Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition.

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S-adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.

Neutron diffraction studies towards deciphering the protonation state of catalytic residues in the bacterial KDN9P phosphatase.

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Šresolution using only neutron data showed significant density for catalytically important residues.

Ring1B contains a ubiquitin-like docking module for interaction with Cbx proteins.

Polycomb group (PcG) proteins are a special set of repressive transcription factors involved in epigenetic modifications of chromatin. They form two functionally distinct groups of catalytically active complexes: Polycomb repressive complex 1 (PRC1) and 2 (PRC2). The PRC1 complex is an important yet poorly characterized multiprotein histone ubiquitylation machine responsible for maintaining transcriptionally silent states of genes through histone H2A K119 modification. The Ring domain containing subunits of PRC1 also have substrate-targeting domains that interact with Cbx proteins, which have been implicated in chromatin and RNA binding. In this work, we present a high resolution structure of the C-terminal domain of Ring1B, revealing a variant ubiquitin-like fold with a distinct conserved surface region. On the basis of crystal structure and mutational analysis of this domain we show that the conserved surface is responsible for interaction with Cbx members of the PRC1 and homodimer formation. These data suggest a mechanism by which Ring1B serves as an adaptor that mediates binding between the members of the PRC1 complex and the nucleosome.

Rapid evolution and gene-specific patterns of selection for three genes of spermatogenesis in Drosophila.

Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. We analyzed the molecular evolution of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and D. simulans and a phylogenetic comparative analysis among species confirms evidence of positive selection driving the evolution of Bam and suggests an yet unknown germ cell line developmental adaptive change between these two species.