49 research outputs found
Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027
Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis
Charge as a Selection Criterion for Translocation through the Nuclear Pore Complex
Nuclear pore complexes (NPCs) are highly selective filters that control the exchange of material between nucleus and cytoplasm. The principles that govern selective filtering by NPCs are not fully understood. Previous studies find that cellular proteins capable of fast translocation through NPCs (transport receptors) are characterized by a high proportion of hydrophobic surface regions. Our analysis finds that transport receptors and their complexes are also highly negatively charged. Moreover, NPC components that constitute the permeability barrier are positively charged. We estimate that electrostatic interactions between a transport receptor and the NPC result in an energy gain of several kBT, which would enable significantly increased translocation rates of transport receptors relative to other cellular proteins. We suggest that negative charge is an essential criterion for selective passage through the NPC.Merck Research LaboratoriesNational Science Foundation (U.S.) (Division of Mathematical Sciences)Kavli Institute for Bionano Science & Technology at Harvard UniversityNational Centers for Systems Biology (U.S.) (NIGMS grant GM068763)National Institute of General Medical Sciences (U.S.
NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan
A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases
Advances on the Transfer of Lipids by Lipid Transfer Proteins
Transfer of lipid across the cytoplasm is an essential process for intracellular lipid traffic. Lipid transfer proteins (LTPs) are defined by highly controlled in vitro experiments. The functional relevance of these is supported by evidence for the same reactions inside cells. Major advances in the LTP field have come from structural bioinformatics identifying new LTPs, and from the development of countercurrent models for LTPs. However, the ultimate aim is to unite in vitro and in vivo data, and this is where much progress remains to be made. Even where in vitro and in vivo experiments align, rates of transfer tend not to match. Here we set out some of the advances that might test how LTPs work
In silico analyses of maleidride biosynthetic gene clusters
Maleidrides are a family of structurally related fungal natural products, many of which possess diverse, potent bioactivities. Previous identification of several maleidride biosynthetic gene clusters, and subsequent experimental work, has determined the âcoreâ set of genes required to construct the characteristic medium-sized alicyclic ring with maleic anhydride moieties. Through genome mining, this work has used these core genes to discover ten entirely novel putative maleidride biosynthetic gene clusters, amongst both publicly available genomes, and encoded within the genome of the previously un-sequenced epiheveadride producer Wicklowia aquatica CBS 125634. We have undertaken phylogenetic analyses and comparative bioinformatics on all known and putative maleidride biosynthetic gene clusters to gain further insights regarding these unique biosynthetic pathways. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40694-022-00132-z
Structure-function study of the enzymes of the cyanuric acid catabolic pathways
Up to now the degradation of atrazine by Pseudomonas sp. strain
ADP1 bacterium was thought to involve six steps successively
catalysed by enzymes: AtzA, AtzB, AtzC, degrading atrazine into
cyanuric acid, and AtzD, AtzE and AtzF, successively mineralising
cyanuric acid to ammonia and carbon dioxide. The genes
atzD-aztE-atzF are arranged in an operon called the cyanuric acid
degradation operon. The exploration of cyanuric acid degradation
pathways in different bacteria showed that substantial
differences exist in the cyanuric acid degradation pathways
between microorganisms. In Rhizobium leguminasorum bv. viciae
3841, for example, a biuret hydrolase (BiuH) belonging to the
isochorismatase family performs the deamination of biuret to
produce allophanate (118); whereas, in the model s-triazine
degrading bacterium Pseudomonas sp. strain ADP, it is an amidase,
AtzE, that is thought to perform that step. The characterisation
of AtzE revealed the existence of two new enzymes in the
Pseudomonas sp. strain ADP1 cyanuric acid operon.
The first part of this PhD, reports the structure-function study
of the BiuH in Rhizobium leguminasorum bv. viciae 3841. The
atomic structure of BiuH was solved and site-directed mutagenesis
was used to gain a better understanding of the BiuH catalytic
mechanism. Additionally, molecular dynamics simulations
highlighted the presence of three channels from the active site
to the enzyme surface forming a potential substrate channel, a
co-product (ammonia) channel and a co-substrate (water) channel.
Although the cyanuric acid degradation pathway in Pseudomonas sp.
strain ADP1 has been known and studied for more than twenty
years, no one had purified and characterised AtzE. The second
part of this PhD reports the purification of the native AtzE from
Pseudomonas sp. strain ADP, allowing its biochemical and
structural characterisation. The structure revealed the presence
of a small, essential protein (AtzG), with which AtzE forms a
heterotetramer. Biochemical characterisation and molecular
dynamics experiments revealed AtzE acts as a 1-carboxybiuret
hydrolase, not as a biuret hydrolase as previously thought.
Finally, this work suggests that AtzE might have evolved from the
GatCAB transamidosome complex.
The final part of my PhD presents the discovery and the study of
AtzH, a previously unknown small protein encoded by a gene
located in the Pseudomonas sp. strain ADPâs cyanuric acid
degradation operon. The structural characterisation of AtzH
determined it belonged to the versatile NFT2 protein superfamily.
A combination of structural modelling and mutagenesis studies was
used to provide evidence that AtzH is an allophanate forming,
1,3-dicarboxyurea amidohydrolase. Mutagenesis also indicated that
Tyr22 and Arg46 may play an essential role in the catalysis of
1,3-dicarboxyurea. Finally, a comparison of the genomic context
suggests AtzH might be involved more broadly in the catabolism of
nitrogenous compounds in Proteobacteria. Moreover, this
observation also suggests that the atzG-atzE-atzH cluster
predates the formation of the cyanuric acid catabolism operon
Proteomics uncovers novel components of an interactive protein network supporting RNA export in trypanosomes
In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation
Kap-Centric control of nuclear pores based on promiscuous binding to FG nucleoporins
Nuclear pore complexes (NPCs) are remarkable molecular machines that perforate the nuclear envelope (NE) in eukaryotic cells and mediate the rapid bidirectional traffic of hundreds of proteins, ribonucleoproteins, and metabolites across the nuclear envelope. Their enormous structure is composed of multiple copies of 30 different proteins (Nups) that add up to 60 â 120 MDa of mass depending on the organism. Each NPC contains a 50 nm-diameter central channel through which only molecules smaller than ~40 kDa or ~5 nm in size can diffuse passively. The movement of larger molecules is impaired by a permeability barrier generated by ~200 partly intrinsically disordered phenylalanine-glycine (FG)-rich nucleoporins (FG Nups) that are tethered to the NPC transport channel surface. These FG Nups interact promiscuously with nuclear transport receptors (NTRs), such as karyopherins (Kaps; e.g. Kap-beta1) or NTF2, that mediate rapid trafficking of cargoes.
Given that the number of FG repeats per FG Nup also varies from 5 to ~50, NTR-FG Nup binding involves highly multivalent interactions, which are generally known to impart a strong avidity that enhances stability and specificity. However, this is paradoxical in the context of the NPC, because the high submicromolar Kap-beta1-FG domain binding affinities predict slow off rates (given a diffusion-limited on rate) that contradict the rapid (~5 ms) in vivo dwell time. As this implies, Kap-FG binding ought to be sufficiently strong to ensure selectivity but also weak enough to promote fast translocation through the NPC. Nonetheless, an explanation as to how promiscuous binding of FG Nups to NTRs is balanced against the mechanistic control of the FG domain barrier is still lacking.
The purpose of my work was to investigate FG Nup-NTR binding promiscuity and multivalency by measuring the interaction kinetics, binding affinity and in situ associated conformational changes in Nsp1p FG domains when binding NTF2 and Kap-beta1, both separately and together. Experimentally, this was achieved by using a novel surface plasmon resonance technique to correlate in situ mechanistic changes (molecular occupancy and conformational changes) with FG Nup-NTR binding.
The obtained results show that surface-tethered Nsp1p FG domains form molecular brushes at physiological conditions. Kap-beta1 binding provokes brush extension while partitioning into a fast and slow kinetic phase, where the latter may form an integral part of the FG domain barrier. In contrast, NTF2 binding to pristine Nsp1p layers induced collapse, but not under competing interactions from Kap-beta1. Therefore, promiscuous binding of NTF2 to Kap-beta1-preloaded Nsp1p attenuates NTF2 towards higher off rates and more transient interactions.
My work demonstrates that promiscuous binding of NTRs to FG Nups ought to influence nucleocytoplasmic transport. This depends on the concentration, size and binding strength of each NTR. Indeed, some form of hierarchy may exist between different NTRs such that their relative concentrations may impact NPC barrier function. This interpretation departs from the conventional view that the FG Nups alone form the NPC permeability barrier. Rather I conclude that concentrating NTRs in the NPC transport channel also contributes to generating crowding-based selective barrier function of the pore
Analysis of diverse signal transduction pathways using the genetic model system Caenorhabditis elegans
Signal transduction allows cells to respond to signals from their environment and is therefore important for most biological processes. The binding of an extracellular signalling molecule to a cell-surface receptor is the first step in most signal transduction pathways. Cell-surface receptors transduce the extracellular signals by generating a cascade of intracellular signals that alter the behaviour of the cell. The proteins involved in signal transduction include, besides the receptors, GTP-binding proteins, protein kinases and ion channels, and these proteins are conserved between multicellular organisms. In this study, we used the nematode Caenorhabditis elegans, a simple and well-described organism that is very well suited to perform genetic experiments, to analyse conserved signal transduction pathways.
Chapter two of this thesis describes the genetic analysis of an important signaltransducing molecule, the adenylyl cyclase SGS-1. Adenylyl cyclases act downstream of certain heterotrimeric G proteins and convert ATP into the second messenger cAMP. In C. elegans, SGS-1 can be activated by the homologue of mammalian Gas, GSA-1, since mutations in sgs-1 suppress the neuronal degeneration induced by expression of constitutively active GSA-1 from its own or heat-shock promoter. We show that SGS-1 is essential for viability and that it is involved in behaviours as diverse as pharyngeal pumping and locomotion. To regulate this latter behaviour SGS-1 needs to be activated by GSA-1.
In chapter three, nxf-1, another suppressor of the neuronal degeneration induced by expression of constitutively active GSA-1 from a heat-shock promoter, is characterized. nxf-1 encodes a nuclear export factor that is involved in transport of mRNA out of the nucleus. Mutations in nxf-1 also suppress other heat-shock promoter-induced phenotypes, indicating that this locus is not a specific downstream target of Gas, but rather a general suppressor of heat-shock promoter-induced phenotypes. We postulate that the mutations in nxf-1 make the protein inactive during heat-shock, resulting in reduced transport of the activated Gas and other mRNAs out of the nucleus and thus the absence of the activated Gas phenotype after heat-shock. Chapter four reports the analysis of the C. elegans homologue of Ga12/13, GPA- 12. Loss of GPA-12 does not result in any obvious defect in development or behaviour. However, overexpression of constitutively active GPA-12 from its own or heat-shock promoter results in a developmental growth arrest that is caused by a feeding defect. Mutations in tpa-1, which encodes two PKC isoforms, suppress the developmental growth arrest induced by activated GPA-12, indicating that TPA-1 acts downstream of GPA-12. Activation of TPA-1 by the tumour-promoting phorbol ester TPA (or PMA) results in an identical developmental growth arrest, showing that activated GPA-12 and PMA use the same PKC signalling pathway.
In chapter five of this thesis, the DYRK family of protein kinases is described. A human DYRK kinase is implicated in the cognitive defects caused by trisomy of chromosome 21, and we show that in Caenorhabditis elegans overexpression of a DYRK family member, mbk-1, results in signal transduction defects in olfactory neurons. mbk-1 knockout animals do not show any obvious defects. Loss of hpk-1, another member of the DYRK family in C. elegans, does also not result in any obvious phenotype. However, a third member, mbk-2, is essential for viability