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1.
Nucleic Acids Res ; 45(17): 10321-10331, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973475

RESUMO

Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2'-deoxyribose, 2'-O-methyl-ribose, 2'-deoxy-2'-fluoro-ribose, arabinose and 2'-deoxy-2'-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2' modifications gave a variety of effects. Arabinose and 2'-deoxy-2'-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2'-O-methyl and 2'-deoxy-2'-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability.


Assuntos
Pentoses/química , RNA de Cadeia Dupla/química , RNA Mensageiro/química , Pareamento de Bases , Cristalografia por Raios X , Desoxirribose/química , Ligação de Hidrogênio , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Estabilidade de RNA , Temperatura , Água
2.
Org Biomol Chem ; 15(39): 8361-8370, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-28937154

RESUMO

DNA interstrand cross-links (ICL) are among the most cytotoxic lesions found in biological systems. O6-Alkylguanine DNA alkyltransferases (AGTs) are capable of removing alkylation damage from the O6-atom of 2'-deoxyguanosine and the O4-atom of thymidine. Human AGT (hAGT) has demonstrated the ability to repair an interstrand cross-linked duplex where two O6-atoms of 2'-deoxyguanosine were tethered by a butylene (XLGG4) or heptylene (XLGG7) linkage. However, the analogous ICL between the O4-atoms of thymidine was found to evade repair. ICL duplexes connecting the O4-atoms of 2'-deoxyuridine by a butylene (XLUU4) or heptylene (XLUU7) linkage have been prepared to examine the influence of the C5-methyl group on AGT-mediated repair. Both XLUU4 and XLUU7 were refractory to repair by human and E. coli (OGT and Ada-C) AGTs with comparably low µM dissociation constants for 2 : 1 or 4 : 1 AGT/DNA stoichiometries. The solution structures of two heptylene linked DNA duplexes (CGAAAYTTTCG)2, XLUU7 (Y = dU) and XLGG7 (Y = dG), were solved and the global structures were virtually identical with a RMSD of 1.22 Å. The ICL was found to reside in the major groove for both duplexes. The linkage adopts an E conformation about the C4-O4 bond for XLUU7 whereas a Z conformation about the C6-O6 bond was observed for XLGG7. This E versus Z conformation may partially account for hAGTs discrimination towards the repair of these ICL, supported by the crystal structures of hAGT with various substrates which have been observed to adopt a Z conformation. In addition, a higher mobility at the ICL site for XLUU7 is observed relative to XLGG7 that may play a role in repair by hAGT. Taken together, these findings provide insights on the AGT-mediated repair of cytotoxic ICL in terms of its processing capability and substrate specificity.


Assuntos
Alquil e Aril Transferases/metabolismo , Reparo do DNA , DNA/química , DNA/genética , Pareamento de Bases , DNA/metabolismo , Humanos , Modelos Moleculares
3.
Mol Cell Proteomics ; 11(9): 710-23, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22665516

RESUMO

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.


Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Mapas de Interação de Proteínas , Animais , Ciclofilinas/metabolismo , Células Epiteliais , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunoglobulina G/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Peptidilprolil Isomerase/metabolismo , Ratos
4.
J Biol Chem ; 286(23): 20407-12, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21507954

RESUMO

Sacsin is a 520-kDa protein mutated in the early-onset neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The C terminus of the protein contains an HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain of unknown function. Here, we determined the high-resolution 1.9-Å crystal structure of the HEPN domain from human sacsin. The structure is composed of five parallel α-helices with a large loop of several short helical segments. Two HEPN protomers assemble as a dimer to form a large positively charged cavity at the dimer interface that binds GTP and other nucleotides. The crystal structure reveals that the ARSACS N4549D mutation disrupts dimerization and protein folding. This study provides novel insights into the oligomerization state of sacsin and functions that are lost in mutations that cause ARSACS.


Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Mutação de Sentido Incorreto , Multimerização Proteica/genética , Substituição de Aminoácidos , Cristalografia por Raios X , Proteínas de Choque Térmico/metabolismo , Humanos , Espasticidade Muscular/genética , Espasticidade Muscular/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Relação Estrutura-Atividade
5.
J Biomol NMR ; 47(3): 195-204, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20461447

RESUMO

Cellulose nanocrystals (CNCs) form liquid crystals in aqueous solution that confer alignment to macromolecules and permit the measurement of residual dipolar couplings. CNCs possess many attractive features as an alignment medium. They are inexpensive, non-toxic, chemically inert, and robust to denaturants and temperature. Despite these advantages, CNCs are seldom employed as an alignment medium and the range of their applicability has not yet been explored. We have re-examined the use of CNCs in biomolecular NMR by analyzing the effects concentration, ionic strength, and temperature on molecular alignment. Stable alignment was obtained over wide ranges of temperature (10-70 degrees C) and pH (2.5-8.0), which makes CNCs potentially very useful in studies of thermophilic proteins and acid-stabilized molecules. Notably, we find that CNC suspensions are very sensitive to the concentrations of biological buffers, which must be taken into account when they are used in NMR analyses. These results have led us to develop a general procedure for preparing aligned samples with CNCs. Using the SH3 domain from the Fyn tyrosine kinase as a model system, we find that CNCs produce an alignment frame collinear with that of the commonly used Pf1 bacteriophage alignment medium, but of opposite magnitude.


Assuntos
Celulose/química , Nanopartículas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Animais , Galinhas , Detergentes/química , Concentração de Íons de Hidrogênio , Isótopos de Nitrogênio/química , Proteínas Proto-Oncogênicas c-fyn/química , Água/química , Domínios de Homologia de src
6.
FEBS J ; 274(6): 1600-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17480208

RESUMO

Regeneration-induced CNPase homolog (RICH) is an axonal growth-associated protein, which is induced in teleost fish upon optical nerve injury. RICH consists of a highly acidic N-terminal domain, a catalytic domain with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity and a C-terminal isoprenylation site. In vitro RICH and mammalian brain CNPase specifically catalyze the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains unknown. Here, we report the NMR structure of the catalytic domain of goldfish RICH and describe its binding to CNPase inhibitors. The structure consists of a twisted nine-stranded antiparallel beta-sheet surrounded by alpha-helices on both sides. Despite significant local differences mostly arising from a seven-residue insert in the RICH sequence, the active site region is highly similar to that of human CNPase. Likewise, refinement of the catalytic domain of rat CNPase using residual dipolar couplings gave improved agreement with the published crystal structure. NMR titrations of RICH with inhibitors point to a similar catalytic mechanism for RICH and CNPase. The results suggest a functional importance for the evolutionarily conserved phosphodiesterase activity and hint of a link with pre-tRNA splicing.


Assuntos
Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Carpa Dourada , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Homologia de Sequência de Aminoácidos
7.
ChemMedChem ; 9(9): 2099-103, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24931822

RESUMO

The bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross-links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2'-deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high-field NMR experiments. The ethylene linker is well-accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies.


Assuntos
Antineoplásicos Alquilantes/síntese química , Antineoplásicos Alquilantes/farmacologia , Carmustina/farmacologia , DNA/química , DNA/farmacologia , Etilenos/química , Reagentes de Ligações Cruzadas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Termodinâmica
8.
FEBS J ; 276(5): 1440-9, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19187238

RESUMO

Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin-like domains (abb'a'). Cross-linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b'. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b', respectively) by triple-resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b' domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb' fragment. Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substrate-binding site.


Assuntos
Isomerases de Dissulfetos de Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Células Epiteliais/enzimologia , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Soluções
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