RESUMEN
Hydrogen bonding between an amide group and the p-π cloud of an aromatic ring was first identified in a protein in the 1980s. Subsequent surveys of high-resolution X-ray crystal structures found multiple instances, but their preponderance was determined to be infrequent. Hydrogen atoms participating in a hydrogen bond to the p-π cloud of an aromatic ring are expected to experience an upfield chemical shift arising from a shielding ring current shift. We surveyed the Biological Magnetic Resonance Data Bank for amide hydrogens exhibiting unusual shifts as well as corroborating nuclear Overhauser effects between the amide protons and ring protons. We found evidence that Trp residues are more likely to be involved in p-π hydrogen bonds than other aromatic amino acids, whereas His residues are more likely to be involved in in-plane hydrogen bonds, with a ring nitrogen acting as the hydrogen acceptor. The p-π hydrogen bonds may be more abundant than previously believed. The inclusion in NMR structure refinement protocols of shift effects in amide protons from aromatic sidechains, or explicit hydrogen bond restraints between amides and aromatic rings, could improve the local accuracy of sidechain orientations in solution NMR protein structures, but their impact on global accuracy is likely be limited.
RESUMEN
Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hepatocitos/enzimología , Insulina/metabolismo , Sirtuina 1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica , Animales , Sitios de Unión , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Células HCT116 , Hepatocitos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Resistencia a la Insulina , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/enzimología , Obesidad/genética , Obesidad/prevención & control , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Sirtuina 1/genética , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genéticaRESUMEN
Chemical shifts are highly sensitive probes of local conformation and overall structure. Both isotropic shifts and chemical shift tensors are readily accessible from NMR experiments but their quantum mechanical calculations remain challenging. In this work, we report and compare accurately measured and calculated 15NH and 13Cα chemical shift tensors in proteins, using the microcrystalline agglutinin from Oscillatoria agardhii (OAA). Experimental 13Cα and 15NH chemical tensors were obtained by solid-state NMR spectroscopy, employing tailored recoupling sequences, and for their quantum mechanics/molecular mechanics (QM/MM) calculations different sets of functionals were evaluated. We show that 13Cα chemical shift tensors are primarily determined by backbone dihedral angles and dynamics, while 15NH tensors mainly depend on local electrostatic contributions from solvation and hydrogen bonding. In addition, the influence of including crystallographic waters, the molecular mechanics geometry optimization protocol, and the level of theory on the accuracy of the calculated chemical shift tensors is discussed. Specifically, the power of QM/MM calculations in accurately predicting the unusually upfield shifted 1HN G26 and G93 resonances is highlighted. Our integrated approach is expected to benefit structure refinement of proteins and protein assemblies.
Asunto(s)
Aglutininas/química , Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética/métodos , Simulación de Dinámica Molecular , Fenómenos Biomecánicos , Cristalización , Cianobacterias/química , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Conformación Proteica , Teoría Cuántica , Electricidad EstáticaRESUMEN
Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Dispersión del Ángulo Pequeño , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Dominios Proteicos , Difracción de Rayos XRESUMEN
NMR chemical shifts are exquisitely sensitive probes for conformation and dynamics in molecules and supramolecular assemblies. Although isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, the 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations for analysis of 13Cα and 15NH isotropic chemical shifts. The influence of local interactions, quaternary contacts, and dynamics on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies.
Asunto(s)
Aglutininas/química , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Teoría Cuántica , Algoritmos , Cristalografía por Rayos X , Oscillatoria/químicaRESUMEN
Biochemical characterization of aromatic prenyltransferase AmbP1 and its close homologs WelP1/FidP1 in hapalindole-type alkaloid biosynthetic pathways is reported. These enzymes mediate the magnesium-dependent selective formation of 3-geranyl 3-isocyanovinyl indolenine (2) from cis-indolyl vinyl isonitrile and geranyl pyrophosphate. The role of the magnesium cofactor in AmbP1/WelP1/FidP1 catalysis is unusual for a microbial aromatic prenyltransferase, as it not only facilitates the formation of 2 but also prevents its rearrangement to an isomeric 2-geranyl 3-isocyanovinyl indole (3). The discovery of 2 as a cryptically conserved common biosynthetic intermediate to all hapalindole-type alkaloids suggests an enzyme-mediated Cope rearrangement and aza-Prins-type cyclization cascade is required to transform 2 to a polycyclic hapalindole-like scaffold.
Asunto(s)
Alcaloides/metabolismo , Dimetilaliltranstransferasa/metabolismo , Manganeso/metabolismoRESUMEN
MGG_03307 is a lectin isolated from Magnaporte oryzae, a fungus that causes devastating rice blast disease. Its function is associated with protecting M. oryzae from the host immune response in plants. To provide the structural basis of how MGG_03307 protects the fungus, crystal structures of its CVNH-LysM module were determined in the absence and presence of GlcNAc-containing cell wall chitin constituents, which can act as pathogen-associated molecular patterns. Our structures revealed that glycan binding is accompanied by a notable conformational change in the LysM domain and that GlcNAc3 and GlcNAc4 are accommodated similarly. GlcNAc5 and GlcNAc6 interact with the LysM domain in multiple conformations, as evidenced by solution nuclear magnetic resonance studies. No dimerization of MoCVNH3 via its LysM domain was observed upon binding to GlcNAc6, unlike in multiple LysM domain-containing proteins. Importantly, we define a specific consensus binding mode for the recognition of GlcNAc oligomers by single LysM domains.
Asunto(s)
Pared Celular/metabolismo , Proteínas Fúngicas/química , Magnaporthe/química , Lectinas de Unión a Manosa/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Pared Celular/química , Quitina/química , Quitina/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Lectinas de Unión a Manosa/metabolismo , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Sterile α motif (SAM) and histidine/aspartate (HD)-containing protein 1 (SAMHD1) restricts human/simian immunodeficiency virus infection in certain cell types and is counteracted by the virulence factor Vpx. Current evidence indicates that Vpx recruits SAMHD1 to the Cullin4-Ring Finger E3 ubiquitin ligase (CRL4) by facilitating an interaction between SAMHD1 and the substrate receptor DDB1- and Cullin4-associated factor 1 (DCAF1), thereby targeting SAMHD1 for proteasome-dependent down-regulation. Host-pathogen co-evolution and positive selection at the interfaces of host-pathogen complexes are associated with sequence divergence and varying functional consequences. Two alternative interaction interfaces are used by SAMHD1 and Vpx: the SAMHD1 N-terminal tail and the adjacent SAM domain or the C-terminal tail proceeding the HD domain are targeted by different Vpx variants in a unique fashion. In contrast, the C-terminal WD40 domain of DCAF1 interfaces similarly with the two above complexes. Comprehensive biochemical and structural biology approaches permitted us to delineate details of clade-specific recognition of SAMHD1 by lentiviral Vpx proteins. We show that not only the SAM domain but also the N-terminal tail engages in the DCAF1-Vpx interaction. Furthermore, we show that changing the single Ser-52 in human SAMHD1 to Phe, the residue found in SAMHD1 of Red-capped monkey and Mandrill, allows it to be recognized by Vpx proteins of simian viruses infecting those primate species, which normally does not target wild type human SAMHD1 for degradation.
Asunto(s)
Interacciones Huésped-Patógeno , Infecciones por Lentivirus/metabolismo , Lentivirus/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Infecciones por Lentivirus/virología , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/química , Estructura Terciaria de Proteína , Proteína 1 que Contiene Dominios SAM y HD , Alineación de Secuencia , Proteínas Reguladoras y Accesorias Virales/químicaRESUMEN
Lectins from different sources have been shown to interfere with HIV infection by binding to the sugars of viral-envelope glycoproteins. Three-dimensional atomic structures of a number of HIV-inactivating lectins have been determined, both as free proteins and in glycan-bound forms. However, details on the mechanism of recognition and binding to sugars are elusive. Herein we focus on the anti-HIV lectin OAA from Oscillatoria agardhii: We show that in the absence of sugars in solution, both the sugar-free and sugar-bound protein conformations that were observed in the X-ray crystal structures exist as conformational substates. Our results suggest that glycan recognition occurs by conformational selection within the ground state; this model differs from the popular "excited-state" model. Our findings provide further insight into molecular recognition of the major receptor on the HIV virus by OAA. These details can potentially be used for the optimization and/or development of preventive anti-HIV therapeutics.
Asunto(s)
Fármacos Anti-VIH/química , Proteínas Bacterianas/química , Carbohidratos/química , Lectinas/química , Oscillatoria/metabolismo , Polisacáridos/química , Fármacos Anti-VIH/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , VIH/metabolismo , Lectinas/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Lectins from different sources are known to interfere with HIV infection. The anti-viral activity is mediated by binding to high mannose sugars present on the viral envelope, thereby inhibiting cell entry. The lectin from Oscillatoria agardhii agglutinin (OAA) specifically recognizes a unique substructure of high mannose sugars and exhibits broad anti-HIV activity. Here we report the assignment of backbone and side-chain (1)H, (13)C and (15)N resonances of free OAA.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Lectinas/química , Oscillatoria/química , Espectroscopía de Protones por Resonancia Magnética , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) plays a critical role in inhibiting HIV infection, curtailing the pool of dNTPs available for reverse transcription of the viral genome. Recent structural data suggested a compelling mechanism for the regulation of SAMHD1 enzymatic activity and revealed dGTP-induced association of two inactive dimers into an active tetrameric enzyme. Here, we present the crystal structures of SAMHD1 catalytic core (residues 113-626) tetramers, complexed with mixtures of nucleotides, including dGTP/dATP, dGTP/dCTP, dGTP/dTTP, and dGTP/dUTP. The combined structural and biochemical data provide insight into dNTP promiscuity at the secondary allosteric site and how enzymatic activity is modulated. In addition, we present biochemical analyses of GTP-induced SAMHD1 full-length tetramerization and the structure of SAMHD1 catalytic core tetramer in complex with GTP/dATP, revealing the structural basis of GTP-mediated SAMHD1 activation. Altogether, the data presented here advance our understanding of SAMHD1 function during cellular homeostasis.
Asunto(s)
Desoxirribonucleótidos/química , Proteínas de Unión al GTP Monoméricas/química , Nucleósido-Trifosfatasa/química , Multimerización de Proteína , Regulación Alostérica , Dominio Catalítico , Cristalografía por Rayos X , Nucleótidos de Desoxiguanina/química , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/metabolismo , Humanos , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación , Conformación de Ácido Nucleico , Nucleósido-Trifosfatasa/genética , Nucleósido-Trifosfatasa/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteína 1 que Contiene Dominios SAM y HDRESUMEN
A growing class of potential antivirals encompasses carbohydrate-binding proteins, such as antibodies and lectins. They block virus entry into host target cells and halt virus transmission from virus-infected cells to non-infected cells, thereby preventing infection. Here, we review the structural basis for the anti-HIV activity of various lectins, describing their structures and determinants of high-affinity oligosaccharide binding. The mechanism of glycan recognition on the gp120 envelope protein by these antiviral lectins may therefore be exploited for developing agents and alternative strategies to prevent HIV transmission.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Lectinas/farmacología , Animales , Fármacos Anti-VIH/química , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Humanos , Lectinas/químicaRESUMEN
OBJECTIVES: Oscillatoria agardhii agglutinin homologue (OAAH) proteins belong to a recently discovered lectin family. The founding member OAA and a designed hybrid OAAH (OPA) recognize similar but unique carbohydrate structures of Man-9, compared with other antiviral carbohydrate-binding agents (CBAs). These two newly described CBAs were evaluated for their inactivating properties on HIV replication and transmission and for their potential as microbicides. METHODS: Various cellular assays were used to determine antiviral activity against wild-type and certain CBA-resistant HIV-1 strains: (i) free HIV virion infection in human T lymphoma cell lines and PBMCs; (ii) syncytium formation assay using persistently HIV-infected T cells and non-infected CD4+ T cells; (iii) DC-SIGN-mediated viral capture; and (iv) transmission to uninfected CD4+ T cells. OAA and OPA were also evaluated for their mitogenic properties and potential synergistic effects using other CBAs. RESULTS: OAA and OPA inhibit HIV replication, syncytium formation between HIV-1-infected and uninfected T cells, DC-SIGN-mediated HIV-1 capture and transmission to CD4+ target T cells, thereby rendering a variety of HIV-1 and HIV-2 clinical isolates non-infectious, independent of their coreceptor use. Both CBAs competitively inhibit the binding of the Manα(1-2)Man-specific 2G12 monoclonal antibody (mAb) as shown by flow cytometry and surface plasmon resonance analysis. The HIV-1 NL4.3(2G12res), NL4.3(MVNres) and IIIB(GRFTres) strains were equally inhibited as the wild-type HIV-1 strains by these CBAs. Combination studies indicate that OAA and OPA act synergistically with Hippeastrum hybrid agglutinin, 2G12 mAb and griffithsin (GRFT), with the exception of OPA/GRFT. CONCLUSIONS: OAA and OPA are unique CBAs with broad-spectrum anti-HIV activity; however, further optimization will be necessary for microbicidal application.
Asunto(s)
Aglutininas/farmacología , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Lectinas/farmacología , Oscillatoria/metabolismo , Aglutininas/metabolismo , Fármacos Anti-VIH/metabolismo , Proteínas Bacterianas/farmacología , Línea Celular , Farmacorresistencia Viral , Células Gigantes/efectos de los fármacos , Células Gigantes/virología , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Lectinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Pruebas de Sensibilidad Microbiana , Unión ProteicaRESUMEN
The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-Ub(K48) complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Reparación del ADN , Proteínas de Unión al ADN/química , Infecciones por VIH/metabolismo , VIH-1/genética , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Enzimas Activadoras de Ubiquitina/química , Ubiquitinación , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Micro-to-millisecond motions of proteins transmit pivotal signals for protein function. A powerful technique for the measurement of these motions is nuclear magnetic resonance spectroscopy. One of the most widely used methodologies for this purpose is the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment where kinetic and structural information can be obtained at atomic resolution. Extraction of accurate kinetics determined from CT-CPMG data requires refocusing frequencies that are much larger than the nuclei's exchange rate between states. We investigated the effect when fast processes are probed by CT-CPMG experiments via simulation and show that if the intrinsic relaxation rate (R(CT-CPMG)(2,0)) is not known a priori the extraction of accurate kinetics is hindered. Errors on the order of 50 % in the exchange rate are attained when processes become fast, but are minimized to 5 % with a priori (CT-CPMG)(2,0)) information. To alleviate this shortcoming, we developed an experimental scheme probing (CT-CPMG)(2,0)) with large amplitude spin-lock fields, which specifically contains the intrinsic proton longitudinal Eigenrelaxation rate. Our approach was validated with ubiquitin and the Oscillatoria agardhii agglutinin (OAA). For OAA, an underestimation of 66 % in the kinetic rates was observed if (CT-CPMG)(2,0)) is not included during the analysis of CT-CPMG data and result in incorrect kinetics and imprecise amplitude information. This was overcome by combining CT-CPMG with (CT-CPMG)(2,0)) measured with a high power R1ρ experiment. In addition, the measurement of (CT-CPMG)(2,0)) removes the ambiguities in choosing between different models that describe CT-CPMG data.
Asunto(s)
Proteínas Bacterianas/química , Lectinas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Ubiquitina/química , Cinética , Oscillatoria/química , Conformación ProteicaRESUMEN
Up to now, efforts to crystallize the cataract-associated P23T mutant of human γD-crystallin have not been successful. Therefore, insights into the light scattering mechanism of this mutant have been exclusively obtained from solution work. Here we present the first crystal structure of the P23T mutant at 2.5 Šresolution. The protein exhibits essentially the same overall structure as seen for the wild-type protein. Based on our structural data, we confirm that no major conformational changes are caused by the mutation, and that solution phase properties of the mutant appear exclusively associated with cataract formation.
Asunto(s)
Mutación/fisiología , Proteínas Recombinantes/química , gamma-Cristalinas/química , Catarata , Escherichia coli/genética , Humanos , Modelos Moleculares , Mutación/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , gamma-Cristalinas/genética , gamma-Cristalinas/metabolismoRESUMEN
Although it has long been established that the amino acid sequence encodes the fold of a protein, how individual proteins arrive at their final conformation is still difficult to predict, especially for oligomeric structures. Here, we present a comprehensive characterization of oligomeric species of cyanovirin-N that all are formed by a polypeptide chain with the identical amino acid sequence. Structures of the oligomers were determined by X-ray crystallography, and each one exhibits 3D domain swapping. One unique 3D domain-swapped structure is observed for the trimer, while for both dimer and tetramer, two different 3D domain-swapped structures were obtained. In addition to the previously identified hinge-loop region of the 3D domain-swapped dimer, which resides between strands ß5 and ß6 in the middle of the polypeptide sequence, another hinge-loop region is observed between strands ß7 and ß8 in the structures. Plasticity in these two regions allows for variability in dihedral angles and concomitant differences in chain conformation that results in the differently 3D domain-swapped multimers. Based on all of the different structures, we propose possible folding pathways for this protein. Altogether, our results illuminate the amazing ability of cyanovirin-N to proceed down different folding paths and provide general insights into oligomer formation via 3D domain swapping.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Pliegue de Proteína , Cristalografía por Rayos X , Dimerización , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Nostoc/metabolismo , Estructura Terciaria de Proteína , Proteínas/química , Difracción de Rayos XRESUMEN
Structure determination of protein-nucleic acid complexes remains a challenging task. Here we present a simple method for generating crystals of a CsrA-nucleic acid complex, guided entirely by results from nuclear magnetic resonances spectroscopy (NMR) spectroscopy. Using a construct that lacks thirteen non-essential C-terminal residues, efficient binding to DNA could be demonstrated. One CsrA dimer interacts with two DNA oligonucleotides, similar to previous findings with RNA. Furthermore, the NMR study of the CsrA-DNA complex was the basis for successfully homing in on conditions that were suitable for obtaining crystals of the CsrA-DNA complex. Our results may be useful for those cases where RNA in protein-nucleic acid complexes may be replaced by DNA.
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Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , ARN/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Multimerización de Proteína/fisiología , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismoRESUMEN
Structures and sugar binding by members of two lectin families, Cyanovirin-N homolog (CVNH) and Oscillatoria Agardhii agglutinin homolog (OAAH), were determined to elucidate the basis for recognition of high-mannose glycans on the HIV envelope glycoprotein gp120. We solved NMR solution and/or crystal structures for several lectins and delineated their carbohydrate specificity by array screening and direct NMR titrations. Both families recognize different epitopes on high-mannose glycans, namely, Manα(1-2)Man units at the end of the D1 and D3 arms and α3,α6-mannopentaose at the central branch point of Man-8 or Man-9 for CVNH and OAAH lectins, respectively.
Asunto(s)
VIH-1 , Lectinas/metabolismo , Polisacáridos/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Humanos , Lectinas/química , Masculino , Datos de Secuencia Molecular , Polisacáridos/química , Proteínas/química , Alineación de SecuenciaRESUMEN
Some mutants of human γD-crystallin are closely linked to congenital cataracts, although the detailed molecular mechanisms of mutant-associated cataract formation are generally not known. Here we report on a recently discovered γD-crystallin mutant (W42R) that has been linked to autosomal dominant, congenital cataracts in a Chinese family. The mutant protein is much less soluble and stable than wild-type γD-crystallin. We solved the crystal structure of W42R at 1.7 Šresolution, which revealed only minor differences from the wild-type structure. Interestingly, the W42R variant is highly susceptible to protease digestion, suggesting the presence of a small population of partially unfolded protein. This partially unfolded species was confirmed and quantified by NMR spectroscopy. Hydrogen/deuterium exchange experiments revealed chemical exchange between the folded and unfolded species. Exposure of wild-type γD-crystallin to UV caused damage to the N-terminal domain of the protein, resulting in very similar proteolytic susceptibility as observed for the W42R mutant. Altogether, our combined data allowed us to propose a model for W42R pathogenesis, with the W42R mutant serving as a mimic for photodamaged γD-crystallin involved in age-related cataract.