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1.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
2.
Structure ; 27(4): 579-589.e5, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30744994

RESUMO

Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.


Assuntos
Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Apoproteínas/química , Corynebacterium/química , DNA Girase/química , Mycobacterium tuberculosis/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Corynebacterium/enzimologia , Cristalografia por Raios X , DNA/química , DNA/metabolismo , DNA Girase/genética , DNA Girase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
3.
Nucleic Acids Res ; 46(22): 12166-12176, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30357414

RESUMO

The three prime repair exonuclease 2 (TREX2) is an essential 3'-to-5' exonuclease that functions in cell proliferation, genome integrity and skin homeostasis maintenance. The abnormal expression level of TREX2 can result in broken chromosome, increased susceptibility to skin carcinogenesis and Psoriasis. However, the molecular mechanisms of how TREX2 binds and processes its natural substrates, dsDNA or chromosomal DNA, to maintain genome stability remain unclear. In this study, we present four new crystal structures: apo-TREX2, TREX2 in complex with two different dsDNA substrates, and TREX2 in complex with a processed dsDNA product. Analysis of the structures reveals that TREX2 stacks with the 5'-terminal of dsDNA by a Leu20-Pro21-Asn22 cluster for precisely trimming the 3'-overhang. In addition, TREX2 specifically interacts with the non-scissile strand of dsDNA by an α-helix-loop region. The unique interaction patterns of the TREX2-dsDNA complex highlight the requirement of long double-stranded region for TREX2 binding and provide evidence of the functional role of TREX2 in processing chromosomal DNA. Moreover, the non-processive property of TREX2 is elucidated by the structure of TREX2-product complex. Our work discloses the first structural basis of the molecular interactions between TREX2 and its substrates and unravels the mechanistic actions of TREX2.


Assuntos
Apoproteínas/química , DNA/química , Exodesoxirribonucleases/química , Subunidades Proteicas/química , Motivos de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
4.
Proc Natl Acad Sci U S A ; 115(39): E9085-E9094, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30201724

RESUMO

Maturation of iron-sulfur (Fe-S) proteins in eukaryotes requires complex machineries in mitochondria and cytosol. Initially, Fe-S clusters are assembled on dedicated scaffold proteins and then are trafficked to target apoproteins. Within the cytosolic Fe-S protein assembly (CIA) machinery, the conserved P-loop nucleoside triphosphatase Nbp35 performs a scaffold function. In yeast, Nbp35 cooperates with the related Cfd1, which is evolutionary less conserved and is absent in plants. Here, we investigated the potential scaffold function of human CFD1 (NUBP2) in CFD1-depleted HeLa cells by measuring Fe-S enzyme activities or 55Fe incorporation into Fe-S target proteins. We show that CFD1, in complex with NBP35 (NUBP1), performs a crucial role in the maturation of all tested cytosolic and nuclear Fe-S proteins, including essential ones involved in protein translation and DNA maintenance. CFD1 also matures iron regulatory protein 1 and thus is critical for cellular iron homeostasis. To better understand the scaffold function of CFD1-NBP35, we resolved the crystal structure of Chaetomium thermophilum holo-Cfd1 (ctCfd1) at 2.6-Å resolution as a model Cfd1 protein. Importantly, two ctCfd1 monomers coordinate a bridging [4Fe-4S] cluster via two conserved cysteine residues. The surface-exposed topology of the cluster is ideally suited for both de novo assembly and facile transfer to Fe-S apoproteins mediated by other CIA factors. ctCfd1 specifically interacted with ATP, which presumably associates with a pocket near the Cfd1 dimer interface formed by the conserved Walker motif. In contrast, ctNbp35 preferentially bound GTP, implying differential regulation of the two fungal scaffold components during Fe-S cluster assembly and/or release.


Assuntos
Apoproteínas/química , Chaetomium/química , Proteínas Fúngicas/química , Proteínas de Ligação ao GTP/química , Proteína 1 Reguladora do Ferro/química , Proteínas com Ferro-Enxofre/química , Motivos de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Domínio Catalítico , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteínas com Ferro-Enxofre/genética , Proteínas com Ferro-Enxofre/metabolismo
5.
J Biol Chem ; 293(43): 16778-16790, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30206118

RESUMO

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp47, Gln50, and Arg55 on CcmC; Arg73, Asp101, and Glu105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.


Assuntos
Apoproteínas/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Citocromos c/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Heme/metabolismo , Hemeproteínas/metabolismo , Proteínas de Membrana/metabolismo , Substituição de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Sítios de Ligação , Cristalografia por Raios X , Citocromos c/química , Citocromos c/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Heme/química , Heme/genética , Hemeproteínas/química , Hemeproteínas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
6.
J Phys Chem B ; 122(49): 11228-11239, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30133301

RESUMO

Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ⇔ I ⇔ L ⇔ M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (ΔΔ G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ΔΔ G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ⇔ I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ΔΔ G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.


Assuntos
Apoproteínas/química , Mioglobina/química , Animais , Apoproteínas/genética , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutagênese Sítio-Dirigida , Mutação , Mioglobina/genética , Conformação Proteica em alfa-Hélice , Desdobramento de Proteína , Cachalote , Termodinâmica
7.
J Phys Chem B ; 122(31): 7682-7698, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30001623

RESUMO

Anfinsen's thermodynamic hypothesis does not explicitly take into account the possibility of protein aggregation. Here, we introduce a cyclic-perturbation approach to prove that not only the native state but also soluble aggregates of most proteins can be highly populated under mild, physiologically relevant conditions, even at very low concentration. Surprisingly, these aggregates are not necessarily amyloid in nature and are usually not observed in bioactive proteins due to the extremely low kinetic flux from the native state toward a region of the chemical-potential landscape encoding aggregates. We first illustrate this concept for the representative model protein apomyoglobin-at room temperature and no denaturant-and demonstrate kinetic trapping of the native state relative to at least two different types of soluble, predominantly nonamyloid aggregates. The concentration and temperature dependence of aggregation confirm the above scenario. Extension of our analysis to the Escherichia coli proteome shows that the majority of the soluble bacterial proteome is also kinetically trapped in the nonaggregated state. Hence, the existence and low kinetic accessibility of large aggregates at room temperature and pH 6-7 is a general phenomenon. We also show that the average critical protein concentration for aggregation of most of the bacterial proteome is extremely small, much lower than the typical cellular protein concentration. Hence, the thermodynamic driving force for protein aggregation is large even if aggregation does not usually occur in healthy cells due to kinetic trapping. A broader view of Anfinsen's thermodynamic hypothesis encompassing all protein states, including aggregates, is necessary to understand the behavior of proteins in their natural environment.


Assuntos
Apoproteínas/química , Mioglobina/química , Apoproteínas/genética , Apoproteínas/metabolismo , Dicroísmo Circular , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Mioglobina/genética , Mioglobina/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteoma , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura , Termodinâmica
8.
Microbiology ; 164(11): 1457-1470, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29957170

RESUMO

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcriptional activator, NtrC. One of these NtrC-activated genes is yeaG, which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in ΔyeaG bacteria experiencing sustained N starvation. It appears the ability of MetJ, the master transcriptional repressor of methionine biosynthesis genes, to effectively repress transcription of genes under its control is compromised in ΔyeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. Although the aberrant biosynthesis does not appear to be a contributing factor for the compromised viability of ΔyeaG bacteria experiencing sustained N starvation, this study identifies YeaG as a novel regulatory factor in E. coli affecting the transcription of methionine biosynthesis genes under sustained N starvation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Metionina/biossíntese , Nitrogênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transcrição Genética/genética , Apoproteínas/genética , Escherichia coli/genética , Deleção de Genes , Proteínas PII Reguladoras de Nitrogênio/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética
9.
Cell Commun Signal ; 16(1): 25, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29879980

RESUMO

BACKGROUND: Programmed cell death protein 1 (PD-1), a negative co-stimulatory molecule, plays crucial roles in immune escape. Blockade of the interaction between PD-1 and PD-L1 shows exciting clinical responses in a fraction of cancer patients and the success makes PD-1 as a valuable target in immune checkpoint therapy. For the rational design of PD-1 targeting modulators, the ligand binding mechanism of PD-1 should be well understood in prior. METHODS: In this study, we applied 50 ns molecular dynamics simulations to observe the structural properties of PD-1 molecule in both apo and ligand bound states, and we studied the structural features of PD-1 in human and mouse respectively. RESULTS: The results showed that the apo hPD-1 was more flexible than that in PD-L1 bound state. We unexpectedly found that K135 was important for binding energy although it was not at the binding interface. Moreover, the residues which stabilized the interactions with PD-L1 were distinguished. Taking the dynamic features of these residues into account, we identified several residual sites where mutations may gain the function of ligand binding. The in vitro binding experiments revealed the mutants M70I, S87 W, A129L, A132L, and K135 M were better in ligand binding than the wild type PD-1. CONCLUSIONS: The structural information from MD simulation combined with in silico mutagenesis provides guidance to design engineered PD-1 mutants to modulate the PD-1/PD-L1 pathway.


Assuntos
Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Humanos , Ligantes , Camundongos , Mutagênese , Proteínas Mutantes/química , Receptor de Morte Celular Programada 1/química , Domínios Proteicos
10.
Elife ; 72018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29809140

RESUMO

Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Apoproteínas/química , Proteínas de Bactérias/química , Ácido Fólico/química , Lactobacillus delbrueckii/química , Vitamina B 12/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Expressão Gênica , Teste de Complementação Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Lactobacillus delbrueckii/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Vitamina B 12/metabolismo
11.
J Exp Bot ; 69(12): 3157-3169, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29648603

RESUMO

Dormancy cycling controls the seasonal conditions under which seeds germinate, and these conditions strongly influence growth and survival of plants. Several endogenous and environmental signals affect the dormancy status of seeds. Factors such as time, light, and temperature influence the balance between abscisic acid (ABA) and gibberellic acid (GA), two phytohormones that play a key role in seed dormancy and germination. High temperatures have been shown to increase ABA level and prevent seed germination, a process known as thermoinhibition. High temperature can also cause the acquisition of secondary dormancy, preventing germination of seeds upon their return to favorable germination conditions. The mechanisms and conditions linking thermoinhibition and secondary dormancy remain unclear. Phytochromes are photoreceptors known to promote seed germination of many plant species including Arabidopsis thaliana. Here, we demonstrate a role for PHYD in modulating secondary dormancy acquisition in seeds exposed to high temperature. We found that a functional PHYD gene is required for the germination of seeds that experienced high temperature, and that ABA- and GA-related gene expression during and after pre-incubation at high temperatures was altered in a phyD mutant. We further show that the level of PHYD mRNA increased in seeds pre-incubated at high temperature and that this increase correlates with efficient removal of the germination repressor PIL5.


Assuntos
Apoproteínas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Temperatura Alta , Fitocromo/genética , Dormência de Plantas/genética , Apoproteínas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fitocromo/metabolismo , Sementes/genética , Sementes/fisiologia
12.
FEBS J ; 285(12): 2306-2318, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29694705

RESUMO

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.


Assuntos
Adenina Fosforribosiltransferase/química , Adenina/química , Monofosfato de Adenosina/química , Apoproteínas/química , Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Adenina/metabolismo , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Francisella tularensis/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Cinética , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica
13.
Mol Biol (Mosk) ; 52(1): 51-61, 2018.
Artigo em Russo | MEDLINE | ID: mdl-29512636

RESUMO

As has been recently shown, the toxicity of protein aggregates is determined by their structure. Therefore, special attention has been focused on the search for factors that specify the structural features of formed amyloid fibrils. The effect of amino acid substitutions in apomyoglobin on the structural characteristics of its amyloid aggregates has been analyzed. The morphology and secondary structure of amyloids of the wild-type protein and its mutant variants Val10Ala, Val10Phe, and Trp14Phe have been compared, and the regions involved in intermolecular interactions in fibrils have been determined using limited proteolysis and mass spectrometry. No considerable differences have been found in the morphology (shape, length, or diameter) or the content (percentage) of the cross-ß structure of apomyoglobin amyloids and its mutant variants. Amyloid cores of wild-type apomyoglobin and variants with Val10Phe and Trp14Phe substitutions have been formed by different regions of the polypeptide chain. The case study of apomyoglobin demonstrates that the location of amyloidogenic regions in the polypeptide chain of wild-type protein and its mutant forms can differ. Thus, possible structural changes in amyloids resulting from amino acid substitutions should be taken into account when studying phenotype aggregation.


Assuntos
Amiloide/química , Apoproteínas/química , Mioglobina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Mioglobina/genética , Estrutura Secundária de Proteína
14.
J Biol Chem ; 293(10): 3685-3699, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29358323

RESUMO

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.


Assuntos
Modelos Moleculares , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Regulação Alostérica , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Humanos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/química , Proteínas Ativadoras de ras GTPase/genética
15.
Structure ; 26(1): 153-160.e4, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29276033

RESUMO

BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.


Assuntos
Antineoplásicos/química , Apoproteínas/química , Antígenos de Histocompatibilidade Menor/química , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Antineoplásicos/síntese química , Apoproteínas/antagonistas & inibidores , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Cell Calcium ; 67: 53-64, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29029791

RESUMO

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin ß-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16-19% of all PAs. We show that structural instability of apo-ß-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.


Assuntos
Apoproteínas/química , Proteínas de Ligação ao Cálcio/química , Cálcio/química , Proteínas de Peixes/química , Proteínas Intrinsicamente Desordenadas/química , Oncorhynchus kisutch/metabolismo , Parvalbuminas/química , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Parvalbuminas/genética , Parvalbuminas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
17.
J Biol Chem ; 292(44): 18169-18177, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28900031

RESUMO

The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone Atox1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. Atox1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking Atox1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-Atox1 and Atox1-Cu by solution NMR. The results show that copper transfer from Atox1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which Atox1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.


Assuntos
ATPases Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Metalochaperonas/metabolismo , Modelos Moleculares , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , ATPases Transportadoras de Cobre/química , ATPases Transportadoras de Cobre/genética , Ativação Enzimática , Estabilidade Enzimática , Humanos , Metalochaperonas/química , Metalochaperonas/genética , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo Ângulo , Solubilidade , Difração de Raios X
18.
Biophys J ; 113(5): 991-1001, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28877500

RESUMO

Investigation of the molecular mechanisms underlying amyloid-related human diseases attracts close attention. These diseases, the number of which currently is above 40, are characterized by formation of peptide or protein aggregates containing a cross-ß structure. Most of the amyloidogenesis mechanisms described so far are based on experimental studies of aggregation of short peptides, intrinsically disordered proteins, or proteins under denaturing conditions, and studies of amyloid aggregate formations by structured globular proteins under conditions close to physiological ones are still in the initial stage. We investigated the effect of amino acid substitutions on propensity of the completely helical protein sperm whale apomyoglobin (sw ApoMb) for amyloid formation from its structured state in the absence of denaturing agents. Stability and aggregation of mutated sw ApoMb were studied using circular dichroism, Fourier transform infrared spectroscopy, x-ray diffraction, native electrophoresis, and electron microscopy techniques. Here, we demonstrate that stability of the protein native state determines both protein aggregation propensity and structural peculiarities of formed aggregates. Specifically, structurally stable mutants show low aggregation propensity and moderately destabilized sw ApoMb variants form amyloids, whereas their strongly destabilized mutants form both amyloids and nonamyloid aggregates.


Assuntos
Apoproteínas/metabolismo , Mioglobina/metabolismo , Agregação Patológica de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/genética , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Eletroforese , Escherichia coli , Microscopia Eletrônica , Mutação , Mioglobina/química , Mioglobina/genética , Agregação Patológica de Proteínas/genética , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Cachalote , Difração de Raios X
19.
J Biol Chem ; 292(37): 15468-15480, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28765283

RESUMO

Under oxygen-limiting conditions, the marine bacterium Dinoroseobacter shibae DFL12T generates energy via denitrification, a respiratory process in which nitric oxide (NO) is an intermediate. Accumulation of NO may cause cytotoxic effects. The response to this nitrosative (NO-triggered) stress is controlled by the Crp/Fnr-type transcriptional regulator DnrF. We analyzed the response to NO and the mechanism of NO sensing by the DnrF regulator. Using reporter gene fusions and transcriptomics, here we report that DnrF selectively repressed nitrate reductase (nap) genes, preventing further NO formation. In addition, DnrF induced the expression of the NO reductase genes (norCB), which promote NO consumption. We used UV-visible and EPR spectroscopy to characterize heme binding to DnrF and subsequent NO coordination. DnrF detects NO via its bound heme cofactor. We found that the dimeric DnrF bound one molecule of heme per subunit. Purified recombinant apo-DnrF bound its target promoter sequences (napD, nosR2, norC, hemA, and dnrE) in electromobility shift assays, and we identified a specific palindromic DNA-binding site 5'-TTGATN4ATCAA-3' in these target sequences via mutagenesis studies. Most importantly, successive addition of heme as well as heme and NO to purified recombinant apo-DnrF protein increased affinity of the holo-DnrF for its specific binding motif in the napD promoter. On the basis of these results, we propose a model for the DnrF-mediated NO stress response of this marine bacterium.


Assuntos
Organismos Aquáticos/fisiologia , Proteínas de Bactérias/metabolismo , Heme/metabolismo , Nitrato Redutase/metabolismo , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas , Rhodobacteraceae/fisiologia , Transativadores/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Organismos Aquáticos/enzimologia , Organismos Aquáticos/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Dimerização , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Heme/química , Sequências Repetidas Invertidas , Cinética , Família Multigênica , Mutação , Nitrato Redutase/química , Nitrato Redutase/genética , Óxido Nítrico/química , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Regulon , Rhodobacteraceae/enzimologia , Rhodobacteraceae/crescimento & desenvolvimento , Estresse Fisiológico , Transativadores/química , Transativadores/genética
20.
Phys Chem Chem Phys ; 19(29): 19021-19031, 2017 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-28702545

RESUMO

Near the minimum free energy basin of proteins where the native ensemble resides, partly unfolded conformations of slightly higher energy can be significantly populated under native conditions. It has been speculated that they play roles in molecular recognition and catalysis, but they might represent contemporary features of the evolutionary process without functional relevance. Obtaining conclusive evidence on these alternatives is difficult because it requires comparing the performance of a given protein when populating and when not populating one such intermediate, in otherwise identical conditions. Wild type apoflavodoxin populates under native conditions a partly unfolded conformation (10% of molecules) whose unstructured region includes the binding sites for the FMN cofactor and for redox partner proteins. We recently engineered a thermostable variant where the intermediate is no longer detectable. Using the wild type and variant, we assess the relevance of the intermediate comparing folding kinetics, cofactor binding kinetics, cofactor affinity, X-ray structure, intrinsic dynamics, redox potential of the apoflavodoxin-cofactor complex (Fld), its affinity for partner protein FNR, and electron transfer rate within the Fld/FNR physiological complex. Our data strongly suggest the intermediate state, conserved in long-chain apoflavodoxins, is not required for the correct assembly of flavodoxin nor does it contribute to shape its electron transfer properties. This analysis can be applied to evaluate other native basin intermediates.


Assuntos
Apoproteínas/química , Flavodoxina/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Cristalografia por Raios X , Transporte de Elétrons , Mononucleotídeo de Flavina/química , Flavodoxina/genética , Flavodoxina/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutagênese , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Temperatura
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