Accelerated protein synthesis via one–pot ligation–deselenization chemistry

Peptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at β-selenoaspartate and γ-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed ?-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare ?-(1?4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature

Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the Anopheles mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated in vivo. To test this prediction, insect cell expression of two anophelin proteins, from Anopheles albimanus and Anopheles gambiae, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from A. albimanus exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.The authors acknowledge the John A. Lamberton Research Scholarship and Australian Postgraduate Award for Ph.D. funding (E.E.W., X.L., and R.E.T.) and The National Health and Medical Research Council of Australia (Project Grant 1120941) and the Australian Research Council (FT130100150 to R.J.P and DP150100383 to G.O) for funding

Accelerated protein synthesis via one–pot ligation–deselenization chemistry

Peptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at β-selenoaspartate and γ-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future

Regulación enzimática y funcional de TrwD, una proteína esencial en la secreción bacteriana

Los sistemas de secreción tipo IV (T4SS) son usados por las bacterias Gram-negativas bien para el intercambio genético entre ellas (proceso denominado conjugación) o bien, en el caso de bacterias patogénicas, para secretar efectores a sus células hospedadoras. Los T4SS son sistemas multiproteicos, formados por al menos doce proteínas, que se expanden a través de las membranas interna y externa de la célula donadora. Tanto para la biogénesis del sistema como para la transferencia del sustrato, se necesita la energía proporcionada por los tres motores situados en la base del canal (VirD4, VirB4 y VirB11). Este trabajo se ha centrado en el estudio de TrwD, un homólogo de VirB11 en nuestro sistema modelo, el plásmido conjugativo R388. Esta tesis aporta evidencias bioquímicas que apoyan que la familia de las proteínas VirB11 pertenecen a la superfamilia de ATPasas implicadas en el tráfico de proteínas durante la secreción mediada por Sistemas de Tipo IV. En este contexto se ha caracterizado la interacción con las otras dos ATPasas del sistema corroborando el papel esencial de esta proteína regulando tanto la biogénesis del sistema como la transferencia de sustrato. Por último se han evaluado inhibidores de la conjugación bacteriana, como alternativa al uso de antibióticos, que actúan específicamente sobre la actividad de esta proteína

Functional interactions of VirB11 traffic ATPases with VirB4 and VirD4 molecular motors in type IV secretion systems

Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems. © 2013, American Society for Microbiology.This work was supported by Ministerio de Economía y Competitividad (MINECO, Spain) grants BFU2011-22874 (to E.C. and I.A.) and BFU2011-26608 (to F.D.L.C.) and by European VII Framework Program grants 248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2 (to F.D.L.C.). J.R.-R. was supported by a fellowship from the University of Cantabria.Peer Reviewe

Autoinhibitory Regulation of TrwK, an Essential VirB4 ATPase in Type IV Secretion Systems*

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS

Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation

PMCID: PMC4908816Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes. Antibiotic resistance is an emergent threat to human health. Bacterial conjugation is the main mechanism for the wide spread dissemination of antibiotic resistance genes. Here, we found that conjugative traffic ATPases are the molecular target for the inhibition of conjugation by unsaturated fatty acids. Identification of this molecular target will provide us with a new tool for the rational design of more potent and efficient drugs to stop the transmission of antibiotic resistance genes.This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) grants BFU2011-22874 (to E.C and I A) and BFU2014-55534 (to FDLC) and EU VII Framework Program projects 282004/FP7-HEALTH-2011-2.3.1-2 and 612146/ICT-2013-10 (to FDLC). DSR thanks the support of the National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 5P20GM103475-13 and the Inter American University of Puerto Rico.Peer Reviewe

Regulation of the type IV secretion ATPase TrwD by magnesium: implications for catalytic mechanism of the secretion ATPase superfamily.

TrwD, the VirB11 homologue in conjugative plasmid R388, is a member of the large secretion ATPase superfamily, which includes ATPases from bacterial type II and type IV secretion systems, type IV pilus, and archaeal flagellae assembly. Based on structural studies of the VirB11 homologues in Helicobacter pylori and Brucella suis and the archaeal type II secretion ATPase GspE, a unified mechanism for the secretion ATPase superfamily has been proposed. Here, we have found that the ATP turnover of TrwD is down-regulated by physiological concentrations of magnesium. This regulation is exerted by increasing the affinity for ADP, hence delaying product release. Circular dichroism and limited proteolysis analysis indicate that magnesium induces conformational changes in the protein that promote a more rigid, but less active, form of the enzyme. The results shown here provide new insights into the catalytic mechanism of the secretion ATPase superfamily

Inhibition of Type IV secretion ATPase TrwD by unsaturated fatty acids as a potential tool to prevent wide spread dissemination of antibiotic resistance genes

Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.Antibiotic resistance has become a major health issue. Unfortunately, the development of new antibiotics is being very slow and expensive and we are running out of weapons to fi ght against the appearance of multi-resistant bugs. Bacterial conjugation is the main mechanism for the wide spread dissemination of antibiotic resistance genes and, therefore, the search of specific inhibitors of conjugation (COINs) could become a brand new strategy in this warfare. In the search of potential COINs, previous studies have reported that unsaturated fatty acids (uFAs) were able to inhibit conjugation. Based on these studies we have looked for the molecular targets of these compounds and we have found that the Type IV secretion ATPase TrwD is inhibited by linoleic acid and 2-alkynoic fatty acids, such as the 2-hexadecynoic acid. These two uFAs compounds were the most effective inhibitors in R388 plasmid conjugation. The inhibitory effect is specific for the traffic ATPase TrwD, as the remaining ATPases of the Type IV secretion system are unaffected by both uFAs and 2-AFAs. We have characterized the inhibition mechanism and we have found that in both cases it is a non-competitive inhibition, as the affinities for the substrate (ATP) and product (ADP) are not altered in the presence of the fatty acids. Altogether, these results could open new avenues in the development of new strategies to prevent the dissemination of antibiotic resistance genes.Peer Reviewe