Insights into the potential function and membrane organization of the TP0435 (Tp17) lipoprotein from Treponema pallidum derived from structural and biophysical analyses

The sexually transmitted disease syphilis is caused by the bacterial spirochete Treponema pallidum. This microorganism is genetically intractable, accounting for the large number of putative and undercharacterized members of the pathogen's proteome. In an effort to ascribe a function(s) to the TP0435 (Tp17) lipoprotein, we engineered a soluble variant of the protein (rTP0435) and determined its crystal structure at a resolution of 2.42 Å. The structure is characterized by an eight‐stranded β‐barrel protein with a shallow “basin” at one end of the barrel and an α‐helix stacked on the opposite end. Furthermore, there is a disulfide‐linked dimer of the protein in the asymmetric unit of the crystals. Solution hydrodynamic experiments established that purified rTP0435 is monomeric, but specifically forms the disulfide‐stabilized dimer observed in the crystal structure. The data herein, when considered with previous work on TP0435, imply plausible roles for the protein in either ligand binding, treponemal membrane architecture, and/or pathogenesis.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

Not as easy as π: An insertional residue does not explain the π‐helix gain‐of‐function in two‐component FMN reductases

The π‐helix located at the tetramer interface of two‐component FMN‐dependent reductases contributes to the structural divergence from canonical FMN‐bound reductases within the NADPH:FMN reductase family. The π‐helix in the SsuE FMN‐dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4‐helix. Variants of Tyr118 were generated, and their X‐ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π‐helix. The structure of the Y118A SsuE π‐helix was converted to an α‐helix, similar to the FMN‐bound members of the NADPH:FMN reductase family. Although the π‐helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π‐helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π‐helix contains a His insertional residue. Exchanging the π‐helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure‐based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN‐bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π‐helix. Results from the structural and functional studies of the FMN‐dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π‐helix, and additional structural adaptions occur to provide the altered gain of function.     

The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl‐CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl‐CoA synthetase and 4‐chlorobenzoate:CoA ligase. Most interestingly, the acyl‐binding pocket of the new structure is compared with other acyl‐ and aryl‐CoA synthetases. A comparison of the acyl‐binding pocket of the acyl‐CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl‐binding pocket. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structures of dimeric dihydrodiol dehydrogenase apoenzyme and inhibitor complex: probing the subunit interface with site-directed mutagenesis

Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene.     

Crystal structure of DFA0005 complexed with alpha-ketoglutarate: a novel member of the ICL/PEPM superfamily from alkali-tolerant Deinococcus ficus

The crystal structure of the DFA0005 protein complexed with alpha-ketoglutarate (AKG) from an alkali-tolerant bacterium Deinococcus ficus has been determined to a resolution of 1.62 A. The monomer forms an incomplete alpha7/beta8 barrel with a protruding alpha8 helix that interacts extensively with another subunit to form a stable dimer of two complete alpha8/beta8 barrels. The dimer is further stabilized by four glycerol molecules situated at the interface. One unique AKG ligand binding pocket per subunit is detected. Fold match using the DALI and SSE servers identifies DFA0005 as belonging to the isocitrate lyase/phosphoenolpyruvate mutase (ICL/PEPM) superfamily. However, further detailed structural and sequence comparison with other members in this superfamily and with other families containing AKG ligand indicate that DFA0005 protein exhibits considerable distinguishing features of its own and can be considered a novel member in this ICL/PEPM superfamily.     

Structure of the Y. pseudotuberculosis adhesin InvasinE

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin‐family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin‐family of proteins. The sequence of the C‐terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin‐family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin‐like domains. The structure reveals a rod shaped architecture, confirmed by small angle X‐ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C‐type lectin‐like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C‐type lectin‐like domain in InvasinE lacks motifs required for Ca²⁺/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β₁‐integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host‐cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin‐family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.     

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.     

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP‐IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP‐IV is highly specific for the male mouse pheromone 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X‐ray crystal structure of MUP‐IV bound to three mouse pheromones: SBT, 2,5‐dimethylpyrazine, and 2‐heptanone. We also obtained the structure of MUP‐IV with 2‐ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water‐mediated hydrogen bond network in the MUP‐I and MUP‐II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP‐IV.     

Open and compressed conformations of Francisella tularensis ClpP

Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder‐order transition of a β‐strand α‐helix combination. This transition occurs at a subunit‐subunit interface. Our study may now underpin future efforts in a structure‐based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188–194. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the carbon monoxide complex of human cytoglobin

Cytoglobin (Cgb) is a vertebrate heme‐containing globin‐protein expressed in a broad range of mammalian tissues. Unlike myoglobin, Cgb displays a hexa‐coordinated (bis‐hystidyl) heme iron atom, having the heme distal His81(E7) residue as the endogenous sixth ligand. In the present study, we crystallized human Cgb in the presence of a reductant Na₂S₂O₄ under a carbon monoxide (CO) atmosphere, and determined the crystal structure at 2.6 Å resolution. The CO ligand occupies the sixth axial position of the heme ferrous iron. Eventually, the imidazole group of His81(E7) is expelled from the sixth position and swings out of the distal heme pocket. The flipping motion of the His81 imidazole group accompanies structural readjustments of some residues (Gln62, Phe63, Gln72, and Ser75) in both the CD‐corner and D‐helix regions of Cgb. On the other hand, no significant structural changes were observed in other Cgb regions, for example, on the proximal side. These structural alterations that occurred as a result of exogenous ligand (CO) binding are clearly different from those observed in other vertebrate hexa‐coordinated globins (mouse neuroglobin, Drosophila melanogaster hemoglobin) and penta‐coordinated sperm whale myoglobin. The present study provides the structural basis for further discussion of the unique ligand‐binding properties of Cgb. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Mcl‐1–Bim complexes accommodate surprising point mutations via minor structural changes

Mcl‐1 is an antiapoptotic Bcl‐2‐family protein that protects cells against death. Structures of Mcl‐1, and of other anti‐apoptotic Bcl‐2 proteins, reveal a surface groove into which the α‐helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl‐2 family function. We report the crystal structure of human Mcl‐1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl‐1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine‐to‐alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix α3 accommodating an isoleucine‐to‐tyrosine mutation at this same position. We surveyed the variation in available Mcl‐1 and Bcl‐x_L structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3‐only proteins with Mcl‐1. With the antiapoptotic Bcl‐2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl‐1.     

Structure of the Yersinia type III secretory system chaperone SycE.

In the type III secretory system of bacterial pathogens, a large number of sequence-divergent but characteristically small (approximately 14-19 kDa), acidic (pI approximately 4-5) chaperone proteins have been identified. We present the 1.74 A resolution crystal structure of the Yersinia pseudotuberculosis chaperone SycE, whose action in promoting translocation of YopE into host macrophages is essential to Yersinia pathogenesis. SycE, a compact, globular dimer with a novel fold, has two large hydrophobic surface patches that may form binding sites for YopE or other type III components. These patches are formed by structurally key residues that are conserved among many chaperones, suggesting shared structural and functional relationships. A negative electrostatic potential covers almost the entire surface of SycE and is likely conserved in character, but not in detail, among chaperones. The structure provides the first structural insights into possible modes of action of SycE and type III chaperones in general.     

Crystal structures of the PLP- and PMP-bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis

The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.     

Compact reduced thioredoxin structure from the thermophilic bacteria Thermus thermophilus

The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the "thioredoxin fold" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources.     

Reversible substrate-induced domain motions in ribonuclease A.

Despite the increasing number of successful determinations of complex protein structures the understanding of their dynamics properties is still rather limited. Using X-ray crystallography, we demonstrate that ribonuclease A (RNase A) undergoes significant domain motions upon ligand binding. In particular, when cytidine 2'-monophosphate binds to RNase A, the structure of the enzyme becomes more compact. Interestingly, our data also show that these structural alterations are fully reversible in the crystal state. These findings provide structural bases for the dynamic behavior of RNase A in the binding of the substrate shown by Petsko and coworkers (Rasmussen et al. Nature 1992;357:423-424). These subtle domain motions may assume functional relevance for more complex system and may play a significant role in the cooperativity of oligomeric enzymes.     

Crystal structure of Bacillus anthracis phosphoglucosamine mutase, an enzyme in the peptidoglycan biosynthetic pathway

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme that participates in the cytoplasmic steps of peptidoglycan biosynthesis. As peptidoglycan is essential for bacterial survival and is absent in humans, enzymes in this pathway have been the focus of intensive inhibitor design efforts. Many aspects of the structural biology of the peptidoglycan pathway have been elucidated, with the exception of the PNGM structure. We present here the crystal structure of PNGM from the human pathogen and bioterrorism agent Bacillus anthracis. The structure reveals key residues in the large active site cleft of the enzyme which likely have roles in catalysis and specificity. A large conformational change of the C-terminal domain of PNGM is observed when comparing two independent molecules in the crystal, shedding light on both the apo- and ligand-bound conformers of the enzyme. Crystal packing analyses and dynamic light scattering studies suggest that the enzyme is a dimer in solution. Multiple sequence alignments show that residues in the dimer interface are conserved, suggesting that many PNGM enzymes adopt this oligomeric state. This work lays the foundation for the development of inhibitors for PNGM enzymes from human pathogens.     

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50^NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11^RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.