Contribution of Sonication to the Identification of Megaprosthesis Infection in Culture Negative Oncologic Patient.

Conventional central osteosarcoma mainly affects the metaphysis of long bones in young people. The use of megaprostheses in oncological patients has increased in recent years. However, this type of surgery is not exempt from complications, with infections being the most common. In recent years, the presence of biofilm-forming bacteria has increased. Biofilm characteristics allow bacteria to resist hostile environmental conditions. The application of long wave ultrasound (process known as sonication) on the rescued inert material before culture interrupts the biofilm and generates a significantly higher recovery of bacterial growth compared to conventional tissue culture. We present the case of a 12-year-old patient with osteosarcoma of the femur, who, after surgery, developed a prosthetic infection detected by sonication, with negative soft tissue culture.

Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.

Tumor mesenquimático fosfatúrico de pelvis: abordaje multidisciplinario / Phosphaturic mesenchymal tumor of the pelvis: A multidisciplinary approach

El tumor mesenquimático fosfatúrico es una entidad clinicopatológica sumamente infrecuente. Además de provocar dolor óseo insidioso y polimialgias, se acompaña de alteraciones del metabolismo fosfocálcico de difícil manejo clínico. El abordaje multidisciplinario resulta la clave del éxito en esta enfermedad. Presentamos una paciente de 52 años de edad con antecedente de tumor mesenquimático fosfatúrico en la hemipelvis derecha con extensión a la cadera homolateral de 10 años de evolución. Clínicamente presentaba osteomalacia oncogénica (hipofosfatemia e hiperfosfaturia) que no se corregía, pese a un agente de última generación, el burosumab, un inhibidor del factor de crecimiento fibroblástico 23, que aumenta la reabsorción tubular renal de fosfatos. En un comité multidisciplinario, se decidió la resección con márgenes oncológicos y se logró una mejoría clínica franca. Comunicamos este caso, debido a que es un cuadro infrecuente. Nivel de Evidencia: IV

Phosphaturic mesenchymal tumor (PMT) is an infrequent clinicopathological entity. It presents insidious bone pain and polymyalgia, accompanied by alterations in calcium and phosphorus metabolism that are difficult to resolve clinically. A multidisciplinary approach is a key to success in this pathology. We present the case of a 52-year-old female patient with a 10-year history of PMT in the right hemipelvis with ipsilateral hip extension. From the clinical point of view, she presented oncogenic osteomalacia (hypophosphatemia and hyperphosphaturia) that did not correct despite being administered the latest generation medication, burosumab, an FGF-23 inhibitor that increases renal tubular phosphate reabsorption. Resection with oncological margins was decided by a multidisciplinary committee resolving her clinical condition. Due to the rarity of this pathology, we decided to report the case. Level of Evidence: IV

Alternative conformation of the C-domain of the P140 protein from Mycoplasma genitalium.

The human pathogen Mycoplasma genitalium is responsible for urethritis in men, and for cervicitis and pelvic inflammatory disease in women. The adherence of M. genitalium to host target epithelial cells is mediated through an adhesion complex called Nap, which is essential for infectivity. Nap is a transmembrane dimer of heterodimers of the immunodominant proteins P110 and P140. The M. genitalium genome contains multiple copies of portions that share homology with the extracellular regions of P140 and P110 encoded by the genes mg191 and mg192, respectively. Homologous recombination between the genes and the copies allows the generation of a large diversity of P140 and P110 variants to overcome surveillance by the host immune system. Interestingly, the C-terminal domain (C-domain) of the extracellular region of P140, which is essential for the function of Nap by acting as a flexible stalk anchoring the protein to the mycoplasma membrane, presents a low degree of sequence variability. In the present work, the X-ray crystal structures of two crystal forms of a construct of the P140 C-domain are reported. In both crystal forms, the construct forms a compact octamer with D4 point-group symmetry. The structure of the C-domain determined in this work presents significant differences with respect to the structure of the C-domain found recently in intact P140. The structural plasticity of the C-domain appears to be a possible mechanism that may help in the functioning of the mycoplasma adhesion complex.

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

Structure and mechanism of the Nap adhesion complex from the human pathogen Mycoplasma genitalium.

Mycoplasma genitalium is a human pathogen adhering to host target epithelial cells and causing urethritis, cervicitis and pelvic inflammatory disease. Essential for infectivity is a transmembrane adhesion complex called Nap comprising proteins P110 and P140. Here we report the crystal structure of P140 both alone and in complex with the N-terminal domain of P110. By cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) we find closed and open Nap conformations, determined at 9.8 and 15 Å, respectively. Both crystal structures and the cryo-EM structure are found in a closed conformation, where the sialic acid binding site in P110 is occluded. By contrast, the cryo-ET structure shows an open conformation, where the binding site is accessible. Structural information, in combination with functional studies, suggests a mechanism for attachment and release of M. genitalium to and from the host cell receptor, in which Nap conformations alternate to sustain motility and guarantee infectivity.

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction.

L-amino acid transporters (LATs) play key roles in human physiology and are implicated in several human pathologies. LATs are asymmetric amino acid exchangers where the low apparent affinity cytoplasmic side controls the exchange of substrates with high apparent affinity on the extracellular side. Here, we report the crystal structures of an LAT, the bacterial alanine-serine-cysteine exchanger (BasC), in a non-occluded inward-facing conformation in both apo and substrate-bound states. We crystallized BasC in complex with a nanobody, which blocks the transporter from the intracellular side, thus unveiling the sidedness of the substrate interaction of BasC. Two conserved residues in human LATs, Tyr 236 and Lys 154, are located in equivalent positions to the Na1 and Na2 sites of sodium-dependent APC superfamily transporters. Functional studies and molecular dynamics (MD) calculations reveal that these residues are key for the asymmetric substrate interaction of BasC and in the homologous human transporter Asc-1.

Cis-trans proline isomers in the catalytic domain of calcineurin.

Calcineurin is an essential calcium-activated serine/threonine phosphatase. The six NMR-observable methionine methyl groups in the catalytic domain of human calcineurin Aα (CNA) were assigned and used as reporters of the presence of potential cis-trans isomers in solution. Proline 84 is found in the cis conformation in most calcineurin X-ray structures, and proline 309, which is part of a highly conserved motif in phosphoprotein phosphatases, was modeled with a cis peptide bond in one of the two molecules present in the asymmetric unit of CNA. We mutated each of the two prolines to alanine to force the trans conformation. Solution NMR shows that the P84A CNA mutant exists in two forms, compatible with cis-trans isomers, while the P309A mutant is predominantly in the trans conformation. DATABASE: PDB depositions mentioned PDB 5C1V and 2JOG.

Mycoplasma genitalium adhesin P110 binds sialic-acid human receptors.

Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.

Structural Insights into Subunits Assembly and the Oxyester Splicing Mechanism of Neq pol Split Intein.

Split inteins are expressed as two separated subunits (N-intein and C-intein) fused to the corresponding exteins. The specific association of both intein subunits precedes protein splicing, which results in excision of the intein subunits and in ligation, by a peptide bond, of the concomitant exteins. Catalytically active intein precursors are typically too reactive for crystallization or even isolation. Neq pol is the trans-intein of the B-type DNA polymerase I split gene from hyperthermophile Nanoarchaeum equitans. We have determined the crystal structures of both the isolated NeqN and the complex of NeqN and NeqC subunits carrying the wild-type sequences, including the essential catalytic residues Ser1 and Thr+1, in addition to seven and three residues of the N- and C-exteins, respectively. These structures provide detailed information on the unique oxyester chemistry of the splicing mechanism of Neq pol and of the extensive rearrangements that occur in NeqN during the association step.

KatG-Mediated Oxidation Leading to Reduced Susceptibility of Bacteria to Kanamycin.

Resistance to antibiotics has become a serious problem for society, and there are increasing efforts to understand the reasons for and sources of resistance. Bacterial-encoded enzymes and transport systems, both innate and acquired, are the most frequent culprits for the development of resistance, although in Mycobacterium tuberculosis, the catalase-peroxidase, KatG, has been linked to the activation of the antitubercular drug isoniazid. While investigating a possible link between aminoglycoside antibiotics and the induction of oxidative bursts, we observed that KatG reduces susceptibility to aminoglycosides. Investigation revealed that kanamycin served as an electron donor for the peroxidase reaction, reducing the oxidized ferryl intermediates of KatG to the resting state. Loss of electrons from kanamycin was accompanied by the addition of a single oxygen atom to the aminoglycoside. The oxidized form of kanamycin proved to be less effective as an antibiotic. Kanamycin inhibited the crystallization of KatG, but the smaller, structurally related glycoside maltose did cocrystallize with KatG, providing a suggestion as to the possible binding site of kanamycin.

The nucleotide-bound/substrate-bound conformation of the Mycoplasma genitalium DnaK chaperone.

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains-nucleotide binding domain (NBD) and substrate binding domain (SBD)-connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide-bound/substrate-bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDß-SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.

Theory Uncovers the Role of the Methionine-Tyrosine-Tryptophan Radical Adduct in the Catalase Reaction of KatGs: O2 Release Mediated by Proton-Coupled Electron Transfer.

Catalase-peroxidases (KatGs) are bifunctional enzymes exhibiting both peroxidase and substantial catalase activities. It is widely recognized from experiments that the catalatic activity of KatGs is correlated with a unique covalent adduct (M-Y-W) formed in the active site, but the exact role of this adduct was elusive up to now. Here, quantum mechanical/molecular mechanical (QM/MM) calculations and QM/MM metadynamics are employed to elucidate the molecular mechanism and the role of M-Y-W adduct in the catalase reaction. It is shown that O2 formation proceeds through a mechanism involving proton-coupled electron transfer (PCET). The M-Y-W cation radical adduct, which is close to the heme, His112 and the HOO. radical intermediate, acts as an electron sink during the PCET process. The present study also highlights the structural differences and functional similarities between KatGs and monofunctional catalases.

Prediction of Protein Configurational Entropy (Popcoen).

A knowledge-based method for configurational entropy prediction of proteins is presented; this methodology is extremely fast, compared to previous approaches, because it does not involve any type of configurational sampling. Instead, the configurational entropy of a query fold is estimated by evaluating an artificial neural network, which was trained on molecular-dynamics simulations of ∼1000 proteins. The predicted entropy can be incorporated into a large class of protein software based on cost-function minimization/evaluation, in which configurational entropy is currently neglected for performance reasons. Software of this type is used for all major protein tasks such as structure predictions, proteins design, NMR and X-ray refinement, docking, and mutation effect predictions. Integrating the predicted entropy can yield a significant accuracy increase as we show exemplarily for native-state identification with the prominent protein software FoldX. The method has been termed Popcoen for Prediction of Protein Configurational Entropy. An implementation is freely available at http://fmc.ub.edu/popcoen/ .

Entropic Stabilization of Cas4 Protein SSO0001 Predicted with Popcoen.

Popcoen is a method for configurational entropy estimation of proteins based on machine-learning. Entropy is predicted with an artificial neural network which was trained on simulation trajectories of a large set of representative proteins. Popcoen is extremely fast compared to other approaches based on the sampling of a multitude of microstates. Consequently, Popcoen can be incorporated into a large class of protein software which currently neglects configurational entropy for performance reasons. Here, we apply Popcoen to various conformations of the Cas4 protein SSO0001 of Sulfolobus solfataricus, a protein that assembles to a decamer of known toroidal shape. We provide numerical evidence that the native state (NAT) of a SSO0001 monomer has a similar structure to the protomers of the oligomer, where NAT of the monomer is stabilized mainly entropically. Due to its large amount of configurational entropy, NAT has lower free energy than alternative conformations of very low enthalpy and solvation free-energy. Hence, SSO0001 serves as an example case where neglecting configurational entropy leads to incorrect conclusion. Our results imply that no refolding of the subunits is required during oligomerization which suggests that configurational entropy is employed by nature to largely enhance the rate of assembly.

Testing the mutual information expansion of entropy with multivariate Gaussian distributions.

The mutual information expansion (MIE) represents an approximation of the configurational entropy in terms of low-dimensional integrals. It is frequently employed to compute entropies from simulation data of large systems, such as macromolecules, for which brute-force evaluation of the full configurational integral is intractable. Here, we test the validity of MIE for systems consisting of more than m = 100 degrees of freedom (dofs). The dofs are distributed according to multivariate Gaussian distributions which were generated from protein structures using a variant of the anisotropic network model. For the Gaussian distributions, we have semi-analytical access to the configurational entropy as well as to all contributions of MIE. This allows us to accurately assess the validity of MIE for different situations. We find that MIE diverges for systems containing long-range correlations which means that the error of consecutive MIE approximations grows with the truncation order n for all tractable n ≪ m. This fact implies severe limitations on the applicability of MIE, which are discussed in the article. For systems with correlations that decay exponentially with distance, MIE represents an asymptotic expansion of entropy, where the first successive MIE approximations approach the exact entropy, while MIE also diverges for larger orders. In this case, MIE serves as a useful entropy expansion when truncated up to a specific truncation order which depends on the correlation length of the system.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium.

Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and ß lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The ß lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.

The Catalase Activity of Catalase-Peroxidases Is Modulated by Changes in the pKa of the Distal Histidine.

The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu ∼7 and ∼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKa of the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct.

Inhibitory properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives acting on glycogen metabolising enzymes.

Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.

Structure-Guided Mutations in the Terminal Organelle Protein MG491 Cause Major Motility and Morphologic Alterations on Mycoplasma genitalium.

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle.