RESUMO
X-ray crystallography provides excellent structural data on protein-DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein-DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein-DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG-DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210-220 and 251-264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG-DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Uracila-DNA Glicosidase/química , Algoritmos , Domínio Catalítico , Biologia Computacional/métodos , Cristalografia por Raios X , Medição da Troca de Deutério , Espectrometria de Massas/métodos , Modelos MolecularesRESUMO
Escherichia coli MutS forms a mispair-dependent ternary complex with MutL that is essential for initiating mismatch repair (MMR) but is structurally uncharacterized, in part owing to its dynamic nature. Here, we used hydrogen/deuterium exchange mass spectrometry and other methods to identify a region in the connector domain (domain II) of MutS that binds MutL and is required for mispair-dependent ternary complex formation and MMR. A structurally conserved region in Msh2, the eukaryotic homolog, was required for formation of a mispair-dependent Msh2-Msh6-Mlh1-Pms1 ternary complex. These data indicate that the connector domain of MutS and Msh2 contains the interface for binding MutL and Mlh1-Pms1, respectively, and support a mechanism whereby mispair and ATP binding induces a conformational change that allows the MutS and Msh2 interfaces to interact with their partners.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Modelos Moleculares , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SolventesRESUMO
We have performed deuterium exchange mass spectrometry (DXMS) to probe the conformational changes that the bacterial MutS homodimer and the homologous eukaryotic heterodimer Msh2-Msh6 undergo when binding to ATP or DNA. The DXMS data support the view that high affinity binding to mispair-containing DNA and low affinity binding to fully base-paired DNA both involve forming rings by MutS protein family dimers around the DNA; however, mispair binding protects additional regions from deuterium exchange. DXMS also reveals two distinct conformations upon binding one or two ATP molecules and that binding of two ATP molecules propagates conformational changes to other regions of the protein complexes. The regions showing major changes in deuterium exchange upon ATP binding tend to occur in regions distinct from those involved in DNA binding, suggesting that although communication occurs between DNA and nucleotide binding, sliding clamps formed by binding both ATP and mispairs could result from the simultaneous action of two independent conformational changes.