Structural and Regulatory Functions of the NH2- and COOH-terminal Regions of Skeletal Muscle Troponin I

O complexo troponina-tropomiosina regula a contração muscular esquelética e cardíaca. A ligação do cálcio nos sítios regulatórios localizados no domínio N-terminal da troponina C (TnC) induz uma mudança conformacional que remove a ação inibitória da troponina I (TnI) e inicia a contração muscular. Nós usamos fragmentos recombinantes da TnI e uma série de mutantes da TnC para estudar as interações estruturais e regulatórias das diferentes regiões da TnI com os domínios da TnC, TnT e actina-tropomiosina. Nossos resultados indicam que a TnI é organizada em regiões que apresentam funções estruturais e regulatórias e que se ligam de modo antiparalelo com os correspondentes domínios estruturais e regulatórios da TnC. Estudos funcionais mostram que a região inibitória (aminoácidos 103-116) em combinação com a região C-terminal da TnI (TnI103-182) pode regular a atividade ATPásica da acto-miosina de maneira dependente de Ca2+. A regulação não é observada com a região inibitória em combinação com a região N-terminal (TnI116) Estudos de ligação mostram que a região N-terminal da TnI (TnI1-98) interage com o domínio C-terminal da TnC na presença e na ausência de Ca2+ e também interage com a TnT. A região inibitória/C-terminal da TnI (TnI103-182) interage com o domínio N-terminal da TnC de maneira dependente de Ca2+. Baseados nestes resultados, propomos um modelo para a mudança conformacional induzida pelo Ca2+. Neste modelo, a região N-terminal da TnI está ligada fortemente com o domínio C-terminal da TnC na presença ou na ausência de Ca2+. As regiões inibitórias e C-terminal da TnI ligam-se à actina-tropomiosina na ausência de Ca2+ e nos domínios N-terminal e C-terminal da TnC na presença de Ca2+.The troponin-tropomyosin complex regulates skeletal and cardiac muscle contraction. Calcium binding to the regulatory sites in the N-terminal domain of troponin C (TnC). induces a conformational change which removes the inhibitory action of troponin I (TnI) and initiates muscular contraction. We used recombinant TnI fragments and a series of TnC mutants to study the structural and regulatory interactions between different TnI regions and the domains of TnC, TnT and actin-tropomyosin. Our results indicate that TnI is organized into regions with distinct structural and regulatory functions which bind, in an antiparallel manner, with the corresponding structural and regulatory domains of TnC. Functional studies show that a fragment containing the inhibitory and C-terminal regions of TnI (TnIl03-182) can regulate the actomyosin ATPase in a Ca2+- dependent manner. Regulation was not observed with a fragment containing the N-terminal and inhibitory regions (TnIl-116). Binding studies show that the N-terminal region of TnI (TnI1-98) interacts with the C-terminal domain of TnC in the presence of Ca2+ or Mg2+. The inhibitory/C-terminal region of TnI (TnI103-182) binds to the N-terminal domain of TnC in a Ca2+-dependent manner. Based on these results, we propose a model for the Ca2+ -induced conformational change. In this model the N-terminal region of TnI is bound strongly to the C-terminal domain of TnC in the presence or absence of Ca2+. The inhibitory and C-terminal regions of TnI bind to actin-tropomyosin in the absence of Ca2+ and to tne N- and C-terminal domains of TnC in the presence of Ca2+

Structural and Regulatory Functions of the NH2- and COOH-terminal Regions of Skeletal Muscle Troponin I

O complexo troponina-tropomiosina regula a contração muscular esquelética e cardíaca. A ligação do cálcio nos sítios regulatórios localizados no domínio N-terminal da troponina C (TnC) induz uma mudança conformacional que remove a ação inibitória da troponina I (TnI) e inicia a contração muscular. Nós usamos fragmentos recombinantes da TnI e uma série de mutantes da TnC para estudar as interações estruturais e regulatórias das diferentes regiões da TnI com os domínios da TnC, TnT e actina-tropomiosina. Nossos resultados indicam que a TnI é organizada em regiões que apresentam funções estruturais e regulatórias e que se ligam de modo antiparalelo com os correspondentes domínios estruturais e regulatórios da TnC. Estudos funcionais mostram que a região inibitória (aminoácidos 103-116) em combinação com a região C-terminal da TnI (TnI103-182) pode regular a atividade ATPásica da acto-miosina de maneira dependente de Ca2+. A regulação não é observada com a região inibitória em combinação com a região N-terminal (TnI116) Estudos de ligação mostram que a região N-terminal da TnI (TnI1-98) interage com o domínio C-terminal da TnC na presença e na ausência de Ca2+ e também interage com a TnT. A região inibitória/C-terminal da TnI (TnI103-182) interage com o domínio N-terminal da TnC de maneira dependente de Ca2+. Baseados nestes resultados, propomos um modelo para a mudança conformacional induzida pelo Ca2+. Neste modelo, a região N-terminal da TnI está ligada fortemente com o domínio C-terminal da TnC na presença ou na ausência de Ca2+. As regiões inibitórias e C-terminal da TnI ligam-se à actina-tropomiosina na ausência de Ca2+ e nos domínios N-terminal e C-terminal da TnC na presença de Ca2+.The troponin-tropomyosin complex regulates skeletal and cardiac muscle contraction. Calcium binding to the regulatory sites in the N-terminal domain of troponin C (TnC). induces a conformational change which removes the inhibitory action of troponin I (TnI) and initiates muscular contraction. We used recombinant TnI fragments and a series of TnC mutants to study the structural and regulatory interactions between different TnI regions and the domains of TnC, TnT and actin-tropomyosin. Our results indicate that TnI is organized into regions with distinct structural and regulatory functions which bind, in an antiparallel manner, with the corresponding structural and regulatory domains of TnC. Functional studies show that a fragment containing the inhibitory and C-terminal regions of TnI (TnIl03-182) can regulate the actomyosin ATPase in a Ca2+- dependent manner. Regulation was not observed with a fragment containing the N-terminal and inhibitory regions (TnIl-116). Binding studies show that the N-terminal region of TnI (TnI1-98) interacts with the C-terminal domain of TnC in the presence of Ca2+ or Mg2+. The inhibitory/C-terminal region of TnI (TnI103-182) binds to the N-terminal domain of TnC in a Ca2+-dependent manner. Based on these results, we propose a model for the Ca2+ -induced conformational change. In this model the N-terminal region of TnI is bound strongly to the C-terminal domain of TnC in the presence or absence of Ca2+. The inhibitory and C-terminal regions of TnI bind to actin-tropomyosin in the absence of Ca2+ and to tne N- and C-terminal domains of TnC in the presence of Ca2+

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao ButantanFundacao ButantanConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Structure of the PilZ-FimXEAL-c-di-GMP complex responsible for the regulation of bacterial type IV pilus biogenesis

Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein-protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins β-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation encountered in c-di-GMP-protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational statesFundação de Amparo à Pesquisa do Estado de São Paulo, 2011/07777-5Fundação de Amparo à Pesquisa do Estado de São Paulo, 2009/14477-8Fundação de Amparo à Pesquisa do Estado de São Paulo, 2011/22571-

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved

Distribution, function and regulation of type 6 secretion systems of xanthomonadales

Members of the Xanthomonadales order include several plant pathogens of significant economic and agricultural impact, such as Xanthomonas spp. Type 6 secretion systems (T6SSs) are contractile nanomachines used by many bacterial species to inject protein effectors into target prokaryotic and eukaryotic cells and provide a competitive advantage for bacteria in different environments. Effectors with antibacterial properties include peptidoglycan hydrolases, lipases and phospholipases that break down structural components of the cell envelope, promoting target-cell lysis; and RNases, DNAses, and NADases that affect target-cell metabolism, arresting growth. Effectors with anti-eukaryotic properties are functionally more diverse. The T6SS of Xanthomonas citri is the only example experimentally characterized so far within the Xanthomonadales order and displays anti-eukaryotic function by providing resistance to predation by amoeba. This T6SS is regulated at the transcriptional level by a signaling cascade involving a Ser/Thr kinase and an extracytoplasmic function (ECF) sigma factor. In this review, we performed in silico analyses of 35 genomes of Xanthomonadales and showed that T6SSs are widely distributed and phylogenetically classified into three major groups. In silico predictions identified a series of proteins with known toxic domains as putative T6SS effectors, suggesting that the T6SSs of Xanthomonadales display both anti-prokaryotic and anti-eukaryotic properties depending on the phylogenetic group and bacterial species10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação2017/02178-2; 2017/17303-7; 2018/01852-4; 2018/04553-8; 2017/02318-

Analysis of three Xanthomonas axonopodis pv. citri effector proteins in pathogenicity and their interactions with host plant proteins

Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, uses effector proteins secreted by a type III protein secretion system to colonize its hosts. Among the putative effector proteins identified for this bacterium, we focused on the analysis of the roles of AvrXacE1, AvrXacE2 and Xac3090 in pathogenicity and their interactions with host plant proteins. Bacterial deletion mutants in avrXacE1, avrXacE2 and xac3090 were constructed and evaluated in pathogenicity assays. The avrXacE1 and avrXacE2 mutants presented lesions with larger necrotic areas relative to the wild-type strain when infiltrated in citrus leaves. Yeast two-hybrid studies were used to identify several plant proteins likely to interact with AvrXacE1, AvrXacE2 and Xac3090. We also assessed the localization of these effector proteins fused to green fluorescent protein in the plant cell, and observed that they co-localized to the subcellular spaces in which the plant proteins with which they interacted were predicted to be confined. Our results suggest that, although AvrXacE1 localizes to the plant cell nucleus, where it interacts with transcription factors and DNA-binding proteins, AvrXacE2 appears to be involved in lesion-stimulating disease 1-mediated cell death, and Xac3090 is directed to the chloroplast where its function remains to be clarified.Argentine Federal Government (ANPCyT) [PICT2010-1507, PICT2010-0300]Argentine Federal Government (ANPCyT)Fundacion Josefina PratsFundacion Josefina PratsFundacao de Amparo a Pesquisa do Estado de Sao Paulo [2005/59243-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paul

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[98/14138-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[00/10266-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[03/08316-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[07/06686-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing.

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings

VirB7 and VirB9 Interactions Are Required for the Assembly and Antibacterial Activity of a Type IV Secretion System

The type IV secretion system (T4SS) from the phytopathogen Xanthomonas citri (Xac) is a bactericidal nanomachine. The T4SS core complex is a ring composed of multiple copies of VirB7-VirB9-VirB10 subunits. Xac-VirB7 contains a disordered N-terminal tail (VirB7(NT)) that recognizes VirB9, and a C-terminal domain (VirB7(CT)) involved in VirB7 self-association. Here, we show that VirB7(NT) forms a short β strand upon binding to VirB9 and stabilizes it. A tight interaction between them is essential for T4SS assembly and antibacterial activity. Abolishing VirB7 self-association or deletion of the VirB7 C-terminal domain impairs this antibacterial activity without disturbing T4SS assembly. These findings reveal protein interactions within the core complex that are critical for the stability and activity of a T4SS