RESUMO
Mutations in the protein dysferlin, a member of the ferlin family, lead to limb girdle muscular dystrophy type 2B and Myoshi myopathy. The ferlins are large proteins characterised by multiple C2 domains and a single C-terminal membrane-spanning helix. However, there is sequence conservation in some of the ferlin family in regions outside the C2 domains. In one annotation of the domain structure of these proteins, an unusual internal duplication event has been noted where a putative domain is inserted in between the N- and C-terminal parts of a homologous domain. This domain is known as the DysF domain. Here, we present the solution structure of the inner DysF domain of the dysferlin paralogue myoferlin, which has a unique fold held together by stacking of arginine and tryptophans, mutations that lead to clinical disease in dysferlin.
Assuntos
Proteínas de Membrana/química , Proteínas Musculares/química , Distrofia Muscular do Cíngulo dos Membros/etiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Ligação ao Cálcio , Disferlina , Humanos , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/classificação , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade Estática , TermodinâmicaRESUMO
UvrB is the damage recognition element of the highly conserved UvrABC pathway that functions in the removal of bulky DNA adducts. Pivotal to this is the formation of a damage detection complex that relies on the ability of UvrB to locate and sequester diverse lesions. Whilst structures of UvrB bound to DNA have recently been reported, none address the issue of lesion recognition. Here, we describe the crystal structure of UvrB bound to a pentanucleotide containing a single fluorescein-adducted thymine that reveals a unique mechanism for damage detection entirely dependent on the exclusion of lesions larger than an undamaged nucleotide.
Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , Oligodesoxirribonucleotídeos/metabolismo , Cristalografia por Raios X/métodos , Adutos de DNA/química , Adutos de DNA/metabolismo , DNA Helicases/química , Endodesoxirribonucleases/química , Proteínas de Escherichia coli/química , Oligodesoxirribonucleotídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The UvrABC pathway is a ubiquitously occurring mechanism targeted towards the repair of bulky base damage. Key to this process is UvrB, a DNA-dependent limited helicase that acts as a lesion recognition element whilst part of a tracking complex involving UvrA, and as a DNA-binding platform required for the presentation of damage to UvrC for subsequent processing. We have been able to determine the structure of a ternary complex involving UvrB* (a C-terminal truncation of full-length UvrB), a polythymine trinucleotide and ADP. This structure has highlighted the roles of key conserved residues in DNA binding distinct from those of the beta-hairpin, where most of the attention in previous studies has been focussed. We are also the first to report the structural basis underlying conformational re-modelling of the beta-hairpin that is absolutely required for DNA binding and how this event results in an ATPase primed for catalysis. Our data provide the first insights at the molecular level into the transformation of UvrB into an active helicase.
Assuntos
Adenosina Trifosfatases/química , Bacillus subtilis/enzimologia , Bacillus/enzimologia , Proteínas de Bactérias/química , DNA Helicases/química , Estrutura Terciária de Proteína , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Bacillus/genética , Bacillus subtilis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA(A) receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 A resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the gamma2 subunit of the GABA(A) receptor and the acidic carboxyl-terminal tails of human alpha- and beta-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.