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1.
Trends Biochem Sci ; 32(2): 63-70, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17210253

RESUMEN

Zinc-fingers (ZnFs) are extremely abundant in higher eukaryotes. Once considered to function exclusively as sequence-specific DNA-binding motifs, ZnFs are now known to have additional activities such as the recognition of RNA and other proteins. Here we discuss recent advances in our understanding of ZnFs as specific modules for protein recognition. Structural studies of ZnF complexes reveal considerable diversity in terms of protein partners, binding modes and affinities, and highlight the often underestimated versatility of ZnF structure and function. An appreciation of the structural features of ZnF-protein interactions will contribute to our ability to engineer and to use ZnFs with tailored protein-binding properties.


Asunto(s)
Secuencias de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Dedos de Zinc/fisiología , Animales , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína
2.
J Mol Biol ; 366(2): 382-90, 2007 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-17174978

RESUMEN

The THAP (Thanatos-associated protein) domain is a recently discovered zinc-binding domain found in proteins involved in transcriptional regulation, cell-cycle control, apoptosis and chromatin modification. It contains a single zinc atom ligated by cysteine and histidine residues within a Cys-X(2-4)-Cys-X(35-53)-Cys-X(2)-His consensus. We have determined the NMR solution structure of the THAP domain from Caenorhabditis elegans C-terminal binding protein (CtBP) and show that it adopts a fold containing a treble clef motif, bearing similarity to the zinc finger-associated domain (ZAD) from Drosophila Grauzone. The CtBP THAP domain contains a large, positively charged surface patch and we demonstrate that this domain can bind to double-stranded DNA in an electrophoretic mobility-shift assay. These data, together with existing reports, indicate that THAP domains might exhibit a functional diversity similar to that observed for classical and GATA-type zinc fingers.


Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Unión al ADN/química , Oxidorreductasas de Alcohol/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Relación Estructura-Actividad
3.
Structure ; 13(2): 257-66, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15698569

RESUMEN

Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.


Asunto(s)
Alanina/genética , Proteínas Portadoras/química , Mutagénesis , Zinc/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Molecular , Mutación/genética , Péptidos/química , Péptidos/genética , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Soluciones/química
4.
Sci Rep ; 6: 19352, 2016 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-26785754

RESUMEN

Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.


Asunto(s)
Expresión Génica , Multimerización de Proteína , Canales Catiónicos TRPM/química , Canales Catiónicos TRPM/genética , Proteínas Fluorescentes Verdes , Humanos , Liposomas/química , Imagen Molecular , Transporte de Proteínas , Proteolisis , Proteínas Recombinantes de Fusión , Canales Catiónicos TRPM/metabolismo
5.
Sci Signal ; 9(423): ra36, 2016 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-27072655

RESUMEN

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.


Asunto(s)
Metaloproteasas/metabolismo , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas ADAM/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células HEK293 , Humanos , Immunoblotting , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas Mutantes/genética , Proteolisis , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Homología de Secuencia de Aminoácido
6.
FEBS Lett ; 571(1-3): 221-6, 2004 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-15280046

RESUMEN

Surface proteins in Gram-positive bacteria are anchored to the cell wall by the action of sortase enzymes. The Staphylococcus aureus sortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG. To understand how SrtA binds this sequence, we carried out NMR studies of new peptidyl-cyanoalkene and peptidyl-sulfhydryl inhibitors that contain the sorting signal sequence LPAT. These studies combined with amino acid mutagenesis identified a catalytically important and conserved binding surface formed by residues A118, T180, and I182. Compatible with its recently proposed role as a general base, R197 is also shown to be required for catalysis.


Asunto(s)
Aminoaciltransferasas/química , Aminoaciltransferasas/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Aminoaciltransferasas/genética , Proteínas Bacterianas , Sitios de Unión , Pared Celular/enzimología , Cisteína Endopeptidasas , Inhibidores Enzimáticos/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Transducción de Señal , Staphylococcus aureus/genética
7.
Protein Sci ; 21(9): 1376-87, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22811351

RESUMEN

Cardiac troponin C (cTnC) is the calcium binding subunit of the troponin complex that triggers the thin filament response to calcium influx into the sarcomere. cTnC consists of two globular EF-hand domains (termed the N- and C-domains) connected by a flexible linker. While the conformation of each domain of cTnC has been thoroughly characterized through NMR studies involving either the isolated N-domain (N-cTnC) or C-domain (C-cTnC), little attention has been paid to the range of interdomain orientations possible in full-length cTnC that arises as a consequence of the flexibility of the domain linker. Flexibility in the domain linker of cTnC is essential for effective regulatory function of troponin. We have therefore utilized paramagnetic relaxation enhancement (PRE) NMR to assess the interdomain orientation of cTnC. Ensemble fitting of our interdomain PRE measurements reveals that isolated cTnC has considerable interdomain flexibility and preferentially adopts a bent conformation in solution, with a defined range of relative domain orientations.


Asunto(s)
Miocardio/química , Resonancia Magnética Nuclear Biomolecular , Troponina C/química , Animales , Cisteína/química , Cisteína/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Miocardio/metabolismo , Estructura Terciaria de Proteína , Ratas , Troponina C/genética
8.
J Biol Chem ; 284(36): 24465-77, 2009 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-19592495

RESUMEN

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.


Asunto(s)
Aminoaciltransferasas/química , Proteínas Bacterianas/química , Cisteína Endopeptidasas/química , Modelos Químicos , Modelos Moleculares , Señales de Clasificación de Proteína , Staphylococcus aureus/enzimología , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico/fisiología , Cisteína Endopeptidasas/metabolismo , Mapeo Peptídico/métodos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína/fisiología , Staphylococcus aureus/patogenicidad
9.
Protein Sci ; 17(9): 1630-5, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18556474

RESUMEN

Glutathione-S-transferase (GST)-fusion proteins are used extensively for structural, biochemical, and functional analyses. Although the conformation of the target protein is of critical importance, confirmation of the folded state of the target is often not undertaken or is cumbersome because of the requirement to first remove the GST tag. Here, we demonstrate that it is possible to record conventional (15)N-HSQC NMR spectra of small GST-fusion proteins and that the observed signals arise almost exclusively from the target protein. This approach constitutes a rapid and straightforward means of assessing the conformation of a GST-fusion protein without having to cleave the GST and should prove valuable, both to biochemists seeking to check the conformation of their proteins prior to functional studies and to structural biologists screening protein constructs for suitability as targets for structural studies.


Asunto(s)
Estudios de Evaluación como Asunto , Glutatión Transferasa/química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Animales , Tampones (Química) , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Dimerización , Glutatión Transferasa/aislamiento & purificación , Peso Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Factores de Tiempo
10.
J Biol Chem ; 281(3): 1817-26, 2006 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-16269411

RESUMEN

Many virulence factors in gram-positive bacteria are covalently anchored to the cell-wall peptidoglycan by sortase enzymes, a group of widely distributed cysteine transpeptidases. The Staphylococcus aureus Sortase A protein (SrtA) is the archetypal member of the Sortase family and is activated by Ca2+, an adaptation that may facilitate host colonization as elevated concentrations of this ion are encountered in human tissue. Here we show that a single Ca2+ ion bound to an ordered pocket on SrtA allosterically activates catalysis by modulating both the structure and dynamics of a large active site loop. Detailed nitrogen-15 relaxation measurements indicate that Ca2+ may facilitate the adaptive recognition of the substrate by inducing slow micro- to millisecond time-scale dynamics in the active site. Interestingly, relaxation compensated Carr-Purcell-Meiboom-Gill experiments suggest that the time scale of these motions is directly correlated with ion binding. The results of site-directed mutagenesis indicate that this motional coupling is mediated by the side chain of Glu-171, which is positioned within the beta6/beta7 loop and shown to contribute to Ca2+ binding. The available structural and dynamics data are compatible with a loop closure model of Ca2+ activation, in which the beta6/beta7 loop fluctuates between a binding competent closed form that is stabilized by Ca2+, and an open, highly flexible state that removes key substrate contacting residues from the active site.


Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Calcio/fisiología , Peptidil Transferasas/metabolismo , Staphylococcus aureus/enzimología , Regulación Alostérica , Sustitución de Aminoácidos , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cisteína Endopeptidasas , Cartilla de ADN , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Peptidil Transferasas/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Virulencia
11.
Bioorg Med Chem Lett ; 15(22): 5076-9, 2005 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-16169722

RESUMEN

L-Threonine 2 was converted in seven steps into the protected aminomercaptoalcohol 8, a threonine mimic. This compound 8 was coupled to various oligopeptides to produce two different tetrapeptide analogues, for example, 11 and 17, which were shown to inhibit the Sortase enzymes (SrtA and SrtB) via covalent attachment of the thiol groups of 11 and 17 to the catalytically active cysteine residue of the Sortase enzymes.


Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Butanoles/síntesis química , Butanoles/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Treonina/análogos & derivados , Butanoles/química , Cromatografía Líquida de Alta Presión , Cisteína Endopeptidasas , Inhibidores Enzimáticos/química , Estructura Molecular , Treonina/química
12.
Proc Natl Acad Sci U S A ; 102(3): 583-8, 2005 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-15644435

RESUMEN

GATA-1 and friend of GATA (FOG) are zinc-finger transcription factors that physically interact to play essential roles in erythroid and megakaryocytic development. Several naturally occurring mutations in the GATA-1 gene that alter the FOG-binding domain have been reported. The mutations are associated with familial anemias and thrombocytopenias of differing severity. To elucidate the molecular basis for the GATA-1/FOG interaction, we have determined the three-dimensional structure of a complex comprising the interaction domains of these proteins. The structure reveals how zinc fingers can act as protein recognition motifs. Details of the architecture of the contact domains and their physical properties provide a molecular explanation for how the GATA-1 mutations contribute to distinct but related genetic diseases.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Unión al ADN/química , Proteínas Nucleares/química , Factores de Transcripción/química , Dedos de Zinc , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Enfermedades Hematológicas/tratamiento farmacológico , Enfermedades Hematológicas/genética , Humanos , Modelos Moleculares , Estructura Molecular , Mutación/fisiología , Proteínas Nucleares/metabolismo , Unión Proteica/genética , Conformación Proteica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
13.
J Biol Chem ; 277(38): 35720-9, 2002 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-12110675

RESUMEN

The N-terminal zinc finger (ZnF) from GATA transcription factors mediates interactions with FOG family proteins. In FOG proteins, the interacting domains are also ZnFs; these domains are related to classical CCHH fingers but have an His --> Cys substitution at the final zinc-ligating position. Here we demonstrate that different CCHC fingers in the FOG family protein U-shaped contact the N-terminal ZnF of GATA-1 in the same fashion although with different affinities. We also show that these interactions are of moderate affinity, which is interesting given the presumed low concentrations of these proteins in the nucleus. Furthermore, we demonstrate that the variant CCHC topology enhances binding affinity, although the His --> Cys change is not essential for the formation of a stably folded domain. To ascertain the structural basis for the contribution of the CCHC arrangement, we have determined the structure of a CCHH mutant of finger nine from U-shaped. The structure is very similar overall to the wild-type domain, with subtle differences at the C terminus that result in loss of the interaction in vivo. Taken together, these results suggest that the CCHC zinc binding topology is required for the integrity of GATA-FOG interactions and that weak interactions can play important roles in vivo.


Asunto(s)
Secuencia Conservada , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Dedos de Zinc
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