RESUMO
Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Humanos , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Ligantes , Família da Proteína 8 Relacionada à Autofagia/química , Autofagossomos/metabolismo , Mamíferos/metabolismoRESUMO
We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an â¼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.
RESUMO
MOTIVATION: Conformational rearrangements during molecular interactions are observed in a wide range of biological systems. However, computational methods that aim at simulating and predicting molecular interactions are still largely ignoring the flexible nature of biological macromolecules as the number of degrees of freedom is computationally intractable when using brute force representations. RESULTS: In this article, we present a computational data structure called the Flexibility Tree (FT) that enables a multi-resolution and hierarchical encoding of molecular flexibility. This tree-like data structure allows the encoding of relatively small, yet complex sub-spaces of a protein's conformational space. These conformational sub-spaces are parameterized by a small number of variables and can be searched efficiently using standard global search techniques. The FT structure makes it straightforward to combine and nest a wide variety of motion types such as hinge, shear, twist, screw, rotameric side chains, normal modes and essential dynamics. Moreover, the ability to assign shapes to the nodes in a FT allows the interactive manipulation of flexible protein shapes and the interactive visualization of the impact of conformational changes on the protein's overall shape. We describe the design of the FT and illustrate the construction of such trees to hierarchically combine motion information obtained from a variety of sources ranging from experiment to user intuition, and describing conformational changes at different biological scales. We show that the combination of various types of motion helps refine the encoded conformational sub-spaces to include experimentally determined structures, and we demonstrate searching these sub-spaces for specific conformations.
Assuntos
Biologia Computacional/métodos , Proteínas/química , Algoritmos , Animais , Simulação por Computador , Interpretação Estatística de Dados , Humanos , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteômica/métodos , Proteínas Quinases p38 Ativadas por Mitógeno/químicaRESUMO
This report focuses on nanotools based on the scanning force microscope (SFM) for imaging, measuring, and manipulating biological matter at the sub-micron scale. Because pathophysiological processes often occur at the (sub-) cellular scale, the SFM has opened the exciting possibility to spot diseases at a stage before they become symptomatic and cause functional impairments in the affected part of the body. Such presymptomatic detection will be key to developing effective therapies to slow or halt disease progression.
Assuntos
Equipamentos para Diagnóstico/tendências , Microscopia de Varredura por Sonda/métodos , Nanotecnologia/métodos , Instrumentos Cirúrgicos/tendências , Cartilagem Articular/patologia , Cartilagem Articular/ultraestrutura , Vasos Coronários/patologia , Vasos Coronários/ultraestrutura , Humanos , Microscopia de Varredura por Sonda/instrumentação , Nanotecnologia/instrumentaçãoRESUMO
The evolving technology of computer autofabrication makes it possible to produce physical models for complex biological molecules and assemblies. Augmented reality has recently developed as a computer interface technology that enables the mixing of real-world objects and computer-generated graphics. We report an application that demonstrates the use of autofabricated tangible models and augmented reality for research and communication in molecular biology. We have extended our molecular modeling environment, PMV, to support the fabrication of a wide variety of physical molecular models, and have adapted an augmented reality system to allow virtual 3D representations to be overlaid onto the tangible molecular models. Users can easily change the overlaid information, switching between different representations of the molecule, displays of molecular properties, or dynamic information. The physical models provide a powerful, intuitive interface for manipulating the computer models, streamlining the interface between human intent, the physical model, and the computational activity.
Assuntos
Desenho Assistido por Computador , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Modelos Moleculares , Software , Estrutura MolecularRESUMO
To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.
Assuntos
Núcleo Celular/ultraestrutura , Poro Nuclear/ultraestrutura , Tomografia/métodos , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/química , Microscopia Crioeletrônica , Citoplasma/ultraestrutura , Feminino , Gelo , Imageamento Tridimensional , Microscopia de Força Atômica , Modelos Biológicos , Oócitos/ultraestrutura , Xenopus laevisRESUMO
The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.
Assuntos
Actinas/metabolismo , Actinas/ultraestrutura , Timosina/química , Timosina/metabolismo , Actinas/química , Animais , Biopolímeros/química , Biopolímeros/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Gelsolina/metabolismo , Humanos , Cinética , Microscopia Eletrônica , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Peso Molecular , Músculo Esquelético/química , Faloidina/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades Proteicas , CoelhosRESUMO
The binding of uropathogenic Escherichia coli to the urothelial surface is a critical initial event for establishing urinary tract infection, because it prevents the bacteria from being removed by micturition and it triggers bacterial invasion as well as host cell defense. This binding is mediated by the FimH adhesin located at the tip of the bacterial type 1-fimbrium and its urothelial receptor, uroplakin Ia (UPIa). To localize the UPIa receptor on the 16 nm particles that form two-dimensional crystals of asymmetric unit membrane (AUM) covering >90 % of the apical urothelial surface, we constructed a 15 A resolution 3-D model of the mouse 16 nm AUM particle by negative staining and electron crystallography. Similar to previous lower-resolution models of bovine and pig AUM particles, the mouse 16 nm AUM particle consists of six inner and six outer domains that are interconnected to form a twisted ribbon-like structure. Treatment of urothelial plaques with 0.02-0.1 % (v/v) Triton X-100 allowed the stain to penetrate into the membrane, revealing parts of the uroplakin transmembrane moiety with an overall diameter of 14 nm, which was much bigger than the 11 nm value determined earlier by quick-freeze deep-etch. Atomic force microscopy of native, unfixed mouse and bovine urothelial plaques confirmed the overall structure of the luminal 16 nm AUM particle that was raised by 6.5 nm above the luminal membrane surface and, in addition, revealed a circular, 0.5 nm high, cytoplasmic protrusion of approximately 14 nm diameter. Finally, a difference map calculated from the mouse urothelial plaque images collected in the presence and absence of recombinant bacterial FimH/FimC complex revealed the selective binding of FimH to the six inner domains of the 16 nm AUM particle. These results indicate that the 16 nm AUM particle is anchored by a approximately 14 nm diameter transmembrane stalk, and suggest that bacterial binding to UPIa that resides within the six inner domains of the 16 nm AUM particle may preferentially trigger transmembrane signaling involved in bacterial invasion and host cell defense.
Assuntos
Adesinas Bacterianas/metabolismo , Adesinas de Escherichia coli , Membrana Celular/metabolismo , Proteínas de Fímbrias , Glicoproteínas de Membrana/metabolismo , Urotélio/química , Urotélio/ultraestrutura , Adesinas Bacterianas/ultraestrutura , Animais , Aderência Bacteriana , Bovinos , Membrana Celular/ultraestrutura , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Glicoproteínas de Membrana/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Ligação Proteica , Estrutura Terciária de Proteína , Tetraspaninas , Infecções Urinárias/microbiologia , Uroplaquina Ia , Urotélio/metabolismoRESUMO
Drug-resistant strains are rapidly selected during AIDS therapy because of the high rate of mutation in HIV. In this report, we present an evolutionary simulation method for analysis of viral mutation and its use for optimization of HIV-1 protease drugs to improve their robustness in the face of resistance mutation. We first present an analysis of the range of resistant mutants that produce viable viruses by using a volume-based viral fitness model. Then, we analyze how this range of mutant proteases allows development of resistance to an optimal inhibitor previously designed by computational coevolution techniques. Finally, we evaluate the resistance patterns of commercially available drugs, and we discuss how resistance might be overcome by optimizing the size of specific side-chains of these inhibitors.
Assuntos
Biologia Computacional/métodos , Desenho de Fármacos , Farmacorresistência Viral , Inibidores da Protease de HIV/química , HIV-1/genética , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Sítios de Ligação , Evolução Biológica , Simulação por Computador , Desenho Assistido por Computador , Quimioterapia Combinada , Protease de HIV/química , Protease de HIV/genética , Inibidores da Protease de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Indinavir/química , Modelos Moleculares , Mutação , Nelfinavir/química , Ritonavir/química , Saquinavir/químicaRESUMO
Small ions and molecules can traverse the nuclear pore complex (NPC) simply by diffusion, whereas larger proteins and RNAs require specific signals and factors that facilitate their passage through the NPC. Our understanding of the factors that participate and regulate nucleocytoplasmic transport has increased tremendously over the past years, whereas the actual translocation step through the NPC has remained largely unclear. Here, we present and discuss recent findings on the interaction between the NPC and transport receptors and provide new evidence that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargos.
Assuntos
Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Modelos BiológicosRESUMO
Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the double-membraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of high-resolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.