RESUMO
Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.
Assuntos
Actinobacteria/enzimologia , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes/métodos , Actinobacteria/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cristalografia por Raios X , Citosina , DNA/química , DNA/genética , DNA/metabolismo , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Metilação , Modelos Moleculares , Conformação Proteica , RNA Guia de Cinetoplastídeos/químicaRESUMO
Interpretation of EPR measurables from spin labels in terms of structure and dynamics requires knowledge of label behavior. General strategies were developed for simulation of labels used in EPR of proteins. The criteria for those simulations are (a) exhaustive sampling of rotamer space, (b) consensus of results independent of starting points, and (c) inclusion of entropy. These criteria are satisfied only when the number of transitions in any dihedral angle exceeds 100 and the simulation maintains thermodynamic equilibrium. Methods such as conventional MD do not efficiently cross energetic barriers, simulated anealing, Monte Carlo or popular Rotamer Library methodologies are potential energy based and ignore entropy (in addition to their specific shortcomings: environment fluctuations, fixed environment, or electrostatics). The Simulated scaling method avoids the above flaws by modulating the force fields between a reduced (allowing crossing energy barriers) and full potential (sampling minima). Spin label diffuses on this surface while remaining in thermodynamic equilibrium. Simulations show that (a) adopting a single conformation is rare, often there are two to four populated rotamers and (b) position of the NO varies up to 16 Å. These results illustrate necessity for caution when interpreting EPR signals in terms of molecular structure. For example, the 10-16 Å distance change in DEER should not be interpreted as a large conformational change, it can well be a flip about Cα-Cß bond. Rigorous exploration of possible rotamer structures of a spin label is paramount in signal interpretation. We advocate use of bifunctional labels, motion of which is restricted 10,000-fold and the NO position is restricted to 2-5 Å.