To successfully mount infections, nearly all bacterial pathogens must acquire iron, a key metal cofactor that primarily resides within human hemoglobin. Corynebacterium diphtheriae causes the life-threatening respiratory disease diphtheria and captures hemoglobin for iron scavenging using the surface-displayed receptor HbpA. Here, we show using X-ray crystallography, NMR, and in situ binding measurements that C. diphtheriae selectively captures iron-loaded hemoglobin by partially ensconcing the heme molecules of its α subunits. Quantitative growth and heme release measurements are compatible with C. diphtheriae acquiring heme passively released from hemoglobin’s β subunits. We propose a model in which HbpA and heme-binding receptors collectively function on the C. diphtheriae surface to capture hemoglobin and its spontaneously released heme. Acquisition mechanisms that exploit the propensity of hemoglobin’s β subunit to release heme likely represent a common strategy used by bacterial pathogens to obtain iron during infections.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3 -phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phospho-mevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference be-tween reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP -indepen-dent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate- binding site impaired this activation, suggesting the physio-logical importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil.

Imaging protein crystals and distinguishing them from salt crystals is an important task for protein crystallographers. The conventional tool used for this purpose is a dual-mode microscope composed of bright-field and ultraviolet (UV) induced fluorescence modes. The distinction between a protein and a salt crystal is made based upon the fluorescence response to the UV excitation, where most protein crystals absorb the UV excitation and emit fluorescence, unlike salt crystals. These dual-mode optical microscopes are sensitive; however, they are relatively bulky and expensive as they require UV-grade optics. As an alternative, here we demonstrate that on-chip UV holographic imaging offers a low-cost, portable, and robust technique to image and distinguish protein crystals from salt crystals, without the need for any expensive and bulky optical components. Only composed of a UV light-emitting-diode at 280 nm and a consumer-grade complementary metal–oxide–semiconductor image sensor de-capped and interfaced to a Raspberry Pi single-board computer, the necessary information from the crystal samples (placed very close to the sensor active area) is captured in the form of in-line holograms and extracted through digital back-propagation. In these holographic amplitude reconstructions, protein crystals appear significantly darker compared to the background due to the strong UV absorption, unlike salt crystals which do not show any contrast, enabling us to clearly distinguish between them. We believe that the on-chip UV holographic microscope could serve as a low-cost, sensitive, and robust alternative to conventional lens-based UV-microscopes used in protein crystallography.

In order to proliferate and mount an infection, many bacterial pathogens need to acquire iron from their host. The most abundant iron source in the body is the oxygen transporter hemoglobin (Hb). Streptococcus pyogenes, a potentially lethal human pathogen, uses the Shr protein to capture Hb on the cell surface. Shr is an important virulence factor, yet the mechanism by which it captures Hb and acquires its heme is not well-understood. Here, we show using NMR and biochemical methods that Shr binds Hb using two related modules that were previously defined as domains of unknown function (DUF1533). These hemoglobin-interacting domains (HIDs), called HID1 and HID2, are autonomously folded and independently bind Hb. The 1.5 Å resolution crystal structure of HID2 revealed that it is a structurally unique Hb-binding domain. Mutagenesis studies revealed a conserved tyrosine in both HIDs that is essential for Hb binding. Our biochemical studies indicate that HID2 binds Hb with higher affinity than HID1 and that the Hb tetramer is engaged by two Shr receptors. NMR studies reveal the presence of a third autonomously folded domain between HID2 and a heme-binding NEAT1 domain, suggesting that this linker domain may position NEAT1 near Hb for heme capture.

Our research seeks to learn how Gram‐positive pathogens acquire iron from the human host, a key process required for bacterial pathogenesis and a potential drug target. In particular, the focus of our work is the mechanism of heme extraction from hemoglobin (Hb), the main source of iron in the human body. In an effort to decipher this process, we are using a combination of biochemical and biophysical methods including X‐ray crystallography, isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), analytical ultra‐centrifugation (AUC), and heme transfer assays. Current efforts are devoted to studying this process in Streptococcus pyogenes (S. pyogenes) given its relevance as a human pathogen. We have recently characterized a novel Hb receptor from S. pyogenes ‐ the Streptococcal hemoprotein receptor (Shr). Shr appears to contain a similar domain architecture to the well‐characterized Hb receptors IsdH and IsdB found in Staphylococcus aureus. Unlike IsdH and IsdB, Shr mediates Hb binding through a domain of unknown function (DUF). Here we present the structure of this DUF domain in isolation and progress toward determining its structure in complex with Hb. The crystal structure of the DUF domain in isolation reveals a novel Hb‐binding domain structurally unrelated to Hb‐binding domains from other Gram‐positive pathogens. In addition, we have collected biochemical data on this domain suggesting that it slows the rate of heme release from Hb. We hypothesize that the DUF domain may slow the spontaneous process of heme release by Hb after erythrocyte lysis, allowing heme to be subsequently utilized downstream in the S. pyogenes heme acquisition pathway. This work is significant as it describes a novel mechanism of heme acquisition by a Gram‐positive pathogen. Future studies will be devoted to understanding the mechanism of heme extraction by the full‐length Shr protein.Support or Funding InformationThis work was supported by National Institutes of Health Grant AI52217 (to R.T.C.). R.M. was supported by a Cellular and Molecular Biology Training Grant (Ruth L. Kirschstein National Research Service Award GM007185). NMR equipment used in this research was purchased using funds from shared equipment grant NIH S10OD016336.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IS607-family transposons are unusual because they do not have terminal inverted repeats or generate target site duplications. They encode two protein-coding genes, but only tnpA is required for transposition. Our X-ray structures confirm that TnpA is a member of the serine recombinase (SR) family, but the chemically-inactive quaternary structure of the dimer, along with the N-terminal location of the DNA binding domain, are different from other SRs. TnpA dimers from IS1535 cooperatively associate with multiple subterminal repeats, which together with additional nonspecific binding, form a nucleoprotein filament on one transposon end that efficiently captures a second unbound end to generate the paired-end complex (PEC). Formation of the PEC does not require a change in the dimeric structure of the catalytic domain, but remodeling of the C-terminal α-helical region is involved. We posit that the PEC recruits a chemically-active conformer of TnpA to the transposon end to initiate DNA chemistry.

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.

Glucans such as starch serve as energy storage compounds and renewable and biodegradable carbon sources for use in industry. ADP‐Glucose Pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step of glucan biosynthesis in plants and bacteria, respectively. The novel Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase, from a thermophilic, sulfate‐reducing bacteria, has potential properties that could lead to engineering a very stable and highly active form to increase biomass yield. Sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal and C‐terminal regions that are important for regulation in other ADPG PPases that may be responsible for its altered effector specificity, including activation by PEP. To probe the roles of these regions, several variants (including F23R, S25A, K365G, and R397A) were generated and purified for kinetic analyses. The F23R demonstrated a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for the substrate ATP. However, in the presence of PEP, ATP affinity was restored to WT values. The variant S25 showed a Vmax ~3 fold higher than WT with a10‐fold higher apparent affinity for ATP as well as desensitization to effectors. The K365G enzyme displayed two fold lower activity than WT and desensitization to PEP. The R397A enzyme exhibited a ~20 fold activity reduction versus WT and PEP inhibition. Initial crystallization trials identified 0.2M di‐ammonium citrate (pH 5.0) and 20% polyethylene glycol as a condition that resulted in crystals of the S25A variant that diffracted; subsequently, an x‐ray structure was determined by molecular replacement to a nominal resolution of 2.9 Å, only the third known ADPG PPase structure. The structure indicates that the activated state of the S25A enzyme may result from an alteration in the positioning of Arg24 which participates in a salt bridge with Asp364, linking the N and C‐terminal domains by a functional relay switch mechanism. Initial results probing ligand binding with Isothermal Titration Calorimetry are promising, with good correlation between some kinetic parameters and KD values. Taken together, the kinetic data as well as the new structure and previous homology modeling confirm and extend understanding of the conserved regions for activity, regulation, and allosteric specificity and suggest additional mutagenesis targets.Support or Funding InformationSupported in part by NSF BIO MCB grant #0448676 and NSF BIO DBI.

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

Autotrophic bacteria rely on various mechanisms to increase intracellular concentrations of inorganic forms of carbon (i.e., bicarbonate and CO2) in order to improve the efficiency with which they can be converted to organic forms. Transmembrane bicarbonate transporters and carboxysomes play key roles in accumulating bicarbonate and CO2, but other regulatory elements of carbon concentration mechanisms in bacteria are less understood. In this study, after analyzing the genomic regions around α-type carboxysome operons, we characterize a protein that is conserved across these operons but has not been previously studied. On the basis of a series of apo- and ligand-bound crystal structures and supporting biochemical data, we show that this protein, which we refer to as the carboxysome-associated PII protein (CPII), represents a new and distinct subfamily within the broad superfamily of previously studied PII regulatory proteins, which are generally involved in regulating nitrogen metabolism in bacteria. CPII undergoes dramatic conformational changes in response to ADP binding, and the affinity for nucleotide binding is strongly enhanced by the presence of bicarbonate. CPII therefore appears to be a unique type of PII protein that senses bicarbonate availability, consistent with its apparent genomic association with the carboxysome and its constituents.

The attempt to elucidate the biological function of enzymes involved in the biosynthesis of tetrahydromethanopterin for the use in one‐carbon metabolism by archaea and select bacteria has resulted in the crystal structure of the hypothetical protein A2617 from Methylibium petroleiphilum. The structure was initially solved at 2.0 Å resolution using a selenomethionine multi‐wavelength anomalous diffraction experiment, and further refined at 1.8 Å resolution using a native dataset. The Rfactor of the current model is 0.19 and the Rfree is 0.22. The crystal structure of A2617 is a homodimer with each monomer consisting of four α‐helices connected to a barrel fold of six anti‐parallel β‐sheets with an α‐helix by a pair of anti‐parallel β‐sheets. The monomers interface to form the homodimer at the region containing four α‐helices with the barrel folds flanking both ends of the structure. A deep cleft is present at the interface of the two monomers. The overall structure of A2617 resembles that of uncharacterized archaeal homologs; however, the structure is distinct due to the lack of bound FMN in the binding pocket that is found in the archaeal homolog crystal structures. Ligand docking studies using intermediates in the tetrahydromethanopterin biosynthetic pathway indicated that A2617 has the greatest binding affinity for 6‐hydroxymethyl‐7,8‐dihydropterin‐ribofuranosylaminobenzene‐5‐phosphate. This metabolite is the substrate for the third step of tetrahydromethanopterin side chain biosynthesis, which is an oxidoreductase reaction. The homology of A2617 with FMN‐containing archaeal proteins and the findings of the ligand binding studies provide support for the role of A2617 in catalyzing a reductive reaction in the biosynthesis of the tetrahydromethanopterin side chain.Support or Funding InformationThis research was supported by National Science Foundation grant number CHE‐1508801 and by a grant from the California State University Program for Education and Research in Biotechnology (CSUPERB).

In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.