ABSTRACT
ß barrel outer membrane proteins (ß-OMPs) play vital roles in mitochondria, chloroplasts, and Gram-negative bacteria. Evolutionarily conserved complexes such as the mitochondrial sorting and assembly machinery (SAM) mediate the assembly of ß-OMPs. We investigated the SAM-mediated assembly of the translocase of the outer membrane (TOM) core complex. Cryoelectron microscopy structures of SAMfully folded Tom40 and the SAM-Tom40/Tom5/Tom6 complexes at ~3-angstrom resolution reveal that Sam37 stabilizes the mature Tom40 mainly through electrostatic interactions, thus facilitating subsequent TOM assembly. These results support the ß barrel switching model and provide structural insights into the assembly and release of ß barrel complexes.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Cell Line , Cryoelectron Microscopy , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Models, Molecular , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/metabolism , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Static ElectricitySubject(s)
Cryoelectron Microscopy/methods , Mitochondrial Precursor Protein Import Complex Proteins/chemistry , Carrier Proteins/metabolism , HEK293 Cells , Humans , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins/metabolism , Molecular Chaperones/metabolism , Protein Conformation, alpha-Helical , Protein DomainsABSTRACT
Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.
Subject(s)
Arabidopsis/enzymology , Chloroplasts/enzymology , DNA, Cruciform/metabolism , Glycine max/enzymology , Oryza/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Recombinases/chemistry , Recombinases/metabolism , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/metabolism , Base Sequence , Chloroplasts/chemistry , Chloroplasts/genetics , DNA, Cruciform/chemistry , DNA, Cruciform/genetics , DNA, Plant/chemistry , DNA, Plant/genetics , DNA, Plant/metabolism , Oryza/chemistry , Oryza/genetics , Oryza/metabolism , Recombinases/genetics , Glycine max/chemistry , Glycine max/genetics , Glycine max/metabolismABSTRACT
Members of the pentatricopeptide repeat (PPR) protein family are sequence-specific RNA-binding proteins that play crucial roles in organelle RNA metabolism. Each PPR protein consists of a tandem array of PPR motifs, each of which aligns to one nucleotide of the RNA target. The di-residues in the PPR motif, which are referred to as the PPR codes, determine nucleotide specificity. Numerous PPR codes are distributed among the vast number of PPR motifs, but the correlation between PPR codes and RNA bases is poorly understood, which hinders target RNA prediction and functional investigation of PPR proteins. To address this issue, we developed a modular assembly method for high-throughput construction of designer PPRs, and by using this method, 62 designer PPR proteins containing various PPR codes were assembled. Then, the correlation between these PPR codes and RNA bases was systematically explored and delineated. Based on this correlation, the web server PPRCODE (http://yinlab.hzau.edu.cn/pprcode) was developed. Our study will not only serve as a platform for facilitating target RNA prediction and functional investigation of the large number of PPR family proteins but also provide an alternative strategy for the assembly of custom PPRs that can potentially be used for plant organelle RNA manipulation.
