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1.
Cell Syst ; 12(2): 128-140.e4, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33373583

RESUMO

Systematic perturbation of cells followed by comprehensive measurements of molecular and phenotypic responses provides informative data resources for constructing computational models of cell biology. Models that generalize well beyond training data can be used to identify combinatorial perturbations of potential therapeutic interest. Major challenges for machine learning on large biological datasets are to find global optima in a complex multidimensional space and mechanistically interpret the solutions. To address these challenges, we introduce a hybrid approach that combines explicit mathematical models of cell dynamics with a machine-learning framework, implemented in TensorFlow. We tested the modeling framework on a perturbation-response dataset of a melanoma cell line after drug treatments. The models can be efficiently trained to describe cellular behavior accurately. Even though completely data driven and independent of prior knowledge, the resulting de novo network models recapitulate some known interactions. The approach is readily applicable to various kinetic models of cell biology. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

2.
bioRxiv ; 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33200136

RESUMO

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, has poor compatibility with certain antigens ( e . g ., integral membrane proteins), and suffers from self-tolerance and immunodominance, which limit the functional spectrum of antibodies that can be obtained. Here, we describe A utonomous H ypermutation y E ast surf A ce D isplay (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. In AHEAD, antibody fragments are encoded on an error-prone orthogonal DNA replication system, resulting in Saccharomyces cerevisiae populations that continuously mutate surface-displayed antibody repertoires. Simple cycles of yeast culturing and enrichment for antigen binding drive the evolution of high-affinity antibody clones in a readily parallelizable process that takes as little as 2 weeks. We applied AHEAD to generate nanobodies against the SARS-CoV-2 S glycoprotein, a GPCR, and other targets. The SARS-CoV-2 nanobodies, concurrently evolved from an open-source naïve nanobody library in 8 independent experiments, reached subnanomolar affinities through the sequential fixation of multiple mutations over 3-8 AHEAD cycles that saw ∼580-fold and ∼925-fold improvements in binding affinities and pseudovirus neutralization potencies, respectively. These experiments highlight the defining speed, parallelizability, and effectiveness of AHEAD and provide a template for streamlined antibody generation at large with salient utility in rapid response to current and future viral outbreaks.

3.
Elife ; 92020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32870157

RESUMO

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.

4.
Nat Microbiol ; 5(6): 813-820, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32152588

RESUMO

The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1-6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS-bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.


Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Proteínas de Ligação às Penicilinas/química , Peptidoglicano Glicosiltransferase/química , Multimerização Proteica , Sítios de Ligação , Parede Celular/metabolismo , Estrutura Molecular , Complexos Multiproteicos/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
5.
Cell Syst ; 10(1): 15-24.e5, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31838147

RESUMO

Natural evolution encodes rich information about the structure and function of biomolecules in the genetic record. Previously, statistical analysis of co-variation patterns in natural protein families has enabled the accurate computation of 3D structures. Here, we explored generating similar information by experimental evolution, starting from a single gene and performing multiple cycles of in vitro mutagenesis and functional selection in Escherichia coli. We evolved two antibiotic resistance proteins, ß-lactamase PSE1 and acetyltransferase AAC6, and obtained hundreds of thousands of diverse functional sequences. Using evolutionary coupling analysis, we inferred residue interaction constraints that were in agreement with contacts in known 3D structures, confirming genetic encoding of structural constraints in the selected sequences. Computational protein folding with interaction constraints then yielded 3D structures with the same fold as natural relatives. This work lays the foundation for a new experimental method (3Dseq) for protein structure determination, combining evolution experiments with inference of residue interactions from sequence information. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

6.
Proc Natl Acad Sci U S A ; 116(36): 17825-17830, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31431536

RESUMO

Clustered protocadherins, a large family of paralogous proteins that play important roles in neuronal development, provide an important case study of interaction specificity in a large eukaryotic protein family. A mammalian genome has more than 50 clustered protocadherin isoforms, which have remarkable homophilic specificity for interactions between cellular surfaces. A large antiparallel dimer interface formed by the first 4 extracellular cadherin (EC) domains controls this interaction. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and undergo binding and unbinding events, but together they form a stable complex through polyvalency. Strongly evolutionarily coupled residue pairs interacted more frequently in our simulations, suggesting that sequence coevolution can inform the frequency of interaction and biochemical nature of a residue interaction. With these simulations and sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs at the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions.


Assuntos
Caderinas/química , Caderinas/metabolismo , Caderinas/genética , Evolução Molecular , Variação Genética , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade
7.
Nat Genet ; 51(7): 1170-1176, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209393

RESUMO

We describe an experimental method of three-dimensional (3D) structure determination that exploits the increasing ease of high-throughput mutational scans. Inspired by the success of using natural, evolutionary sequence covariation to compute protein and RNA folds, we explored whether 'laboratory', synthetic sequence variation might also yield 3D structures. We analyzed five large-scale mutational scans and discovered that the pairs of residues with the largest positive epistasis in the experiments are sufficient to determine the 3D fold. We show that the strongest epistatic pairings from genetic screens of three proteins, a ribozyme and a protein interaction reveal 3D contacts within and between macromolecules. Using these experimental epistatic pairs, we compute ab initio folds for a GB1 domain (within 1.8 Å of the crystal structure) and a WW domain (2.1 Å). We propose strategies that reduce the number of mutants needed for contact prediction, suggesting that genomics-based techniques can efficiently predict 3D structure.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Bactérias/química , Epistasia Genética , Mutação , Proteínas de Ligação a Poli(A)/química , Conformação Proteica , RNA Catalítico/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Bactérias/genética , Humanos , Proteínas de Ligação a Poli(A)/genética , Domínios Proteicos , Dobramento de Proteína , RNA Catalítico/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
8.
Methods Enzymol ; 614: 363-392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30611430

RESUMO

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. This hybrid "EC-NMR" method has been developed using NMR data for several soluble proteins and validated by comparison with corresponding reference structures determined by X-ray crystallography and/or conventional NMR methods. For small proteins, only backbone resonance assignments are utilized, while for larger proteins both backbone and some sidechain methyl resonance assignments are generally required. ECs can be combined with sparse NMR data obtained on deuterated, selectively protonated protein samples to provide structures that are more accurate and complete than those obtained using such sparse NMR data alone. EC-NMR also has significant potential for analysis of protein structures from solid-state NMR data and for studies of integral membrane proteins. The requirement that ECs are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.


Assuntos
Algoritmos , Proteínas de Escherichia coli/química , Escherichia coli/química , Evolução Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Periplásmicas de Ligação/química , Software , Análise de Variância , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Deutério/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Marcação por Isótopo , Modelos Moleculares , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Termodinâmica
9.
Nat Microbiol ; 4(2): 328-338, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30510172

RESUMO

Genome analysis should allow the discovery of interdependent loci that together cause antibiotic resistance. In practice, however, the vast number of possible epistatic interactions erodes statistical power. Here, we extend an approach that has been successfully used to identify epistatic residues in proteins to infer genomic loci that are strongly coupled. This approach reduces the number of tests required for an epistatic genome-wide association study of antibiotic resistance and increases the likelihood of identifying causal epistasis. We discovered 38 loci and 240 epistatic pairs that influence the minimum inhibitory concentrations of 5 different antibiotics in 1,102 isolates of Neisseria gonorrhoeae that were confirmed in a second dataset of 495 isolates. Many known resistance-affecting loci were recovered; however, the majority of associations occurred in unreported genes, such as murE. About half of the discovered epistasis involved at least one locus previously associated with antibiotic resistance, including interactions between gyrA and parC. Still, many combinations involved unreported loci and genes. While most variation in minimum inhibitory concentrations could be explained by identified loci, epistasis substantially increased explained phenotypic variance. Our work provides a systematic identification of epistasis affecting antibiotic resistance in N. gonorrhoeae and a generalizable approach for epistatic genome-wide association studies.


Assuntos
Resistência Microbiana a Medicamentos/genética , Epistasia Genética , Genoma Bacteriano/genética , Genômica/métodos , Neisseria gonorrhoeae/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Variação Biológica da População , Loci Gênicos , Estudo de Associação Genômica Ampla , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/classificação , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/isolamento & purificação , Filogenia , Conformação Proteica
10.
Bioinformatics ; 35(9): 1582-1584, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30304492

RESUMO

SUMMARY: Coevolutionary sequence analysis has become a commonly used technique for de novo prediction of the structure and function of proteins, RNA, and protein complexes. We present the EVcouplings framework, a fully integrated open-source application and Python package for coevolutionary analysis. The framework enables generation of sequence alignments, calculation and evaluation of evolutionary couplings (ECs), and de novo prediction of structure and mutation effects. The combination of an easy to use, flexible command line interface and an underlying modular Python package makes the full power of coevolutionary analyses available to entry-level and advanced users. AVAILABILITY AND IMPLEMENTATION: https://github.com/debbiemarkslab/evcouplings.


Assuntos
Análise de Sequência , Software , Proteínas , RNA , Alinhamento de Sequência
11.
PLoS Genet ; 14(11): e1007753, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30403663

RESUMO

During the morphological process of sporulation in Bacillus subtilis two adjacent daughter cells (called the mother cell and forespore) follow different programs of gene expression that are linked to each other by signal transduction pathways. At a late stage in development, a signaling pathway emanating from the forespore triggers the proteolytic activation of the mother cell transcription factor σK. Cleavage of pro-σK to its mature and active form is catalyzed by the intramembrane cleaving metalloprotease SpoIVFB (B), a Site-2 Protease (S2P) family member. B is held inactive by two mother-cell membrane proteins SpoIVFA (A) and BofA. Activation of pro-σK processing requires a site-1 signaling protease SpoIVB (IVB) that is secreted from the forespore into the space between the two cells. IVB cleaves the extracellular domain of A but how this cleavage activates intramembrane proteolysis has remained unclear. Structural studies of the Methanocaldococcus jannaschii S2P homolog identified closed (substrate-occluded) and open (substrate-accessible) conformations of the protease, but the biological relevance of these conformations has not been established. Here, using co-immunoprecipitation and fluorescence microscopy, we show that stable association between the membrane-embedded protease and its substrate requires IVB signaling. We further show that the cytoplasmic cystathionine-ß-synthase (CBS) domain of the B protease is not critical for this interaction or for pro-σK processing, suggesting the IVB-dependent interaction site is in the membrane protease domain. Finally, we provide evidence that the B protease domain adopts both open and closed conformations in vivo. Collectively, our data support a substrate-gating model in which IVB-dependent cleavage of A on one side of the membrane triggers a conformational change in the membrane-embedded protease from a closed to an open state allowing pro-σK access to the caged interior of the protease.


Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica , Transporte Proteico , Proteólise , Esporos
12.
J Cell Biol ; 217(12): 4080-4091, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30327422

RESUMO

Metabolic energy is stored in cells primarily as triacylglycerols in lipid droplets (LDs), and LD dysregulation leads to metabolic diseases. The formation of monolayer-bound LDs from the endoplasmic reticulum (ER) bilayer is poorly understood, but the ER protein seipin is essential to this process. In this study, we report a cryo-electron microscopy structure and functional characterization of Drosophila melanogaster seipin. The structure reveals a ring-shaped dodecamer with the luminal domain of each monomer resolved at ∼4.0 Å. Each luminal domain monomer exhibits two distinctive features: a hydrophobic helix (HH) positioned toward the ER bilayer and a ß-sandwich domain with structural similarity to lipid-binding proteins. This structure and our functional testing in cells suggest a model in which seipin oligomers initially detect forming LDs in the ER via HHs and subsequently act as membrane anchors to enable lipid transfer and LD growth.


Assuntos
Proteínas de Drosophila , Subunidades gama da Proteína de Ligação ao GTP , Gotículas Lipídicas , Modelos Biológicos , Modelos Moleculares , Animais , Microscopia Crioeletrônica , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Domínios Proteicos , Relação Estrutura-Atividade
14.
Nat Methods ; 15(10): 816-822, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30250057

RESUMO

The functions of proteins and RNAs are defined by the collective interactions of many residues, and yet most statistical models of biological sequences consider sites nearly independently. Recent approaches have demonstrated benefits of including interactions to capture pairwise covariation, but leave higher-order dependencies out of reach. Here we show how it is possible to capture higher-order, context-dependent constraints in biological sequences via latent variable models with nonlinear dependencies. We found that DeepSequence ( https://github.com/debbiemarkslab/DeepSequence ), a probabilistic model for sequence families, predicted the effects of mutations across a variety of deep mutational scanning experiments substantially better than existing methods based on the same evolutionary data. The model, learned in an unsupervised manner solely on the basis of sequence information, is grounded with biologically motivated priors, reveals the latent organization of sequence families, and can be used to explore new parts of sequence space.


Assuntos
Biologia Computacional/métodos , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Teóricos , Mutação , Algoritmos , Humanos
15.
Evolution ; 72(10): 2234-2243, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30152871

RESUMO

Viral gain-of-function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ's J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative of ecological diversification. Our results show that viral evolution in the laboratory mirrors that in nature and that laboratory experiments can be coupled with protein sequence analyses to identify the causes of viral evolution in the real world. Furthermore, our results provide evidence for widespread host-shift evolution in lambdoid viruses.


Assuntos
Bacteriófago lambda/genética , Evolução Molecular , Mutação com Ganho de Função/genética , Seleção Genética , Filogenia
16.
Mol Cell ; 71(1): 178-190.e8, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29979965

RESUMO

The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major "hotspot" codons, which account for only ∼30% of cancer-associated p53 mutations. To comprehensively characterize the consequences of the p53 mutation spectrum, we created a synthetically designed library and measured the functional impact of ∼10,000 DNA-binding domain (DBD) p53 variants in human cells in culture and in vivo. Our results highlight the differential outcome of distinct p53 mutations in human patients and elucidate the selective pressure driving p53 conservation throughout evolution. Furthermore, while loss of anti-proliferative functionality largely correlates with the occurrence of cancer-associated p53 mutations, we observe that selective gain-of-function may further favor particular mutants in vivo. Finally, when combined with additional acquired p53 mutations, seemingly neutral TP53 SNPs may modulate phenotypic outcome and, presumably, tumor progression.


Assuntos
Evolução Molecular , Biblioteca Gênica , Mutação , Neoplasias/genética , Proteína Supressora de Tumor p53/genética , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , Domínios Proteicos , Proteína Supressora de Tumor p53/metabolismo
17.
Proc Natl Acad Sci U S A ; 115(26): 6709-6714, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891673

RESUMO

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transferência de Fosfolipídeos/química , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Evolução Molecular , Biblioteca Gênica , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade
18.
Nature ; 556(7699): 118-121, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29590088

RESUMO

The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.


Assuntos
Cristalografia por Raios X/métodos , Nucleotidiltransferases/química , Peptidoglicano/metabolismo , Thermus thermophilus/enzimologia , Bacillus subtilis/genética , Biocatálise , Parede Celular/enzimologia , Parede Celular/metabolismo , Escherichia coli/genética , Modelos Moleculares , Nucleotidiltransferases/metabolismo , Domínios Proteicos , Dobramento de Proteína , Relação Estrutura-Atividade , Thermus thermophilus/genética
19.
Adv Exp Med Biol ; 1105: 153-169, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30617828

RESUMO

While 3D structure determination of small (<15 kDa) proteins by solution NMR is largely automated and routine, structural analysis of larger proteins is more challenging. An emerging hybrid strategy for modeling protein structures combines sparse NMR data that can be obtained for larger proteins with sequence co-variation data, called evolutionary couplings (ECs), obtained from multiple sequence alignments of protein families. This hybrid "EC-NMR" method can be used to accurately model larger (15-60 kDa) proteins, and more rapidly determine structures of smaller (5-15 kDa) proteins using only backbone NMR data. The resulting structures have accuracies relative to reference structures comparable to those obtained with full backbone and sidechain NMR resonance assignments. The requirement that evolutionary couplings (ECs) are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, potentially also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.


Assuntos
Evolução Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/química , Alinhamento de Sequência
20.
Genome Biol Evol ; 9(4): 1072-1083, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379360

RESUMO

Bacteria can evolve rapidly under positive selection owing to their vast numbers, allowing their genes to diversify by adapting to different environments. We asked whether the same genes that evolve rapidly in the long-term evolution experiment (LTEE) with Escherichia coli have also diversified extensively in nature. To make this comparison, we identified ∼2000 core genes shared among 60 E. coli strains. During the LTEE, core genes accumulated significantly more nonsynonymous mutations than flexible (i.e., noncore) genes. Furthermore, core genes under positive selection in the LTEE are more conserved in nature than the average core gene. In some cases, adaptive mutations appear to modify protein functions, rather than merely knocking them out. The LTEE conditions are novel for E. coli, at least in relation to its evolutionary history in nature. The constancy and simplicity of the environment likely favor the complete loss of some unused functions and the fine-tuning of others.

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