RESUMEN
Coassembly of peptide biomaterials offers a compelling avenue to broaden the spectrum of hierarchically ordered supramolecular nanoscale structures that may be relevant for biomedical and biotechnological applications. In this work coassemblies of amphiphilic and oppositely charged, anionic and cationic, ß-sheet peptides are studied, which may give rise to a diverse range of coassembled forms. Mixtures of the peptides show significantly lower critical coassembly concentration (CCC) values compared to those of the individual pure peptides. Intriguingly, the highest formation of coassembled fibrils is found to require excess of the cationic peptide whereas equimolar mixtures of the peptides exhibited the maximum folding into ß-sheet structures. Mixtures of the peptides coassembled sequentially from solutions at concentrations surpassing each peptide's intrinsic critical assembly concentration (CAC), are also found to require a higher portion of the cationic peptide to stabilize hydrogels. This study illuminates a systematic investigation of oppositely charged ß-sheet peptides over a range of concentrations, in solutions and in hydrogels. The results may be relevant to the fundamental understanding of such intricate charge-driven assembly systems and to the formulation of peptide-based nanostructures with diverse functionalities.
RESUMEN
We have successfully created self-assembled membranes by combining positively charged (Pro-X-(Phe-X)5-Pro) PFX peptides with negatively charged alginate. These PFX/alginate membranes were formed by three different peptides that contain either X = Arginine (R), Histidine (H), or Ornithine (O) as their charged amino acid. The assemblies were compared to membranes that were previously reported by us composed of X = lysine (K). This study enabled us to elucidate the impact of amino acids' specific interactions on membrane formation. SEM, SAXS, and cryo-TEM measurements show that although K, R, H, and O may have a similar net charge, the specific traits of the charged amino acid is an essential factor in determining the hierarchical structure of alginate/PFX self-assembled membranes.
Asunto(s)
Alginatos , Alginatos/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Péptidos/química , Cationes/química , Membranas Artificiales , Arginina/químicaRESUMEN
Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of ß-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.
Asunto(s)
Cinesinas , Proteínas de Saccharomyces cerevisiae , Cinesinas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Microscopía por Crioelectrón , Microtúbulos/químicaRESUMEN
The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.
Asunto(s)
Aminopeptidasas , Plasmodium falciparum/enzimología , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Dominio Catalítico , Metales/metabolismo , Plasmodium falciparum/metabolismoRESUMEN
In this study, we analyzed intracellular functions and motile properties of neck-linker (NL) variants of the bi-directional S. cerevisiae kinesin-5 motor, Cin8. We also examined - by modeling - the configuration of H-bonds during NL docking. Decreasing the number of stabilizing H-bonds resulted in partially functional variants, as long as a conserved backbone H-bond at the N-latch position (proposed to stabilize the docked conformation of the NL) remained intact. Elimination of this conserved H-bond resulted in production of a non-functional Cin8 variant. Surprisingly, additional H-bond stabilization of the N-latch position, generated by replacement of the NL of Cin8 by sequences of the plus-end directed kinesin-5 Eg5, also produced a nonfunctional variant. In that variant, a single replacement of N-latch asparagine with glycine, as present in Cin8, eliminated the additional H-bond stabilization and rescued the functional defects. We conclude that exact N-latch stabilization during NL docking is critical for the function of bi-directional kinesin-5 Cin8.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Cinesinas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Enlace de Hidrógeno , Cinesinas/química , Cinesinas/clasificación , Cinesinas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/metabolismoRESUMEN
Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.
Asunto(s)
Apoptosis/fisiología , Citocromos c/química , Mutación Missense , Mutación Puntual , Sustitución de Aminoácidos , Apoptosomas , Cristalografía por Rayos X , Citocromos c/genética , Citocromos c/aislamiento & purificación , Citocromos c/metabolismo , Humanos , Enlace de Hidrógeno , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Células U937RESUMEN
Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir's specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range of conformations that are not apparent from static crystallographic snapshots, but observed no difference in either the conformations, nor degree of flexibility when compared to abacavir-unbound systems. Our results support hypersensitivity models in which abacavir-binding alters the conformational ensemble of neopeptides, so as to favour exposed peptide surfaces that are no longer recognized as self by circulating CD8+ T cells, and are conducive to TCR binding. Our findings highlight the need to also consider the role of dynamics in understanding drug-induced hypersensitivities at the molecular and mechanistic level. This additional insight can help inform the chemical modification of abacavir to prevent hypersensitivity reactions in HLA-B*57:01+ HIV patients whilst retaining potent antiretroviral activity.
Asunto(s)
Fármacos Anti-VIH/efectos adversos , Didesoxinucleósidos/efectos adversos , Hipersensibilidad a las Drogas/etiología , Antígenos HLA-B/metabolismo , Secuencia de Aminoácidos , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Sitios de Unión , Cristalografía por Rayos X , Didesoxinucleósidos/metabolismo , Didesoxinucleósidos/farmacología , Hipersensibilidad a las Drogas/genética , Predisposición Genética a la Enfermedad , Antígenos HLA-B/efectos de los fármacos , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Oligopéptidos/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacosRESUMEN
Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as α1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from α1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.
Asunto(s)
Serpinas/metabolismo , Secuencia de Aminoácidos , Escherichia coli , Humanos , Elastasa de Leucocito/metabolismo , Simulación de Dinámica Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Pliegue de Proteína , Serpinas/química , Serpinas/genética , Electricidad Estática , Tripsina/metabolismoRESUMEN
Cation diffusion facilitator (CDF) proteins are a conserved family of transmembrane transporters that ensure cellular homeostasis of divalent transition metal cations. Metal cations bind to CDF protein's cytoplasmic C-terminal domain (CTD), leading to closure from its apo open V-shaped dimer to a tighter packed structure, followed by a conformational change of the transmembrane domain, thus enabling transport of the metal cation. By implementing a comprehensive range of biochemical and biophysical methods, we studied the molecular mechanism of metal binding to the magnetotactic bacterial CDF protein MamM CTD. Our results reveal that the CTD is rather dynamic in its apo form, and that two dependent metal-binding sites, a single central binding site and two symmetrical, peripheral sites, are available for metal binding. However, only cation binding to the peripheral sites leads to conformational changes that lock the protein in a compact state. Thus, this work reveals how metal binding is regulating the sequential uptakes of metal cations by MamM, and extends our understanding of the complex regulation mechanism of CDF proteins. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6G64, 6G55, 6G5E and 6G6I (for CS, C267S, CS-C267S and W247A, respectively).
Asunto(s)
Proteínas Bacterianas/química , Cationes/metabolismo , Magnetospirillum/química , Zinc/metabolismo , Apoproteínas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios ProteicosRESUMEN
The M1 metallo-aminopeptidase from Plasmodium falciparum, PfA-M1, is an attractive drug target for the design of new antimalarials. Bestatin, a broad-spectrum metalloprotease inhibitor, is a moderate inhibitor of PfA-M1, and has been used to provide structure-activity relationships to inform drug design. The crystal structure of PfA-M1 with bestatin bound within its active site has been determined; however, dynamics of the inhibitor and the association or dissociation pathway have yet to be characterized. Here we present an all-atom molecular dynamics study where we have generated a hidden Markov state model from 2.3â µs of molecular dynamics simulation. Our hidden Markov state model identifies five macrostates that clearly show the events involved in bestatin dissociation from the PfA-M1 active site. The results show for the first time that bestatin can escape the substrate specificity pockets of the enzyme, primarily due to weak interactions within the pockets. Our approach identifies relevant conformational sampling of the inhibitor inside the enzyme and the protein dynamics that could be exploited to produce potent and selective inhibitors that can differentiate between similar members of the M1 aminopeptidase superfamily.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Leucina/análogos & derivados , Plasmodium falciparum/enzimología , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Dominio Catalítico/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Leucina/farmacología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Unión ProteicaRESUMEN
Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term "structural capacitance," which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the potential for evolutionary change conferred by structural capacitance are consequently counterbalanced by this accompanying risk. The phenomenon of structural capacitance has implications ranging from the ancestral diversification of protein folds to the engineering of synthetic proteins with enhanced evolvability.
Asunto(s)
Susceptibilidad a Enfermedades , Evolución Molecular , Proteínas/química , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
The M1 and M17 aminopeptidases are metallo-exopeptidases that rely on the presence of divalent cations, usually zinc, in their active site for proteolytic activity. They are from separate protease superfamilies, however, members often have overlapping substrate specificity. Inhibitors of one or both enzymes can be used to modulate hypertension, reduce proliferation of certain types of cancers and control malaria parasites. Current inhibitors act to chelate the zinc ions in the active site, locking the enzymes in an inactive transition state. We were interested in using a computational approach to understand the structure and dynamics of the M1 and M17 aminopeptidases, however, the presence of the essential metal ions in the proteases presents a challenge to classical molecular dynamics (MD) simulation. The zinc amber force field does not contain applicable descriptions of the zinc coordination environment present in either of these two protease families. To provide tools for the study of these two enzymes, we have used the metal centre parameter builder to generate new hybrid bonded/nonbonded force field (FF) parameters to correctly describe the active site architecture for each enzyme. The new parameters were evaluated by fitting the normal mode frequencies of molecular mechanics to the quantum mechanics frequencies and validated by performing short MD simulations. The new FF parameters now enable more accurate and reliable MD simulations for any member of the M1 or M17 aminopeptidase superfamilies.
Asunto(s)
Aminopeptidasas/química , Simulación de Dinámica Molecular , Plasmodium falciparum/enzimología , Zinc/química , Factores de TiempoRESUMEN
The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.
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Calicreínas/antagonistas & inhibidores , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Regulación de la Expresión Génica , Helianthus , Humanos , Enlace de Hidrógeno , Metales/química , Simulación de Dinámica Molecular , Níquel/química , Péptidos Cíclicos/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Serina Proteasas/química , Tripsina/químicaRESUMEN
The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.
RESUMEN
Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mutación , Transportador 8 de Zinc/química , Transportador 8 de Zinc/genética , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Clonación Molecular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Transportador 8 de Zinc/metabolismoRESUMEN
Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto's disease--the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The "trans" model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the "cis" model favours it slightly over the "trans" model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.
Asunto(s)
Autoantígenos/inmunología , Autoantígenos/metabolismo , Yoduro Peroxidasa/inmunología , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/inmunología , Proteínas de Unión a Hierro/metabolismo , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Autoantígenos/química , Membrana Celular/enzimología , Estabilidad de Enzimas , Espacio Extracelular/enzimología , Humanos , Yoduro Peroxidasa/química , Proteínas de Unión a Hierro/química , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency.
Asunto(s)
Bacteriófagos/enzimología , Enzimas/química , Streptococcus/virología , Bacteriófagos/química , Dominio Catalítico , Cristalografía por Rayos X/métodos , Enzimas/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo PequeñoRESUMEN
Dynamics plays an important but underappreciated role in the interaction between the T cell receptor (TCR) and peptide-bound major histocompatibility complex (pMHC). Crystallographic studies have provided key molecular insights into this interaction; however, due to inherent features of the structural approach, the image of TCR-pMHC interactions that has emerged is a static one. In this review, we discuss how molecular dynamics (MD) simulations can complement and extend current experimental methods aimed at examining TCR-pMHC dynamics. We review the insights obtained from studies that leverage MD approaches, and propose that an integrative strategy that harnesses both MD simulations and structural and biophysical methods will provide new inroads into understanding the transitory and dynamic molecular events that dictate TCR signaling and T cell activation.
RESUMEN
Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein-protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from Plasmodium falciparum strain FVO. We compare our new structure to those of AMA1 from P. falciparum 3D7 and Plasmodium vivax. A combination of normalized B factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting P. falciparum AMA1.
Asunto(s)
Antígenos de Protozoos/química , Malaria Falciparum/parasitología , Proteínas de la Membrana/química , Plasmodium falciparum/química , Proteínas Protozoarias/química , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Plasmodium vivax/química , Estructura Terciaria de ProteínaRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.