Chromatin organization in relation to the nuclear periphery

AbstractIn the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery

Fusidic acid resistance through changes in the dynamics of the drug target

Antibiotic resistance in clinically important bacteria can be mediated by target protection mechanisms, whereby a protein binds to the drug target and protects it from the inhibitory effects of the antibiotic. The most prevalent source of clinical resistance to the antibiotic fusidic acid (FA) is expression of the FusB family of proteins that bind to the drug target (Elongation factor G [EF-G]) and promote dissociation of EF-G from FA-stalled ribosome complexes. FusB binding causes changes in both the structure and conformational flexibility of EF-G, but which of these changes drives FA resistance was not understood. We present here detailed characterization of changes in the conformational flexibility of EF-G in response to FusB binding and showthat these changes are responsible for conferring FA resistance. Binding of FusB to EF-G causes a significant change in the dynamics of domain III of EF-GC3 that leads to an increase in a minor, more disordered state of EF-G domain III. This is sufficient to overcome the steric block of transmission of conformational changes within EF-G by which FA prevents release of EF-G from the ribosome. This study has identified an antibiotic resistance mechanism mediated by allosteric effects on the dynamics of the drug target

Crystal structure of monomeric human β-2- microglobulin reveals clues to its amyloidogenic properties

Dissociation of human β-2-microglobulin (β(2)m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β(2)m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β(2)m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J.&Richardson, D. (2002) Proc. Natl. Acad. Sci. USA 99, 2754–2759] and replaces them with an aggregationcompetent surface. In combination with solution studies using (1)H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-?-D-glucoside (?-OG), n-dodecyl-?-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR

Exo70-Mediated Recruitment of Nucleoporin Nup62 at the Leading Edge of Migrating Cells is Required for Cell Migration

Nucleoporin Nup62 localizes at the central channel of the nuclear pore complex and is essential for nucleocytoplasmic transport. Through its FG-repeat domain, Nup62 regulates nuclear pore permeability and binds nuclear transport receptors. Here, we report that Nup62 interacts directly with Exo70 and colocalizes with Exo70 at the leading edge of migrating cells. Nup62 binds the N-terminal domain of Exo70 through its coiled-coil domain but not through its FG-repeat domain. Selective inhibition of leading edge Nup62 using RNA interference significantly reduces cell migration. Furthermore, Exo70 recruits Nup62 at the plasma membrane and at filopodia. Removal of the Exo70-binding domain of Nup62 prevents leading edge localization of Nup62. Analogous to Exo70, Nup62 cycles between the plasma membrane and the perinuclear recycling compartment. Altogether, we propose that Nup62 not solely regulates access to the cell nucleus, but additionally functions in conjunction with Exo70, a key regulator of exocytosis and actin dynamics, at the leading edge of migrating cells

Dynamic ion pair behavior stabilizes single alpha helices in proteins.

Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs

A population shift between sparsely populated folding intermediates determines amyloidogenicity

The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of non-uniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and non-amyloidogenic variants of ?2-microglobulin (?2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the ITrans state and population of a less stable, more dynamic species ablates amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding

Grb2 binding induces phosphorylation-independent activation of Shp2.

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation