RESUMEN
This compound is a synthetic macrocycle comprising three pivaloyl-protected resorcinarene units connected by six pentylene chains. We conducted a conformational study using 1H-NMR, X-ray diffraction (XRD), and computational analyses. The macrocycle adopts two conformers, one open, the other closed. The ratio of the open to closed forms depended on the solvent used. Only the open form existed in [D8]toluene, both forms coexisted in [D6]benzene, and the closed form was the major conformer in [D1]chloroform. The benzene-solvated open form observed in the solid state suggests that cavity solvation by solvent molecules directs the open form. The open form was the major or only conformer in [D10]o- and [D10]m-xylene and [D12]mesitylene, whereas the closed form was the major conformer in [D6]acetone. The open and closed forms were equally populated in [D10]p-xylene, suggesting that the size, shape, and dimensions of the solvent molecules most likely influenced the conformation of the protected trisresocinarene.
RESUMEN
To develop novel bovine lactoferrin (bLF) peptides targeting bLF-tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) binding sites, we identified two peptides that could target bLF-TRAF6 binding sites using structural analysis. Moreover, another peptide that could bind to the TRAF6 dimerization area was selected from the bLF sequence. The effects of each peptide on cytokine expression in lipopolysaccharide (LPS)-stimulated osteoblasts (ST2) and on osteoclastogenesis were examined using an LPS-treated co-culture of primary bone marrow cells (BMCs) with ST2 cells and a single culture of osteoclast precursor cells (RAW-D) treated with soluble receptor activator of NF-κB ligand. Finally, the effectiveness of these peptides against LPS-induced alveolar bone destruction was assessed. Two of the three peptides significantly suppressed LPS-induced TNF-α and interleukin-1ß expression in ST2 cells. Additionally, these peptides inhibited and reversed LPS-induced receptor activator of NF-κB ligand (RANKL) upregulation and osteoprotegerin (OPG) downregulation, respectively. Furthermore, both peptides significantly reduced LPS-induced osteoclastogenesis in the BMC-ST2 co-culture and RANKL-induced osteoclastogenesis in RAW-D cells. In vivo, topical application of these peptides significantly reduced the osteoclast number by downregulating RANKL and upregulating OPG in the periodontal ligament. It is indicated that the novel bLF peptides can be used to treat periodontitis-associated bone destruction.
Asunto(s)
Lactoferrina , Lipopolisacáridos , Osteoclastos , Péptidos , Animales , Lactoferrina/farmacología , Lactoferrina/química , Lactoferrina/metabolismo , Lipopolisacáridos/farmacología , Ratas , Péptidos/farmacología , Péptidos/química , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Masculino , Pérdida de Hueso Alveolar/tratamiento farmacológico , Pérdida de Hueso Alveolar/metabolismo , Pérdida de Hueso Alveolar/patología , Bovinos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/citología , Ratas Sprague-Dawley , Osteogénesis/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Sitios de Unión , Técnicas de Cocultivo , Osteoprotegerina/metabolismo , Modelos Animales de EnfermedadRESUMEN
Fused in sarcoma (FUS), a DNA/RNA-binding protein, undergoes liquid-liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin-containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time-dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long-lasting VCP colocalization destabilizes the granules. The proteolysis-promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.
Asunto(s)
Esclerosis Amiotrófica Lateral , Degeneración Lobar Frontotemporal , Sarcoma , Adenosina Trifosfato , Esclerosis Amiotrófica Lateral/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
Tau aggregation is a central hallmark of tauopathies such as frontotemporal lobar degeneration and progressive supranuclear palsy as well as of Alzheimer's disease, and it has been a target for therapeutic development. Herein, we unexpectedly found that hepta-histidine (7H), an inhibitor of the interaction between Ku70 and Huntingtin proteins, suppresses aggregation of Tau-R3 peptides in vitro. Addition of the trans-activator of transcription (TAT) sequence (YGRKKRRQRRR) derived from the TAT protein to 7H increased its permeability into cells, and TAT-7H treatment of iPS cell-derived neurons carrying Tau or APP mutations suppressed Tau phosphorylation. These results indicate that 7H is a promising lead compound for developing anti-aggregation drugs against Tau-related neurodegenerative diseases including Alzheimer's disease (AD).
Asunto(s)
Enfermedad de Alzheimer , Degeneración Lobar Frontotemporal , Tauopatías , Enfermedad de Alzheimer/tratamiento farmacológico , Histidina , Humanos , Proteínas tauRESUMEN
Pin1 enzyme protein recognizes specifically phosphorylated serine/threonine (pSer/pThr) and catalyzes the slow interconversion of the peptidyl-prolyl bond between cis and trans forms. Structural dynamics between the cis and trans forms are essential to reveal the underlying molecular mechanism of the catalysis. In this study, we apply the weighted ensemble (WE) simulation method to obtain comprehensive path ensembles for the Pin1-catalyzed isomerization process. Associated rate constants for both cis-to-trans and trans-to-cis isomerization are calculated to be submicroseconds time scales, which are in good agreement with the calculated free energy landscape where the cis form is slightly less favorable. The committor-like analysis indicates the shift of the transition state toward trans form (at the isomerization angle ω â¼ 110°) compared to the intrinsic position for the isolated substrate (ω â¼ 90°). The calculated structural ensemble clarifies a role of both the dual-histidine motif, His59/His157, and the basic residues, Lys63/Arg68/Arg69, to anchor both sides of the peptidyl-prolyl bond, the aromatic ring in Pro, and the phosphate in pSer, respectively. The rotation of the torsion angle is found to be facilitated by relaying the hydrogen-bond partner of the main-chain oxygen in pSer from Cys113 in the cis form to Arg68 in the trans form, through Ser154 at the transition state, which is really the cause of the shift in the transition state. The role of Ser154 as a driving force of the isomerization is confirmed by additional WE and free energy calculations for S154A mutant where the isomerization takes place slightly slower and the free energy barrier increases through the mutation. The present study shows the usefulness of the WE simulation for substantial path samplings between the reactant and product states, unraveling the molecular mechanism of the enzyme catalysis.
Asunto(s)
Simulación de Dinámica Molecular , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Péptidos/metabolismo , Biocatálisis , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA/química , Péptidos/química , EstereoisomerismoRESUMEN
Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Nucleosomas/metabolismo , Animales , Drosophila/química , Simulación de Dinámica Molecular , Fosforilación , Dominios ProteicosRESUMEN
Tau forms intracellular insoluble aggregates as a neuropathological hallmark of Alzheimer's disease. Tau is largely unstructured, which complicates the characterization of the tau aggregation process. Recent studies have demonstrated that tau samples two distinct conformational ensembles, each of which contains the soluble and aggregation-prone states of tau. A shift to populate the aggregation-prone ensemble may promote tau fibrillization. However, the mechanism of this ensemble transition remains elusive. In this study, we explored the conformational dynamics of a tau fragment by using paramagnetic relaxation enhancement (PRE) and interference (PRI) NMR experiments. The PRE correlation map showed that tau is composed of segments consisting of residues in correlated motions. Intriguingly, residues forming the ß-structures in the heparin-induced tau filament coincide with residues in these segments, suggesting that each segment behaves as a structural unit in fibrillization. PRI data demonstrated that the P301L mutation exclusively alters the transiently formed tau structures by changing the short- and long-range correlated motions among residues. The transient conformations of P301L tau expose the amyloid motif PHF6 to promote tau self-aggregation. We propose the correlated motions among residues within tau determine the population sizes of the conformational ensembles, and perturbing the correlated motions populates the aggregation-prone form.
Asunto(s)
Enfermedad de Alzheimer/genética , Mutación , Proteínas tau/genética , Secuencias de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Heparina/química , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Espectroscopía de Resonancia Magnética , Agregado de Proteínas , Dominios Proteicos , Isoformas de Proteínas , Estructura Secundaria de Proteína , Proteínas tau/químicaRESUMEN
The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.
Asunto(s)
Cloruro de Amonio/metabolismo , Glucosa/metabolismo , Schizosaccharomyces/metabolismo , Cuerpos Polares del Huso/metabolismo , Adenosina Trifosfato/metabolismo , Centrómero/metabolismo , Schizosaccharomyces/citologíaRESUMEN
Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.
Asunto(s)
Apoproteínas/química , Peptidilprolil Isomerasa de Interacción con NIMA/química , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Regulación Alostérica , Humanos , Dominios ProteicosRESUMEN
In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA/química , Péptidos/química , Agregación Patológica de Proteínas , Dominios WW , Proteínas tau/química , Amiloide/química , Amiloide/metabolismo , Sitios de Unión/genética , Dominio Catalítico , Humanos , Espectroscopía de Resonancia Magnética , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The stress-induced 70 kDa heat shock protein (Hsp70) functions as a molecular chaperone to maintain protein homeostasis. Hsp70 contains an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). The SBD is divided into the ß subdomain containing the substrate-binding site (ßSBD) and the α-helical subdomain (αLid) that covers the ßSBD. In this report, the solution structures of two different forms of the SBD from human Hsp70 were solved. One structure shows the αLid bound to the substrate-binding site intramolecularly, whereas this intramolecular binding mode is absent in the other structure solved. Structural comparison of the two SBDs from Hsp70 revealed that client-peptide binding rearranges residues at the interdomain contact site, which impairs interdomain contact between the SBD and the NBD. Peptide binding also disrupted the inter-subdomain interaction connecting the αLid to the ßSBD, which allows the binding of the αLid to the NBD. The results provide a mechanism for interdomain communication upon substrate binding from the SBD to the NBD via the lynchpin site in the ßSBD of human Hsp70. In comparison to the bacterial ortholog, DnaK, some remarkable differences in the allosteric signal propagation among residues within the Hsp70 SBD exist.
Asunto(s)
Sitios de Unión , Proteínas HSP70 de Choque Térmico/química , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Regulación Alostérica , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Soluciones , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113-H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Sitios de Unión , Dominio Catalítico , Histidina/química , Humanos , Enlace de Hidrógeno , Isomerismo , Espectroscopía de Resonancia Magnética , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.
Asunto(s)
Edición Génica , Mutación , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Cromatografía en Gel , ADN/metabolismo , Dispersión Dinámica de Luz , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Secuencias Repetitivas de Aminoácido , Termodinámica , Nucleasas de los Efectores Tipo Activadores de la Transcripción/químicaRESUMEN
Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(ß) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35 °C, and then evaluated the H(N), N(H), C(α) and C(ß) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain.
Asunto(s)
Escherichia coli/enzimología , Ácido Fólico/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Tetrahidrofolato Deshidrogenasa/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Intimate cooperativity among active site residues in enzymes is a key factor for regulating elaborate reactions that would otherwise not occur readily. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is the phosphorylation-dependent cis-trans peptidyl-prolyl isomerase (PPIase) that specifically targets phosphorylated Ser/Thr-Pro motifs. Residues C113, H59, H157, and T152 form a hydrogen bond network in the active site, as in the noted connection. Theoretical studies have shown that protonation to thiolate C113 leads to rearrangement of this hydrogen bond network, with switching of the tautomeric states of adjacent histidines (H59 and H157) [Barman, A., and Hamelberg, D. (2014) Biochemistry 53, 3839-3850]. This is called the "dual-histidine motif". Here, C113A and C113S Pin1 mutants were found to alter the protonation states of H59 according to the respective residue type replaced at C113, and the mutations resulted in disruption of the hydrogen bond within the dual-histidine motif. In the C113A mutant, H59 was observed to be in exchange between ε- and δ-tautomers, which widened the entrance of the active site cavity, as seen by an increase in the distance between residues A113 and S154. The C113S mutant caused H59 to exchange between the ε-tautomer and imidazolium while not changing the active site structure. Moreover, the imidazole ring orientations of H59 and H157 were changed in the C113S mutant. These results demonstrated that a mutation at C113 modulates the hydrogen bond network dynamics. Thus, C113 acts as a pivot to drive the concerted function among the residues in the hydrogen bond network, as theoretically predicted.
Asunto(s)
Sitio Alostérico , Dominio Catalítico , Histidina , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Secuencias de Aminoácidos , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genéticaRESUMEN
Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.
Asunto(s)
Histidina/metabolismo , Mutación , Isomerasa de Peptidilprolil/química , Fosfatos/metabolismo , Regulación Alostérica , Sitios de Unión , Calorimetría , Humanos , Espectroscopía de Resonancia Magnética , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Conformación ProteicaRESUMEN
To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl2 concentration-dependence of the (1)H-(15)N HSQC spectra of the wild-type DHFR-folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased Km and Kd values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250MPa together with negative activation volumes of -4.0 or -4.8mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.
Asunto(s)
Sustitución de Aminoácidos , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Mutación Missense , Tetrahidrofolato Deshidrogenasa/química , Compuestos de Bario/química , Dominio Catalítico , Cloruros/química , Estabilidad de Enzimas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Concentración de Iones de Hidrógeno , Solventes/química , Especificidad por Sustrato , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Interactions between the nucleosome and the non-histone chromosomal proteins (HMGN1 and HMGN2) were studied by circular dichroism (CD) spectroscopy to elucidate structural changes in the nucleosome induced by HMGN binding. Unlike previous studies that used a nucleosome extracted from living cells, in this study we utilized a nucleosome reconstituted from unmodified recombinant histones synthesized in Escherichia coli and a 189-bp synthetic DNA fragment harboring a nucleosome positioning sequence. This DNA fragment consists of 5'-TATAAACGCC-3' repeats that has a high affinity to the histone octamer. A nucleosome containing a unique octamer-binding sequence at a specific location on the DNA was produced at sufficiently high yield for spectroscopic analysis. CD data have indicated that both HMGN1 and HMGN2 can increase the winding angle of the nucleosome DNA, but the extent of the structural changes induced by these proteins differs significantly. This suggests HMGN1 and HMGN2 would have different abilities to facilitate nucleosome remodeling.
RESUMEN
The intrinsically disordered region (IDR) of a protein is an important topic in molecular biology. The functional significance of IDRs typically involves gene-regulation processes and is closely related to posttranslational modifications such as phosphorylation. We previously reported that the Drosophila facilitates chromatin transcription (FACT) protein involved in chromatin remodeling contains an acidic ID fragment (AID) whose phosphorylation modulates FACT binding to nucleosomes. Here, we performed dynamic atomic force microscopy and NMR analyses to clarify how the densely phosphorylated AID masks the DNA binding interface of the high-mobility-group domain (HMG). Dynamic atomic force microscopy of the nearly intact FACT revealed that a small globule temporally appears but quickly vanishes within each mobile tail-like image, corresponding to the HMG-containing IDR. The lifespan of the globule increases upon phosphorylation. NMR analysis indicated that phosphorylation induces no ordered structure but increases the number of binding sites in AID to HMG with an adjacent basic segment, thereby retaining the robust electrostatic intramolecular interaction within FACT even in the presence of DNA. These data lead to the conclusion that the inhibitory effect of nucleosome binding is ascribed to the increase in the probability of encounter between HMG and the phosphorylated IDR.