RESUMO
Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins-such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)-impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy ([Formula: see text]) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published [Formula: see text] for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (Funfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (Funfold > 80 pN) and brittle (Δx < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.
Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Negativas/ultraestrutura , Microscopia de Força Atômica , Estabilidade Proteica , Salmonella/fisiologia , Salmonella/ultraestrutura , TermodinâmicaRESUMO
Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.
Assuntos
Lipídeo A , Polimixinas , Polimixinas/farmacologia , Polimixinas/metabolismo , Lipídeo A/metabolismo , Arabinose/metabolismo , Amino Açúcares/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Carboidratos , Proteínas de Bactérias/químicaRESUMO
Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD+-dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of Salmonella enterica serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD+ binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.
Assuntos
NAD , Polimixinas , Humanos , Polimixinas/farmacologia , Polimixinas/química , Ligantes , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismo , OxirredutasesRESUMO
Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.
Assuntos
Abietanos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Dobramento de ProteínaRESUMO
BamA is an essential component of the ß-barrel assembly machine (BAM) that is responsible for insertion and folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. BamA is an OMP itself, and its ß-barrel transmembrane domain is thought to catalyze OMP insertion and folding, although the molecular mechanism remains poorly understood. Crystal structures of BamA and complementary molecular dynamics simulations have shown that its ß-barrel seam (the interface between the first and last barrel strands) is destabilized. This has led to mechanistic models in which the BamA barrel seam functions as a lateral gate that opens and successively accepts ß-hairpins from a nascent OMP such that a nascent barrel can bud from BamA. Consistent with this model, disulfide locking of the BamA barrel seam is lethal in Escherichia coli. Here we show that disulfide locking of the BamA barrel has no effect on its ability to catalyze folding of a model OMP into liposomes. However, disulfide trapping experiments indicate that the BamA barrel is highly dynamic in the liposome membranes, with the ß-strands at the barrel seam undergoing "register sliding" by more than 14 Å both up and down the membrane. Remarkably, these extreme dynamics were also observed in the BamA barrel in the context of the native E. coli outer membrane. These results are consistent with a model in which the BamA barrel dynamics induce defects in the outer membrane that facilitate insertion of nascent OMPs.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Termodinâmica , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cinética , Lipossomos/química , Lipossomos/metabolismo , Dobramento de ProteínaRESUMO
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.
Assuntos
Proteínas/química , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Conformação Proteica em alfa-Hélice , TemperaturaRESUMO
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50-1000 µs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-µs temporal resolution was achieved using an ultrashort (L = 9 µm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-µm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-µs response time while eliminating cantilever ringing (Q â 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.
Assuntos
Microscopia de Força Atômica/métodos , Análise Espectral/métodosRESUMO
We present the first crystallographic insight into the interactions of an ionic liquid (IL) with an enzyme, which has widespread implications for stabilizing enzymes in IL media for biocatalysis. Structures of Bacillus subtilis lipaseâ A (lipA) and an IL-stable variant (QM-lipA) were obtained in the presence of increasing concentrations of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). These studies revealed that the [BMIM] cation interacts with surface residues through hydrophobic and cation-π interactions. Of specific interest was the disruption of internal stacking interactions of aromatic side chains by [BMIM], which provides structural evidence for the mechanism of enzyme denaturation by ILs. The interaction of [BMIM] and Cl ions with lipA was reduced by the stabilizing mutations Y49E and G158E in QM-lipA. Ultimately, these findings present the molecular basis for stabilizing enzymes from IL-induced inactivation, as well as the selection of ILs that are less denaturing.
Assuntos
Imidazóis/química , Líquidos Iônicos/química , Bacillus subtilis/enzimologia , Sítios de Ligação , Biocatálise , Lipase/química , Lipase/genética , Lipase/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de ProteínaRESUMO
Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas , have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar-nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the (4)C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana , Polimixinas/farmacologia , Salmonella typhimurium/enzimologia , Transaminases/química , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação Proteica , Fosfato de Piridoxal/química , Salmonella typhimurium/genética , Especificidade por Substrato , Açúcares de Uridina Difosfato/químicaRESUMO
Metastatic melanoma is among the most intractable cancers to treat; patients show resistance to therapy and limited survival time. A critical step in the development of metastatic melanoma is the acquisition of invasion and transition from thin to thick tumors on the skin, followed by invasion to lymph nodes. Prior studies have shown that metastatic melanoma is associated with dysregulation of RhoA and enhanced expression of a protein named "mediator of RhoA-dependent invasion (MRDI)". Importantly, MRDI is a "moonlighting" enzyme, with two distinct functions in melanoma cells. First, MRDI acts as a methylthioribose-1-phosphate (MTR-1-P) isomerase, catalyzing a critical step in methionine salvage. Second, MRDI promotes and is necessary for melanoma cell invasion, independent of its catalytic activity. This paper demonstrates that MtnA, a bacterial MTR-1-P isomerase, rescues the methionine salvage function of MRDI, but is unable to rescue its role in invasion. The crystal structure of MRDI was solved to a resolution of 2.5 Å to identify structural elements important for its invasion activity. This structure and its comparison with other MTR-1-P isomerases are presented, and mutations within a region separate from the MTR-1-P binding site, which interfere with invasion, are identified. Thus, structural elements in MRDI distal from the MTR-1-P catalytic site are responsible for the invasion phenotype.
Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Western Blotting , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Teste de Complementação Genética , Humanos , Isomerases/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Invasividade Neoplásica , Conformação Proteica , Ribulosefosfatos/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Relação Estrutura-AtividadeRESUMO
Outer membrane proteins (OMPs) of gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded beta-barrel domain and a periplasmic alphabeta domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the beta-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA beta-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of beta-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/química , Chaperonas Moleculares/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Transporte ProteicoRESUMO
RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Melanoma , Metionina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Melanoma/enzimologia , Melanoma/patologia , Metionina/química , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Estrutura Molecular , Invasividade Neoplásica , Metástase Neoplásica , Proteômica/métodos , Interferência de RNA , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Transdução de Sinais/fisiologia , Transplante Heterólogo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The envelope of Gram-negative bacteria consists of inner and outer membranes surrounding the peptidoglycan wall. The outer membrane (OM) is rich in integral membrane proteins (OMPs), which have a characteristic beta barrel domain embedded in the OM. The Omp85 family of proteins, ubiquitous among Gram-negative bacteria and also present in chloroplasts and mitochondria, is required for folding and insertion of OMPs into the outer membrane. Bacterial Omp85 proteins are characterized by a periplasmic domain containing five repeats of polypeptide transport-associated (POTRA) motifs. Here we report the crystal structure of a periplasmic fragment of YaeT (the Escherichia coli Omp85) containing the first four POTRA domains in an extended conformation consistent with recent solution X-ray scattering data. Analysis of the YaeT structure reveals conformational flexibility around a hinge point between POTRA2 and 3 domains. The structure's implications for substrate binding and folding mechanisms are also discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Conformação Proteica , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Cristalização , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Periplasma/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Absorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic-GMP (cGMP). This hydrolysis promotes the closing of cGMP-gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate cyclase-activating proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca2+-dependent manner. At high [Ca2+], typical of the dark-adapted state (approximately 500 nM), GCAPs inhibit retinal GCs. At the low [Ca2+] (approximately 50 nM) that occurs after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark-state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca2+ bound. GCAP3 is an EF-hand Ca2+-binding protein with Ca2+ bound to EF2, 3 and 4, while Ca2+ binding to EF-hand 1 is disabled. GCAP3 contains two domains with the EF-hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca2+-binding proteins. Residues not involved in Ca2+ binding, but conserved in all GCAPs, cluster around EF1 in the N-terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations.
Assuntos
Proteínas Ativadoras de Guanilato Ciclase/química , Estrutura Terciária de Proteína , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/metabolismo , Cálcio/metabolismo , Cristalografia por Raios X , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Células Fotorreceptoras Retinianas Cones/químicaRESUMO
The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 A conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors.
Assuntos
Amino Açúcares/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Polimixinas/farmacologia , Uridina Difosfato Glucose Desidrogenase/química , Trifosfato de Adenosina/metabolismo , Amino Açúcares/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dimerização , Ligação de Hidrogênio , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/metabolismo , Ligantes , Modelos Moleculares , Conformação Molecular , Mutação , Oxirredução , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Análise Espectral Raman , Especificidade por Substrato , Uridina Difosfato Glucose Desidrogenase/genética , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismoRESUMO
The ß-barrel assembly machine (BAM) mediates the biogenesis of outer membrane proteins (OMPs) in Gram-negative bacteria. BamA, the central BAM subunit composed of a transmembrane ß-barrel domain linked to five polypeptide transport-associated (POTRA) periplasmic domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. One model is that conformational flexibility between POTRA domains is part of this conformational cycling. Nuclear magnetic resonance (NMR) spectroscopy was used here to study the flexibility of the POTRA domains 1-5 in solution. NMR relaxation studies defined effective rotational correlational times and together with residual dipolar coupling data showed that POTRA1-2 is flexibly linked to POTRA3-5. Mutants of BamA that restrict flexibility between POTRA2 and POTRA3 by disulfide crosslinking displayed impaired function in vivo. Together these data strongly support a model in which conformational cycling of hinge motions between POTRA2 and POTRA3 in BamA is required for biological function.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Periplasma , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.
Assuntos
Proteínas de Choque Térmico/química , Serina Endopeptidases/química , Proteases Dependentes de ATP , Adenosina Trifosfatases/metabolismo , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Ativação Enzimática , Haemophilus influenzae/enzimologia , Proteínas de Choque Térmico/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Inibidores de Proteases/química , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Sulfonas/químicaRESUMO
The CS-RDC-NOE Rosetta program was used to generate the solution structure of a 27-kDa fragment of the Escherichia coli BamC protein from a limited set of NMR data. The BamC protein is a component of the essential five-protein ß-barrel assembly machine in E. coli. The first 100 residues in BamC were disordered in solution. The Rosetta calculations showed that BamC101â344 forms two well-defined domains connected by an ~18-residue linker, where the relative orientation of the domains was not defined. Both domains adopt a helix-grip fold previously observed in the Bet v 1 superfamily. ¹5N relaxation data indicated a high degree of conformational flexibility for the linker connecting the N-terminal domain and the C-terminal domain in BamC. The results here show that CS-RDC-NOE Rosetta is robust and has a high tolerance for misassigned nuclear Overhauser effect restraints, greatly simplifying NMR structure determinations.