The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis.

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded ß sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded ß sheet MinE dimer with the released ß strands (MinD-binding regions) converted to α helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin.

NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.

HSV-1 ICP0 dimer domain adopts a novel ß-barrel fold.

Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555-767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine ß-strands and two α-helices. Interestingly, two adjacent ß-strands from one monomer "reach" into the adjacent subunit during dimer formation, generating two ß-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two ß-strands of each dimer, creating a "stacking" of the ß-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle.

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor.

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity.

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.

Structure-activity relationship of ipglycermide binding to phosphoglycerate mutases.

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure-activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase-phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

Improving the kinetic parameters of nicotine oxidizing enzymes by homologous structure comparison and rational design.

Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s-1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s-1 to 0.014 s-1), and reduce the Km of the NctB protein by approximately 34% (940 µM-622 µM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.

Crystal structure of human mitochondrial trifunctional protein, a fatty acid ß-oxidation metabolon.

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes ß-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α2ß2 The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two ß-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the ß2 dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3'-AMP-PPi moiety is guided by the positively charged residues located along the "ceiling" of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.

Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis support a role in TCA cycle regulation.

Chlamydia trachomatis is an obligate intracellular bacteria that undergo dynamic morphologic and physiologic conversions upon gaining an access to a eukaryotic cell. These conversions likely require the detection of key environmental conditions and regulation of metabolic activity. Chlamydia encodes homologs to proteins in the Rsb phosphoregulatory partner-switching pathway, best described in Bacillus subtilis. ORF CT588 has a strong sequence similarity to RsbU cytoplasmic phosphatase domain but also contains a unique periplasmic sensor domain that is expected to control the phosphatase activity. A 1.7 Å crystal structure of the periplasmic domain of the RsbU protein from C. trachomatis (PDB 6MAB) displays close structural similarity to DctB from Vibrio and Sinorhizobium. DctB has been shown, both structurally and functionally, to specifically bind to the tricarboxylic acid (TCA) cycle intermediate succinate. Surface plasmon resonance and differential scanning fluorimetry of TCA intermediates and potential metabolites from a virtual screen of RsbU revealed that alpha-ketoglutarate, malate and oxaloacetate bound to the RsbU periplasmic domain. Substitutions in the putative binding site resulted in reduced binding capabilities. An RsbU null mutant showed severe growth defects which could be restored through genetic complementation. Chemical inhibition of ATP synthesis by oxidative phosphorylation phenocopied the growth defect observed in the RsbU null strain. Altogether, these data support a model with the Rsb system responding differentially to TCA cycle intermediates to regulate metabolism and key differentiation processes.

Crystal Structure of VapBC-1 from Nontypeable Haemophilus influenzae and the Effect of PIN Domain Mutations on Survival during Infection.

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivoIMPORTANCE Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in cis wild-type toxin under the same conditions.

Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease.

Human noroviruses are the primary cause of outbreaks of acute gastroenteritis worldwide. The problem is further compounded by the current lack of norovirus-specific antivirals or vaccines. Noroviruses have a single-stranded, positive sense 7 to 8 kb RNA genome which encodes a polyprotein precursor that is processed by a virus-encoded 3C-like cysteine protease (NV 3CLpro) to generate at least six mature nonstructural proteins. Processing of the polyprotein is essential for virus replication, consequently, NV 3CLpro has emerged as an attractive target for the discovery of norovirus therapeutics and prophylactics. We have recently described the structure-based design of macrocyclic transition state inhibitors of NV 3CLpro. In order to gain insight and understanding into the interaction of macrocyclic inhibitors with the enzyme, as well as probe the effect of ring size on pharmacological activity and cellular permeability, additional macrocyclic inhibitors were synthesized and high resolution cocrystal structures determined. The results of our studies tentatively suggest that the macrocyclic scaffold may hamper optimal binding to the active site by impeding concerted cross-talk between the S2 and S4 subsites.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Reconfiguration of the proteasome during chaperone-mediated assembly.

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes, it inserts into the α2/α3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

Single-domain antibodies pinpoint potential targets within Shigella invasion plasmid antigen D of the needle tip complex for inhibition of type III secretion.

Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. Shigella spp. cause shigellosis, also called bacillary dysentery, and invade colonic epithelial cells via the T3SS. The tip complex of Shigella flexneri contains invasion plasmid antigen D (IpaD), which initially regulates secretion and provides a physical platform for the translocon pore. The tip complex represents a promising therapeutic target for many important T3SS-containing pathogens. Here, in an effort to further elucidate its function, we created a panel of single-VH domain antibodies (VHHs) that recognize distinct epitopes within IpaD. These VHHs recognized the in situ tip complex and modulated the infectious properties of Shigella Moreover, structural elucidation of several IpaD-VHH complexes provided critical insights into tip complex formation and function. Of note, one VHH heterodimer could reduce Shigella hemolytic activity by >80%. Our observations along with previous findings support the hypothesis that the hydrophobic translocator (IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens, suggesting potential therapeutic avenues for managing infections by these pathogens.

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein.

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp), and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation, and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicate that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerates the completion of the heme iron coordination.

Structural and inhibitory effects of hinge loop mutagenesis in serpin-2 from the malaria vector Anopheles gambiae.

Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits clip domain serine proteinase 9 (CLIPB9), which functions in a serine proteinase cascade culminating in the activation of prophenoloxidase and melanization. Silencing of SRPN2 in A. gambiae results in spontaneous melanization and decreased life span and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into ß sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site, and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate that the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase.