The structure of a reduced form of OxyR from Neisseria meningitidis

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited - © 2010 Sainsbury et al; licensee BioMed Central Ltd.Background: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. Results: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 Å. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix α3, separated by 18 Å from C208, which is positioned between helices α3 and α4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. Conclusion: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.This work was supported by UK Medical Research Council, the Biotechnology Biological Research Council, and by a MRC Research Studentship

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment

BACKGROUND Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution

Structural mass spectrometry decodes domain interaction and dynamics of the full-length Human Histone Deacetylase 2

Human Histone Deacetylase 2 (HDAC2) belongs to a conserved enzyme superfamily that regulates deacetylation inside cells. HDAC2 is a drug target as it is known to be upregulated in cancers and neurodegenerative disorders. It consists of a globular deacetylase and C-terminus intrinsically-disordered domains [1-3]. To date, there is no full-length structure of HDAC2 available due to the high intrinsic flexibility of its C-terminal domain. The intrinsically-disordered domain, however, is known to be important for the enzymatic function of HDAC2 [1, 4]. Here we combine several structural Mass Spectrometry (MS) methodologies such as denaturing, native, ion mobility and chemical crosslinking, alongside biochemical assays and molecular modelling to study the structure and dynamics of the full-length HDAC2 for the first time. We show that MS can easily dissect heterogeneity inherent within the protein sample and at the same time probe the structural arrangement of the different conformers present. Activity assays combined with data from MS and molecular modelling suggest how the structural dynamics of the C-terminal domain, and its interactions with the catalytic domain, regulate the activity of this enzyme

Clinical disorders affecting mesopic vision

Vision in the mesopic range is affected by a number of inherited and acquired clinical disorders. We review these conditions and summarize the historical background, describing the clinical characteristics alongside the genetic basis and molecular biological mechanisms giving rise to rod and cone dysfunction relevant to twilight vision. The current diagnostic gold standards for each disease are discussed and curative and symptomatic treatment strategies are summarized

Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme

Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications

A red knot as a black swan:How a single bird shows navigational abilities during repeat crossings of the Greenland Icecap

Despite the wealth of studies on seasonal movements of birds between southern nonbreeding locations and High Arctic breeding locations, the key mechanisms of navigation during these migrations remain elusive. A flight along the shortest possible route between pairs of points on a sphere ('orthodrome') requires a bird to be able to assess its current location in relation to its migration goal and to make continuous adjustment of heading to reach that goal. Alternatively, birds may navigate along a vector with a fixed orientation ('loxodrome') based on magnetic and/or celestial compass mechanisms. Compass navigation is considered especially challenging for summer migrations in Polar regions, as continuous daylight and complexity in the geomagnetic field may complicate the use of both celestial and magnetic compasses here. We examine the possible use of orientation mechanisms during migratory flights across the Greenland Icecap. Using a novel 2 g solar-powered satellite transmitter, we documented the flight paths travelled by a female red knotCalidris canutus islandicaduring two northward and two southward migrations. The geometry of the paths suggests that red knots can migrate across the Greenland Icecap along the shortest-, orthodrome-like, path instead of the previously suggested loxodrome path. This particular bird's ability to return to locations visited in a previous year, together with its sudden course changes (which would be appropriate responses to ambient wind fields), suggest a map sense that enables red knots to determine location, so that they can tailor their route depending on local conditions