Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC

7 pags, 3 figs, 1 tabThe first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division. © 2010 Nature America, Inc. All rights reserved.This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711, SAF2006-00390), EU-CP223111 (CAREPNEUMO, European Union), the COMBACT program (S-BIO-0260/2006) and CIBER de Enfermedades Respiratorias (CIBERES). CIBERES is an initiative of Instituto de Salud Carlos III. The work in the US was supported by the US National Institutes of Health. W.V. was supported by the European Commission (EUR-INTAFAR project). I.P.-D. was a fellow of the Consejo Superior de Investigaciones Científicas

Building a molecular machine : heterologous expression and biochemical characterisation of transcription factor IIH

Organisms across Archaea, Bacteria and Eukarya possess a series of mechanisms to protect the integrity of the deoxyribonucleic acid (DNA) from the constant attack of internal and external factors that modify the structure of the double helix, potentially causing an increase in mutagenesis. The Nucleotide Excision Repair (NER) pathway removes bulky lesions in the DNA caused by ultraviolet (UV) radiation and other sources that can introduce a strong distortion in the double helix. A key element in this repair mechanism is the transcription factor IIH (TFIIH), a ten-subunit complex which opens and extends the DNA bubble originated at the damaged site to allow the subsequent repair factors access to the lesion, so the insult can be removed. Mutations in TFIIH subunits xeroderma pigmentosum group B (XPB), xeroderma pigmentosum group D (XPD) or p8 can cause a series of autosomal recessive disorders with symptoms that include mild-to-extreme photosensitivity, progeria, physical and neurological abnormalities, and in some cases an increased susceptibility to cancer. This thesis describes the processes of cloning, expression and purification followed to obtain damage-detector heterodimer XPC-HR23B, the 7-subunit TFIIH Core sub-complex and the 10-subunit TFIIH complex by means of a powerful baculoviral expression vector (BEV) called MultiBacTM, a tool specifically conceived for the obtaining of multi-subunit eukaryotic complexes. The biochemical characterization of TFIIH Core showed that the sub-complex can bind a series of double-stranded DNA (dsDNA) substrates with an affinity in the nM range, and the presence of a bubble or damage at the duplex does not change TFIIH Core’s binding affinity significantly. The study of TFIIH Core’s unwinding ability confirmed that the sub-complex can open a series of dsDNA substrates only when a 5’ overhang end is available, and this activity can be stalled by a lesion located in both the translocating and non-translocating strands."I am very grateful to the School of Biology, EastBio and the Biotechnology and Biological Sciences Research Council (BBSRC) for supporting this work [grant number BB/J01446X/1]" -- Acknowledgement

Facile and scalable expression and purification of transcription factor IIH (TFIIH) core complex

This work was supported by the BBSRC [grant number BB/J01446X/1] and [grant number BB/R015570/1]Transcription factor IIH (TFIIH) plays essential roles in both the initiation of RNA Polymerase II-mediated transcription and the Nucleotide Excision Repair (NER) pathway in eukaryotes. In NER, the 7-subunit TFIIH Core sub-complex is responsible for the opening and extension of the DNA bubble created at the lesion site, utilizing the molecular motors XPB and XPD. Mutations in Core subunits are associated with a series of severe autosomal recessive disorders characterised by symptoms such as mild-to-extreme photosensitivity, premature ageing, physical and neurological anomalies, and in some cases an increased susceptibility to cancer. Although TFIIH Core has been successfully obtained in the past, the process has always remained challenging and laborious, involving many steps that severely hindered the amount of pure, active complex obtained. This has limited biochemical and functional studies of the NER process. Here we describe improved and simplified processes for the cloning, expression and purification of the 7-subunit TFIIH Core sub-complex. The combined use of auto-cleavable 2A-like sequences derived from the Foot-and-Mouth Disease Virus (FMDV) and the MultiBac™ cloning system, a powerful baculoviral expression vector specifically conceived for the obtaining of multi-subunit eukaryotic complexes, allowed us to obtain a single, 7-gene plasmid in a short time using regular restriction cloning strategies. Additionally, expression of the construct in High Five™ insect cells paired with a simple 5-step purification protocol allowed the extraction of a pure, active TFIIH Core sub-complex in milligram quantities.PostprintPeer reviewe

Crystallization of the pneumococcal autolysin LytC: in-house phasing using novel lanthanide complexes

The complete pneumococcal autolysin LytC has been crystallized by the hanging-drop vapor-diffusion method. A SAD data set has been collected in-house from a Gd derivative up to 2.6 Å resolution