Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]x0PxxP, the hydrophobic residue 0 being proline or leucine. We have studied the unusual 0 = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.Peer reviewe

Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite

The authors report here the structure-function analysis of highly divergent actin from Leishmania parasite. The study reveals remarkably rapid dynamics of parasite actin as well as the underlying molecular basis, thus providing insight into evolution of the actin cytoskeleton. Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin 'cofactors', which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.Peer reviewe

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin -binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.Peer reviewe

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo

Biochemical and structural comparison of spleen tyrosine kinase interaction with integrin and the immunoreceptor tyrosine-based activation motif

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in many different signalling pathways activated by immunoreceptors and integrins. The Syk activation mediated by phosphorylated Immunoreceptor Tyrosine-based Activation Motif (pITAM) receptors involves Src homology 2 (SH2) regulatory domains leading to Syk structural rearrangements. Differently, integrin cytoplasmic domains bind to the regulatory domain of Syk and the interaction does not require the phosphorylation, but the molecular mechanism is still unknown. This work focussed on describing the mechanism of integrin-Syk interaction and on how integrins activate Syk. First, using a fluorescent-based kinetic assay we showed that the soluble integrin peptide had no detectable effect on Syk activity, whereas Syk was activated by clustered integrin peptides. This suggests that autophosphorylation is involved in the integrin-induced activation process. Clustered integrins had also a synergic effect combined with pITAM. Next, we wanted to elucidate the molecular mechanism of Syk-integrin interaction. Surface plasmon resonance techniques was used to measure the binding affinities of different Syk constructs towards the integrin peptide. The N-terminal SH2 domain (N-SH2) plus the interdomain A (IA) segment had a comparable binding affinity to the two SH2 domains, whereas N-SH2 was a weak binder. Using nuclear magnetic resonance spectroscopy, we solved the structure of the N-SH2 domain of Syk and analysed the changes on the chemical environments of the N-SH2 domain induced by the integrin peptide and pITAM. The results indicate that integrin and pITAM binding induce changes on the same surface of the protein. In line with this, surface plasmon resonance experiments showed that the integrin and pITAM peptides compete for binding to the regulatory domain of Syk. We compared the binding affinity of pITAM to N-SH2 with C-terminal SH2 (C-SH2) domains observed that N- SH2 had a very low binding affinity compared to C-SH2. These studies suggest that integrin and pITAM-mediated Syk activation are independent of each other but may cause synergistic activation responses at the cellular level.Pernan tyrosiinikinaasi (engl. spleen tyrosine kinase), Syk, on solunsisäinen entsyymi, joka osallistuu immuunireseptoreiden ja integriiniperheen solun tarttumisreseptoreiden välittämään solun toiminnan säätelyyn. Immuunireseptoreiden aiheuttama Syk-entsyymin aktivaatio perustuu reseptoreihin liittyvään fosforyloituvaan proteiinialueeseen nimeltään pITAM (engl. phosphorylated Immunoreceptor Tyrosine-based Activation Motiv). Immunoreseptoreiden kiinnittyessä solun ulkoisiin vastinmolekyyleihinsä, Syk kiinnittyy pITAM-alueeseen reseptorin solunsisäisessä osassa. Tämä kiinnittyminen aiheuttaa Syk-entsyymissä rakennemuutoksen ja aktivoi sen. Tämän tutkimuksen tarkoituksena on ollut selvittää, miten integriinit saavat aikaan Syk:n aktivaation. Tutkimuksessa käytettiin yhdistelmä-DNA tekniikoilla tuotettua ja puhdistettua Syk-proteiinia sekä sen säätelyosan paloja. Entsyymiaktiivisuusmittaukset osoittivat, että liukoinen integriinipeptidi ei aktivoinut Syk-entsyymiä, mutta pITAM-peptidi aktivoi. Sen sijaan analyysikammion pintaan kiinnitetty integriinipeptidi aktivoi Syk-entsyymiä ja pystyi tehostamaan liukoisen pITAM-peptidin aiheuttamaan aktivaatiota. Tästä pääteltiin, että pITAM ja integriinit aktivoivat Syk entsyymiä eri mekanismeilla: pITAM sitoutumisen aiheuttaman rakennemuutoksen kautta ja integriinit klusteroimalla Syk-entsyymiä niin, että se pystyy fosforyloimaan itseään ja näin aktivoitumaan. Toinen lähestymistapa tutkimuksessa oli integriini- ja pITAM- peptidien sitoutumispaikkojen tarkka kartoitus Syk:n säätelyalueessa. Tähän käytettiin pintaplasmoniresonanssiin perustuvaa menetelmää ja ydinmagneettista resonanssispektroskopiaa, NMR-spektroskopiaa (engl. Nuclear Magnetic Resonance). Tutkimuksia varten myös laskettiin NMR-mittauksiin perustuva 3- ulotteinen proteiinimalli Syk:n N-SH2-domeenista. Tulokset osoittivat, että pITAM-alue sitoutuu ensisijaisesti Syk:n C-SH2-domeeniin ja toissijaisesti N- SH2-domeeniin. Integriini sen sijaan sitoutuu ensisijaisesti domeenien väliseen alueeseen ja toissijaisesti N-SH2-domeeniin. Yllätys oli, että N-SH2-domeenissa integriinin sitoutuminen aiheutti kemiallisia muutoksia lähellä pITAM:n sitoutumiskohtaan ja että pITAM-peptidi pystyi inhiboimaan Syk:n säätelyosan sitoutumista integriinipeptidiin. Kokonaisuudessaan nämä tutkimukset osoittavat, että integriinien ja pITAM-reseptoreiden aiheuttama Syk-entsyymin aktivaatimekanismit ovat osittain toisistaan riippumattomia ja voivat siten voivat siten solutasolla toimia yhteistyössä

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called “last-resort” drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete 1 H/13C/15N resonance assignment of the catalytic domain of LytM, residues 185–316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall.peerReviewe

Structure–activity relationships of fraxamoside as an unusual xanthine oxidase inhibitor

Fraxamoside, a macrocyclic secoiridoid glucoside featuring a hydroxytyrosol group, was recently identified as a xanthine oxidase inhibitor (XOI) comparable in potency in vitro to the standard antigout drug allopurinol. However, this activity and its considerably higher value than its derivatives oleuropein, oleoside 11-methyl ester, and hydroxytyrosol are not explained by structure–activity relationships (SARs) of known XOIs. To exclude allosteric mechanisms, we first determined the inhibition kinetic of fraxamoside. The resulting competitive mechanism prompted a computational SAR characterization, combining molecular docking and dynamics, which fully explained the behavior of fraxamoside and its derivatives, attributed the higher activity of the former to conformational properties of its macrocycle, and showed a substantial contribution of the glycosidic moiety to binding, in striking contrast with glycoside derivatives of most other XOIs. Overall, fraxamoside emerged as a lead compound for a new class of XOIs potentially characterized by reduced interference with purine metabolism

Characterization of a Type II L-Asparaginase from the Halotolerant <i>Bacillus subtilis</i> CH11

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg−1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value Vmax and Km were 145.2 µmol mL−1 min−1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications