RESUMO
Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing â¼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.
RESUMO
Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.
Assuntos
Adenosina Trifosfatases/metabolismo , Disenteria Bacilar/metabolismo , Shigella flexneri/fisiologia , Sistemas de Secreção Tipo III/metabolismo , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Shigella flexneri/química , Shigella flexneri/patogenicidadeRESUMO
To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Yersinia/enzimologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/genética , Homologia de SequênciaRESUMO
Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Mutação Puntual , Multimerização Proteica , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sistemas de Secreção Bacterianos/química , Sistemas de Secreção Bacterianos/genética , Humanos , Relação Estrutura-AtividadeRESUMO
Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.
Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Biopolímeros/metabolismo , Cromatografia em Gel , Humanos , Cinética , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Espectrometria de Massas em TandemRESUMO
Many key cellular processes can be regulated by the seemingly simple addition of one, or two, methyl groups to arginine residues by the nine known mammalian protein arginine methyltransferases (PRMTs). The impact that arginine methylation has on cellular well-being is highlighted by the ever growing evidence linking PRMT dysregulation to disease states, which has marked the PRMTs as prominent pharmacological targets. This review is meant to orient the reader with respect to the structural features of the PRMTs that account for catalytic activity, as well as provide a framework for understanding how these enzymes are regulated. An overview of what we understand about substrate recognition and binding is provided. Control of product specificity and enzyme processivity are introduced as necessary but flexible features of the PRMTs. Precise control of PRMT activity is a critical component to eukaryotic cell health, especially given that an arginine demethylase has not been identified. We therefore conclude the review with a comprehensive discussion of how protein arginine methylation is regulated.
Assuntos
Arginina/química , Arginina/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ativação Enzimática , Humanos , Metilação , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteína-Arginina N-Metiltransferases/ultraestrutura , Especificidade por SubstratoRESUMO
The FRH (frequency-interacting RNA helicase) protein is the Neurospora crassa homolog of yeast Mtr4, an essential RNA helicase that plays a central role in RNA metabolism as an activator of the nuclear RNA exosome. FRH is also a required component of the circadian clock, mediating protein interactions that result in the rhythmic repression of gene expression. Here we show that FRH unwinds RNA substrates in vitro with a kinetic profile similar to Mtr4, indicating that while FRH has acquired additional functionality, its core helicase function remains intact. In contrast with the earlier FRH structures, a new crystal form of FRH results in an ATP binding site that is undisturbed by crystal contacts and adopts a conformation consistent with nucleotide binding and hydrolysis. Strikingly, this new FRH structure adopts an arch domain conformation that is dramatically altered from previous structures. Comparison of the existing FRH structures reveals conserved hinge points that appear to facilitate arch motion. Regions in the arch have been previously shown to mediate a variety of protein-protein interactions critical for RNA surveillance and circadian clock functions. The conformational changes highlighted in the FRH structures provide a platform for investigating the relationship between arch dynamics and Mtr4/FRH function.