps to explain why this TR can function without the need for Sec. In contrast, mTR3 shows a narrower pocket. One possible result of this narrower interface is that the mammalian redox-active tetrapeptide Gly-Cys-Sec-Gly may adopt a trans conformation for a better fit. This places the Sec residue farther away from the protonating histidine residue, but the lower pKa of Sec in comparison to that of Cys eliminates the need for Sec to be protonated.

All nucleotide polymerases and transferases catalyze nucleotide addition in a 5' to 3' direction. In contrast, tRNA(His) guanylyltransferase (Thg1) enzymes catalyze the unusual reverse addition (3' to 5') of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3'-5' addition activity to add G-1 to the 5'-end of tRNA(His), a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs) are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5'-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA(His). Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA(His) maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP). The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3'-5' reaction with 5'-monophosphorylated tRNA necessitates first an activation step, generating a 5'-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5'-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.