RESUMEN
Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
RESUMEN
Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of â¼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.
Asunto(s)
Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas/fisiología , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Retículo Endoplásmico/metabolismo , Escherichia coli/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica/fisiología , Biosíntesis de Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
The universally conserved signal recognition particle (SRP) co-translationally delivers newly synthesized membrane and secretory proteins to the target cellular membrane. The only exception is found in the chloroplast of green plants, where the chloroplast SRP (cpSRP) post-translationally targets light-harvesting chlorophyll a/b-binding proteins (LHCP) to the thylakoid membrane. The mechanism and regulation of this post-translational mode of targeting by cpSRP remain unclear. Using biochemical and biophysical methods, here we show that anionic phospholipids activate the cpSRP receptor cpFtsY to promote rapid and stable cpSRP54·cpFtsY complex assembly. Furthermore, the stromal domain of the Alb3 translocase binds with high affinity to and regulates GTP hydrolysis in the cpSRP54·cpFtsY complex, suggesting that cpFtsY is primarily responsible for initial recruitment of the targeting complex to Alb3. These results suggest a new model for the sequential recruitment, remodeling, and unloading of the targeting complex at membrane translocase sites in the post-translational cpSRP pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Aniones , Arabidopsis/crecimiento & desarrollo , Membrana Celular/metabolismo , Cloroplastos/metabolismo , Evolución Molecular , GTP Fosfohidrolasas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
The signal recognition particle (SRP) is an essential ribonucleoprotein particle that mediates the co-translational targeting of newly synthesized proteins to cellular membranes. The SRP RNA is a universally conserved component of SRP that mediates key interactions between two GTPases in SRP and its receptor, thus enabling rapid delivery of cargo to the target membrane. Notably, this essential RNA is bypassed in the chloroplast (cp) SRP of green plants. Previously, we showed that the cpSRP and cpSRP receptor GTPases (cpSRP54 and cpFtsY, respectively) interact efficiently by themselves without the SRP RNA. Here, we explore the molecular mechanism by which this is accomplished. Fluorescence analyses showed that, in the absence of SRP RNA, the M-domain of cpSRP54 both accelerates and stabilizes complex assembly between cpSRP54 and cpFtsY. Cross-linking coupled with mass spectrometry and mutational analyses identified a new interaction between complementarily charged residues on the cpFtsY G-domain and the vicinity of the cpSRP54 M-domain. These residues are specifically conserved in plastids, and their evolution coincides with the loss of SRP RNA in green plants. These results provide an example of how proteins replace the functions of RNA during evolution.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Plastidios/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Proteínas de Cloroplastos/química , Cristalografía por Rayos X , Evolución Molecular , GTP Fosfohidrolasas/química , Filogenia , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/químicaRESUMEN
Interactions between proteins underlie numerous biological functions. Theoretical work suggests that protein interactions initiate with formation of transient intermediates that subsequently relax to specific, stable complexes. However, the nature and roles of these transient intermediates have remained elusive. Here, we characterized the global structure, dynamics, and stability of a transient, on-pathway intermediate during complex assembly between the Signal Recognition Particle (SRP) and its receptor. We show that this intermediate has overlapping but distinct interaction interfaces from that of the final complex, and it is stabilized by long-range electrostatic interactions. A wide distribution of conformations is explored by the intermediate; this distribution becomes more restricted in the final complex and is further regulated by the cargo of SRP. These results suggest a funnel-shaped energy landscape for protein interactions, and they provide a framework for understanding the role of transient intermediates in protein assembly and biological regulation.
Asunto(s)
Escherichia coli/química , Receptores Citoplasmáticos y Nucleares/química , Receptores de Péptidos/química , Partícula de Reconocimiento de Señal/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estructura Cuaternaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Membrane protein biogenesis poses acute challenges to protein homeostasis, and how they are selectively escorted to the target membrane is not well understood. Here we address this question in the guided-entry-of-tail-anchored protein (GET) pathway, in which tail-anchored membrane proteins (TAs) are relayed through an Hsp70-Sgt2-Get3 chaperone triad for targeting to the endoplasmic reticulum. We show that the Hsp70 ATPase cycle and TA substrate drive dimeric Sgt2 from a wide-open conformation to a closed state, in which TAs are protected by both substrate binding domains of Sgt2. Get3 is privileged to receive TA from closed Sgt2, whereas off-pathway chaperones remove TAs from open Sgt2. Sgt2 closing is less favorable with suboptimal GET substrates, which are rejected during or after the Hsp70-to-Sgt2 handover. Our results demonstrate how fine-tuned conformational dynamics in Sgt2 enable hydrophobic TAs to be effectively funneled onto their dedicated targeting factor while also providing a mechanism for substrate selection.
Asunto(s)
Proteínas Portadoras , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Portadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Unión ProteicaRESUMEN
During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Perros , GTP Fosfohidrolasas/química , Modelos Biológicos , Conformación Proteica , Transporte de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP-SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clorofila A , Proteínas de Cloroplastos , Activadores de GTP Fosfohidrolasa/metabolismo , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de la Membrana/genética , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Péptidos/genéticaRESUMEN
Co-translational protein targeting to membranes by the signal recognition particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.
Asunto(s)
Mamíferos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Animales , Bacterias/metabolismo , Microscopía por Crioelectrón , GTP Fosfohidrolasas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Dominios Proteicos , Transporte de Proteínas , ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/ultraestructura , Receptores de Péptidos/química , Receptores de Péptidos/metabolismo , Receptores de Péptidos/ultraestructura , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/ultraestructuraRESUMEN
The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3â¢TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas de la Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Citosol/metabolismo , Retículo Endoplásmico/genética , Mutación/genética , Biosíntesis de Proteínas/genética , Transporte de Proteínas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)-driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.
RESUMEN
Cotranslational protein targeting to membranes is regulated by two GTPases in the signal recognition particle (SRP) and the SRP receptor; association between the two GTPases is slow and is accelerated 400-fold by the SRP RNA. Intriguingly, the otherwise universally conserved SRP RNA is missing in a novel chloroplast SRP pathway. We found that even in the absence of an SRP RNA, the chloroplast SRP and receptor GTPases can interact efficiently with one another; the kinetics of interaction between the chloroplast GTPases is 400-fold faster than their bacterial homologues, and matches the rate at which the bacterial SRP and receptor interact with the help of SRP RNA. Biochemical analyses further suggest that the chloroplast SRP receptor is pre-organized in a conformation that allows optimal interaction with its binding partner, so that conformational changes during complex formation are minimized. Our results highlight intriguing differences between the classical and chloroplast SRP and SRP receptor GTPases, and help explain how the chloroplast SRP pathway can mediate efficient targeting of proteins to the thylakoid membrane in the absence of the SRP RNA, which plays an indispensable role in all the other SRP pathways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cloroplastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , ARN del Cloroplasto/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Proteínas de Cloroplastos , Activación Enzimática , Hidrólisis , Cinética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Nucleótidos/metabolismo , Unión Proteica , Ribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.
Asunto(s)
Chaperonas Moleculares/metabolismo , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Microscopía Fluorescente , Modelos Teóricos , Chaperonas Moleculares/genética , Biosíntesis de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Partícula de Reconocimiento de Señal/química , Imagen Individual de MoléculaRESUMEN
Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Cinética , Transporte de ProteínasRESUMEN
Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon.
Asunto(s)
Señales de Clasificación de Proteína , Receptores Citoplasmáticos y Nucleares/química , Receptores de Péptidos/química , Ribosomas/química , Animales , Microscopía por Crioelectrón , GTP Fosfohidrolasas/química , Guanosina Trifosfato/química , Hidrólisis , Conformación Proteica , Multimerización de Proteína , ARN/química , Receptores Citoplasmáticos y Nucleares/ultraestructura , Receptores de Péptidos/ultraestructura , Ribosomas/ultraestructuraRESUMEN
The signal recognition particle (SRP) pathway mediates co-translational targeting of nascent proteins to membranes. Chloroplast SRP is unique in that it does not contain the otherwise universally conserved SRP RNA, which accelerates the association between the SRP guanosine-5'-triphosphate (GTP) binding protein and its receptor FtsY in classical SRP pathways. Recently, we showed that the SRP and SRP receptor (SR) GTPases from chloroplast (cpSRP54 and cpFtsY, respectively) can interact with one another 400-fold more efficiently than their bacterial homologues, thus providing an explanation as to why this novel chloroplast SRP pathway bypasses the requirement for the SRP RNA. Here we report the crystal structure of cpFtsY from Arabidopsis thaliana at 2.0 A resolution. In this chloroplast SR, the N-terminal "N" domain is more tightly packed, and a more extensive interaction surface is formed between the GTPase "G" domain and the N domain than was previously observed in many of its bacterial homologues. As a result, the overall conformation of apo-cpFtsY is closer to that found in the bacterial SRP*FtsY complex than in free bacterial FtsY, especially with regard to the relative orientation of the N and G domains. In contrast, active-site residues in the G domain are mispositioned, explaining the low basal GTP binding and hydrolysis activity of free cpFtsY. This structure emphasizes proper N-G domain arrangement as a key factor in modulating the efficiency of SRP-receptor interaction and helps account, in part, for the faster kinetics at which the chloroplast SR interacts with its binding partner in the absence of an SRP RNA.
Asunto(s)
Cloroplastos/química , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/química , Receptores de Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/metabolismo , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , GTP Fosfohidrolasas/química , Enlace de Hidrógeno , Hidrólisis , Cinética , Malonatos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Receptores de Péptidos/genética , Receptores de Péptidos/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
In an effort to understand the reaction mechanism of a B2 metallo-beta-lactamase, steady-state and pre-steady-state kinetic and rapid freeze quench electron paramagnetic resonance (EPR) studies were conducted on ImiS and its reaction with imipenem and meropenem. pH dependence studies revealed no inflection points in the pH range of 5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Site-directed mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s(-)(1) and a slower rate of fluorescence regain of 98 s(-)(1). Stopped-flow UV-vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s(-)(1). These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 ms that is assigned to a reaction intermediate that has a five-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 ms. When these results are taken together, they allow for a reaction mechanism to be offered for the B2 metallo-beta-lactamases and demonstrate that the mono- and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.
Asunto(s)
Aeromonas/enzimología , Proteínas Bacterianas/química , Zinc/química , beta-Lactamasas/química , Sitios de Unión , Catálisis , Concentración de Iones de Hidrógeno , Estructura Molecular , Unión Proteica , Especificidad por SustratoRESUMEN
The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The biochemical properties of recombinant ImiS were compared with those of native ImiS. Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures. Gel filtration chromatography revealed that both enzymes exist as monomers in solution. MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca. 0.7 mol of Zn(II). Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc. Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme. This over-expression protocol now allows for detailed spectroscopic and mechanistic studies on ImiS as well as site-directed mutants of ImiS to be prepared for future structure/function studies.