RESUMEN
Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.
Asunto(s)
Carboxipeptidasas , Glutamatos , Microtúbulos , Tubulina (Proteína) , Animales , Humanos , Sitios de Unión , Encéfalo/metabolismo , Carboxipeptidasas/química , Carboxipeptidasas/metabolismo , Carboxipeptidasas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Glutamatos/metabolismo , Glutamatos/química , Homeostasis , Espectroscopía de Resonancia Magnética , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Unión Proteica , Células Sf9 , Especificidad por Sustrato , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructuraRESUMEN
Mitochondrial-derived peptides are encoded by mitochondrial DNA but have biological activity outside mitochondria. Eight of these are encoded by sequences within the mitochondrial 12S and 16S ribosomal genes: humanin, MOTS-c, and the six SHLP peptides, SHLP1-SHLP6. These peptides have various effects in cell culture and animal models, affecting neuroprotection, insulin sensitivity, and apoptosis, and some are secreted, potentially having extracellular signaling roles. However, except for humanin, their importance in normal cell function is unknown. To gauge their importance, their coding sequences in vertebrates have been analyzed for synonymous codon bias. Because they lie in RNA genes, such bias should only occur if their amino acids have been conserved to maintain biological function. Humanin and SHLP6 show strong synonymous codon bias and sequence conservation. In contrast, SHLP1, SHLP2, SHLP3, and SHLP5 show no significant bias and are poorly conserved. MOTS-c and SHLP4 also lack significant bias, but contain highly conserved N-terminal regions, and their biological importance cannot be ruled out. An additional potential mitochondrial-derived peptide sequence was discovered preceding SHLP2, named SHLP2b, which also contains a highly conserved N-terminal region with synonymous codon bias.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Péptidos , Animales , Péptidos/genética , Secuencia de Aminoácidos , Selección GenéticaRESUMEN
Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin ßN-domains including fibrin ß chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the ßN-domains that are involved in the interaction using NMR and site-directed mutagenesis. The results obtained revealed that Lys47 and Lys53 of the second and third positively charged clusters of the ßN-domain, respectively, interact with Trp20 and Asp25 of the CR2 domain and Trp63 and Glu68 of the CR3 domain, respectively. This finding indicates that Lys residues of the ßN-domain interact with the Lys-binding site of the CR domains in a manner proposed earlier for the interaction of other members of the LDL receptor family with their ligands. In addition, Gly15 of the ßN-domain and its first positively charged cluster contribute to the high-affinity interaction with VLDLR. Molecular modeling based on the results obtained and analysis of the previously published structures of such domains complexed with RAP and HRV2 allowed us to propose a model of interaction of fibrin ßN-domains with the fibrin-binding CR domains of the VLDL receptor.
Asunto(s)
Fibrina/química , Fibrina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Mutagénesis Sitio-Dirigida/métodos , Receptores de LDL/química , Receptores de LDL/metabolismo , Acetilación , Sitios de Unión , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Receptores de LDL/genética , Resonancia por Plasmón de SuperficieRESUMEN
Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor. In this study, we have made a next step toward this goal by establishing the nuclear magnetic resonance solution structure of the recombinant VLDLR(2-4) fragment containing all three fibrin-binding CR domains of this receptor. The structure revealed that all three CR domains have a similar general fold. Each domain contains a calcium-binding loop, and the loop in the CR3 domain has a unique conformation relative to the other two. Domains CR2 and CR3 interact with each other, while CR4 is flexible relative to the other two domains. In addition, analysis of the electrostatic potential surface of VLDLR(2-4) revealed extended negatively charged regions in each of its CR domains. The presence of these regions suggests that they may interact with three positively charged clusters of the fibrin ßN domain whose involvement in interaction with the VLDL receptor was demonstrated earlier. Altogether, these findings provide a solid background for our next step toward establishing the structural basis for fibrin-VLDL receptor interaction.
Asunto(s)
Cisteína/química , Fibrina/metabolismo , Receptores de LDL/química , Secuencia de Aminoácidos , Sitios de Unión , Cisteína/metabolismo , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Estructura Secundaria de Proteína , Receptores de LDL/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Neutron reflectometry (NR) is uniquely suited for studying protein interaction with phospholipid bilayers along the bilayer normal on an angstrom scale. However, NR on its own cannot discern specific membrane-bound regions due to a lack of scattering contrast within a protein. Here we report the successful coupling of native chemical ligation (NCL) and NR to study α-synuclein (α-syn), a membrane-binding neuronal protein central in Parkinson's disease. Two α-syn variants were generated where either the first 86 or last 54 residues are deuterated, allowing for region-specific contrast within the protein and the identification of membrane interacting residues by NR. Residues 1-86 are positioned at the hydrocarbon/headgroup interface of the outer leaflet, whereas the density distribution of the 54 C-terminal residues ranges from the hydrocarbon region to the aqueous environment. Coupling of NCL and NR should have broad utility in studies of membrane protein folding.
RESUMEN
Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.
RESUMEN
We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site). We hypothesized cooperative binding of PtdIns(4,5)P2 to the C site and a nonspecific anionic phospholipid to the A site. PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5)P2 binding to the PH domain of PLC δ1 was hyperbolic. Mutation of amino acids in either the C or A site resulted in decreased PtdIns(4,5)P2-dependent binding to vesicles and decreased GAP activity. The results support the idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. We propose that the mechanism underlies rapid switching between active and inactive ASAP1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/química , Unión ProteicaRESUMEN
Apolipoproteins are essential human proteins for lipid metabolism. Together with phospholipids, they constitute lipoproteins, nm to µm sized particles responsible for transporting cholesterol and triglycerides throughout the body. To investigate specific protein-lipid interactions, we produced and characterized three single-Trp containing apolipoprotein C-III (ApoCIII) variants (W42 (W54F/W65F), W54 (W42F/W65F), W65 (W42F/W54F)). Upon binding to phospholipid vesicles, wild-type ApoCIII adopts an α-helical conformation (50% helicity) as determined by circular dichroism spectroscopy with an approximate apparent partition constant of 3×10(4) M(-1). Steady-state and time-resolved fluorescence measurements reveal distinct residue-specific behaviors with W54 experiencing the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements show a converse trend for relative Trp mobility with position 54 being the least immobile. To determine the relative insertion depths of W42, W54, and W65 in the bilayer, fluorescence quenching experiments were performed using three different brominated lipids. W65 had a clear preference for residing near the headgroup while W54 and W42 sample the range of depths ~8-11 Å from the bilayer center. On average, W54 is slightly more embedded than W42. Based on Trp spectral differences between ApoCIII binding to phospholipid vesicles and sodium dodecyl sulfate micelles, we suggest that ApoCIII adopts an alternate helical conformation on the bilayer which could have functional implications.
Asunto(s)
Apolipoproteína C-III/química , Fosfolípidos/química , Estructura Secundaria de Proteína , Triptófano/química , Algoritmos , Secuencia de Aminoácidos , Anisotropía , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Dicroismo Circular , Humanos , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfolípidos/metabolismo , Unión Proteica , Espectrometría de Fluorescencia , Triptófano/genética , Triptófano/metabolismoRESUMEN
Mutations in the GBA1 gene are associated with increased risk of Parkinson's disease, and the protein produced by the gene, glucocerebrosidase, interacts with α-synuclein, the protein at the center of the disease etiology. One possibility is that the mutations disrupt a beneficial interaction between the proteins, and a beneficial interaction would imply that the proteins have coevolved. To explore this possibility, a correlated mutation analysis has been performed for all 72 vertebrate species where complete sequences of α-synuclein and glucocerebrosidase are known. The most highly correlated pair of residue variations is α-synuclein A53T and glucocerebrosidase G115E. Intriguingly, the A53T mutation is a Parkinson's disease risk factor in humans, suggesting the pathology associated with this mutation and interaction with glucocerebrosidase might be connected. Correlations with ß-synuclein are also evaluated. To assess the impact of lowered species number on accuracy, intra and inter-chain correlations are also calculated for hemoglobin, using mutual information Z-value and direct coupling analyses.
Asunto(s)
Evolución Molecular , Glucosilceramidasa/genética , Modelos Genéticos , Mutación Missense , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Sustitución de Aminoácidos , HumanosRESUMEN
Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 µM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 µM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63-66 and 74-76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.
Asunto(s)
Enfermedad de Gaucher/enzimología , Glucosilceramidasa/química , Glucosilceramidasa/metabolismo , Enfermedad de Parkinson/enzimología , Saposinas/metabolismo , Dominio Catalítico , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Estabilidad Proteica , Saposinas/química , alfa-Sinucleína/metabolismoRESUMEN
Mutations in glucocerebrosidase (GCase), the enzyme deficient in Gaucher disease, are a common genetic risk factor for the development of Parkinson disease and related disorders, implicating the role of this lysosomal hydrolase in the disease etiology. A specific physical interaction exists between the Parkinson disease-related protein α-synuclein (α-syn) and GCase both in solution and on the lipid membrane, resulting in efficient enzyme inhibition. Here, neutron reflectometry was employed as a first direct structural characterization of GCase and α-syn·GCase complex on a sparsely-tethered lipid bilayer, revealing the orientation of the membrane-bound GCase. GCase binds to and partially inserts into the bilayer with its active site most likely lying just above the membrane-water interface. The interaction was further characterized by intrinsic Trp fluorescence, circular dichroism, and surface plasmon resonance spectroscopy. Both Trp fluorescence and neutron reflectometry results suggest a rearrangement of loops surrounding the catalytic site, where they extend into the hydrocarbon chain region of the outer leaflet. Taking advantage of contrasting neutron scattering length densities, the use of deuterated α-syn versus protiated GCase showed a large change in the membrane-bound structure of α-syn in the complex. We propose a model of α-syn·GCase on the membrane, providing structural insights into inhibition of GCase by α-syn. The interaction displaces GCase away from the membrane, possibly impeding substrate access and perturbing the active site. GCase greatly alters membrane-bound α-syn, moving helical residues away from the bilayer, which could impact the degradation of α-syn in the lysosome where these two proteins interact.
Asunto(s)
Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/ultraestructura , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/ultraestructura , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/patología , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Mutación , Difracción de Neutrones , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Unión Proteica , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , Triptófano/química , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
In this issue of Structure, Liu and colleagues describe an experimentally rigorous and innovative approach for understanding the role of membranes in the function and regulation of peripheral membrane proteins. This work is the beginning of a new era of experimental work that promises many advances relevant to molecular mechanisms and therapeutic targeting of this important class of proteins.
Asunto(s)
Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , HumanosRESUMEN
Mutations in GBA1, the gene for glucocerebrosidase (GCase), are genetic risk factors for Parkinson disease (PD). α-Synuclein (α-Syn), a protein implicated in PD, interacts with GCase and efficiently inhibits enzyme activity. GCase deficiency causes the lysosomal storage disorder Gaucher disease (GD). We show that saposin C (Sap C), a protein vital for GCase activity in vivo, protects GCase against α-syn inhibition. Using nuclear magnetic resonance spectroscopy, site-specific fluorescence, and Förster energy transfer probes, Sap C was observed to displace α-syn from GCase in solution and on lipid vesicles. Our results suggest that Sap C might play a crucial role in GD-related PD.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Glucosilceramidasa/metabolismo , Enfermedad de Parkinson/metabolismo , Saposinas/metabolismo , alfa-Sinucleína/metabolismo , Inhibidores Enzimáticos/química , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/química , Glucosilceramidasa/genética , Humanos , Cinética , Modelos Moleculares , Mutación , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Unión Proteica , Saposinas/química , Saposinas/genética , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Calmodulin (CaM) is a calcium binding protein that plays numerous roles in Ca-dependent cellular processes, including uptake and release of neurotransmitters in neurons. α-Synuclein (α-syn), one of the most abundant proteins in central nervous system neurons, helps maintain presynaptic vesicles containing neurotransmitters and moderates their Ca-dependent release into the synapse. Ca-Bound CaM interacts with α-syn most strongly at its N-terminus. The N-terminal region of α-syn is important for membrane binding; thus, CaM could modulate membrane association of α-syn in a Ca-dependent manner. In contrast, Ca-free CaM has negligible interaction. The interaction with CaM leads to significant signal broadening in both CaM and α-syn NMR spectra, most likely due to conformational exchange. The broadening is much reduced when binding a peptide consisting of the first 19 residues of α-syn. In neurons, most α-syn is acetylated at the N-terminus, and acetylation leads to a 10-fold increase in binding strength for the α-syn peptide (KD = 35 ± 10 µM). The N-terminally acetylated peptide adopts a helical structure at the N-terminus with the acetyl group contacting the N-terminal domain of CaM and with less ordered helical structure toward the C-terminus of the peptide contacting the CaM C-terminal domain. Comparison with known structures shows that the CaM/α-syn complex most closely resembles Ca-bound CaM in a complex with an IQ motif peptide. However, a search comparing the α-syn peptide sequence with known CaM targets, including IQ motifs, found no homologies; thus, the N-terminal α-syn CaM binding site appears to be a novel CaM target sequence.
Asunto(s)
Calmodulina/química , alfa-Sinucleína/química , Sitios de Unión , Calmodulina/metabolismo , Humanos , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , alfa-Sinucleína/metabolismoRESUMEN
Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.
Asunto(s)
Lipoilación/fisiología , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/metabolismo , Metionina/análogos & derivados , Animales , Metionina/química , Metionina/genética , Metionina/metabolismo , Metionina Sulfóxido Reductasas/genética , Ratones , Estructura Terciaria de ProteínaRESUMEN
Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.
Asunto(s)
Cisteína/química , Metionina Sulfóxido Reductasas/química , Animales , Catálisis , Dominio Catalítico , Cisteína/genética , Cisteína/metabolismo , Enlace de Hidrógeno , Metionina/química , Metionina/genética , Metionina/metabolismo , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Ratones , Oxidación-ReducciónRESUMEN
Brag2, a Sec7 domain (sec7d)-containing guanine nucleotide exchange factor, regulates cell adhesion and tumor cell invasion. Brag2 catalyzes nucleotide exchange, converting Arf·GDP to Arf·GTP. Brag2 contains a pleckstrin homology (PH) domain, and its nucleotide exchange activity is stimulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we determined kinetic parameters for Brag2 and examined the basis for regulation by phosphoinositides. Using myristoylated Arf1·GDP as a substrate, the k(cat) was 1.8 ± 0.1/s as determined by single turnover kinetics, and the K(m) was 0.20 ± 0.07 µm as determined by substrate saturation kinetics. PIP(2) decreased the K(m) and increased the k(cat) of the reaction. The effect of PIP(2) required the PH domain of Brag2 and the N terminus of Arf and was largely independent of Arf myristoylation. Structural analysis indicated that the linker between the sec7d and the PH domain in Brag2 may directly contact Arf. In support, we found that a Brag2 fragment containing the sec7d and the linker was more active than sec7d alone. We conclude that Brag2 is allosterically regulated by PIP(2) binding to the PH domain and that activity depends on the interdomain linker. Thus, the PH domain and the interdomain linker of Brag2 may be targets for selectively regulating the activity of Brag2.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Sitios de Unión/genética , Sitios de Unión/fisiología , Línea Celular , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19).
Asunto(s)
Histonas/química , Histonas/genética , Nucleosomas/química , Nucleosomas/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Cromatina/química , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Lisina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Pmel17 is a human amyloid involved in melanin synthesis. A fragment of Pmel17, the repeat domain (RPT) rich in glutamic acids, forms amyloid only at mildly acidic pH. Unlike pathological amyloids, these fibrils dissolve at neutral pH, supporting a reversible aggregation-disaggregation process. Here, we study RPT dissolution using atomic force microscopy and solution-state nuclear magnetic resonance spectroscopy. Our results reveal asymmetric fibril disassembly proceeding in the absence of intermediates. We suggest that fibril unfolding involves multiple deprotonation events resulting in electrostatic charge repulsion and filament dissolution.
Asunto(s)
Amiloide/química , Amiloide/ultraestructura , Antígeno gp100 del Melanoma/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Estructura Terciaria de ProteínaRESUMEN
The presynaptic protein α-synuclein (α-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between α-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that α-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the α-syn C-terminal residues, 118-137. This α-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 µm in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for α-syn, as does the inhibitor conduritol-ß-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the α-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.