The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that plays a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and the type 1 transmembrane domain protein, receptor activity modifying protein (RAMP) 1. Herein, we report the 3.3 Å structure of the human CGRP receptor in complex with CGRP and the Gs40 protein heterotrimer determined by Volta phase plate cryo-electron microscopy. The RAMP transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilises CLR extracellular loop 2. RAMP1 makes only limited direct interaction with CGRP, consistent with allosteric modulation of CLR as its key function. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly the location of the CLR extracellular domain. The work provides novel insight into the control of G-protein-coupled receptor function

The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß–ß–a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated

Protein nanorings organized by poly(styrene-block-ethylene oxide) self-assembled thin films

This study explores the use of block copolymer self-assembly to organize Lsmα, a protein which forms stable doughnut-shaped heptameric structures. Here, we have explored the idea that 2-D crystalline arrays of protein filaments can be prepared by stacking doughnut shaped Lsmα protein into the poly(ethylene oxide) blocks of a hexagonal microphase-separated polystyrene-b-polyethylene oxide (PS-b-PEO) block copolymer. We were able to demonstrate the coordinated assembly of such a complex hierarchical nanostructure. The key to success was the choice of solvent systems and protein functionalization that achieved sufficient compatibility whilst still promoting assembly. Unambiguous characterisation of these structures is difficult; however AFM and TEM measurements confirmed that the protein was sequestered into the PEO blocks. The use of a protein that assembles into stackable doughnuts offers the possibility of assembling nanoscale optical, magnetic and electronic structures

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable

Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology

\u3cp\u3eThe potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology.\u3c/p\u3

Structures of human peroxiredoxin 3 suggest self-chaperoning assembly that maintains catalytic state

\u3cp\u3ePeroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides in cells. On exposure to various cellular stresses, peroxiredoxins can acquire chaperone activity, manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-Å resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-Å crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress.\u3c/p\u3

A model for chromosomal DNA organization suggested by hexagonal crystal packing.

<p>(A) The N-terminal Lsr2 dimerization domain in the <i>P</i>3₁21 space group in two orientations; (B) Proposed orientation of DNA (in blue) between N-terminal chains and a 90° rotation illustrating this from above. The C-terminal DNA-binding domains (in orange) have been positioned according to Gordon <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038542#pone.0038542-Gordon3" target="_blank">[22]</a>; (C) Our model of full-length Lsr2 binding to DNA and forming chains cross-linking multiple strands of DNA. The dimension at the bottom of the figure is consistent with the compact fibrils seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038542#pone-0038542-g006" target="_blank">Figure 6B</a>.</p