RESUMEN
At low temperatures, protein degradation by the AAA+ HslUV protease is very slow. New crystal structures reveal that residues in the intermediate domain of the HslU6 unfoldase can plug its axial channel, blocking productive substrate binding and subsequent unfolding, translocation, and degradation by the HslV12 peptidase. Biochemical experiments with wild-type and mutant enzymes support a model in which heat-induced melting of this autoinhibitory plug activates HslUV proteolysis.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , CalorRESUMEN
Small ubiquitin-like modifier (SUMO) is commonly used as a protein fusion domain to facilitate expression and purification of recombinant proteins, and a SUMO-specific protease is then used to remove SUMO from these proteins. Although this protease is highly specific, its limited solubility and stability hamper its utility as an in vitro reagent. Here, we report improved SUMO protease enzymes obtained via two approaches. First, we developed a computational method and used it to re-engineer WT Ulp1 from Saccharomyces cerevisiae to improve protein solubility. Second, we discovered an improved SUMO protease via genomic mining of the thermophilic fungus Chaetomium thermophilum, as proteins from thermophilic organisms are commonly employed as reagent enzymes. Following expression in Escherichia coli, we found that these re-engineered enzymes can be more thermostable and up to 12 times more soluble, all while retaining WT-or-better levels of SUMO protease activity. The computational method we developed to design solubility-enhancing substitutions is based on the RosettaScripts application for the macromolecular modeling suite Rosetta, and it is broadly applicable for the improvement of solution properties of other proteins. Moreover, we determined the X-ray crystal structure of a SUMO protease from C. thermophilum to 1.44 Å resolution. This structure revealed that this enzyme exhibits structural and functional conservation with the S. cerevisiae SUMO protease, despite exhibiting only 28% sequence identity. In summary, by re-engineering the Ulp1 protease and discovering a SUMO protease from C. thermophilum, we have obtained proteases that are more soluble, more thermostable, and more efficient than the current commercially available Ulp1 enzyme.
Asunto(s)
Chaetomium/enzimología , Cisteína Endopeptidasas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Cristalografía por Rayos X , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Estabilidad de Enzimas , Mutación , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , TemperaturaRESUMEN
The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.
Asunto(s)
Adenosina Trifosfato/química , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Desplegamiento Proteico , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
The I domain of HslU sits above the AAA+ ring and forms a funnel-like entry to the axial pore, where protein substrates are engaged, unfolded, and translocated into HslV for degradation. The L199Q I-domain substitution, which was originally reported as a loss-of-function mutation, resides in a segment that appears to adopt multiple conformations as electron density is not observed in HslU and HslUV crystal structures. The L199Q sequence change does not alter the structure of the AAA+ ring or its interactions with HslV but increases I-domain susceptibility to limited endoproteolysis. Notably, the L199Q mutation increases the rate of ATP hydrolysis substantially, results in slower degradation of some proteins but faster degradation of other substrates, and markedly changes the preference of HslUV for initiating degradation at the N or C terminus of model substrates. Thus, a structurally dynamic region of the I domain plays a key role in controlling protein degradation by HslUV.
Asunto(s)
Adenosina Trifosfato/química , Endopeptidasa Clp/química , Endopeptidasa Clp/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/enzimología , Mutación , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/genética , Hidrólisis , Microscopía Electrónica , Modelos Moleculares , Dominios Proteicos , Proteolisis , Especificidad por SustratoRESUMEN
ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones.
Asunto(s)
Adenosina Trifosfatasas/química , Endopeptidasa Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Chaperonas Moleculares/química , Desnaturalización Proteica , Proteolisis , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing UâL interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Chaperonas Moleculares/metabolismo , Conformación Proteica , Desplegamiento Proteico , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfato/metabolismo , Sitios de Unión , Modelos Moleculares , Nucleótidos/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismoRESUMEN
The goal of this study was to identify bacterial populations that assimilated methanol in a denitrifying sequencing batch reactor (SBR), using stable isotope probing (SIP) of (13)C labeled DNA and quantitatively track changes in these populations upon changing the electron donor from methanol to ethanol in the SBR feed. Based on SIP derived (13)C 16S rRNA gene clone libraries, dominant SBR methylotrophic bacteria were related to Methyloversatilis spp. and Hyphomicrobium spp. These methylotrophic populations were quantified via newly developed real-time PCR assays. Upon switching the electron donor from methanol to ethanol, Hyphomicrobium spp. concentrations decreased significantly in accordance with their obligately methylotrophic nutritional mode. In contrast, Methyloversatilis spp. concentrations were relatively unchanged, in accordance with their ability to assimilate both methanol and ethanol. Direct assimilation of ethanol by Methyloversatilis spp. but not Hyphomicrobium spp. was also confirmed via SIP. The reduction in methylotrophic bacterial concentration upon switching to ethanol was paralleled by a significant decrease in the methanol supported denitrification biokinetics of the SBR on nitrate. In sum, the results of this study demonstrate that the metabolic capabilities (methanol assimilation and metabolism) and substrate specificity (obligately or facultatively methylotrophic) of two distinct methylotrophic bacterial populations contributed to their survival or washout in denitrifying bioreactors.
Asunto(s)
Reactores Biológicos , Hyphomicrobium/metabolismo , Metanol/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Rhodocyclaceae/metabolismo , Biomasa , Isótopos de Carbono/metabolismo , Etanol/metabolismo , Hyphomicrobium/genética , Cinética , Modelos Lineales , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Rhodocyclaceae/genética , Aguas del Alcantarillado/microbiologíaRESUMEN
Although methanol is a widely employed carbon source for denitrification, relatively little is known on the abundance and diversity of methylotrophic bacteria in activated sludge. The primary aim of this study was to specifically identify bacteria that metabolized methanol in a sequencing batch denitrifying reactor (SBDR), using a novel technique, stable isotope probing (SIP) of 13C labeled DNA. A secondary aim was to quantitatively track dominant methylotrophic bacteria in the SBDR exposed to different terminal electron acceptors. SIP enabled 13C 16S rDNA clone libraries revealed that SBDR methylotrophic populations were related to Methyloversatilis spp. and Hyphomicrobium spp. Based on newly developed quantitative polymerase chain reaction (qPCR) assays, Hyphomicrobium spp. were more abundant than Methyloversatilis spp. throughout the period of SBDR operation. The relative population abundance was stable despite a shift in electron acceptor from nitrate to nitrite (keeping the same methanol dose). However, the shift to nitrite resulted in a significant decrease in denitrification biokinetics on both nitrate and nitrite.