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

Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs.Peer reviewe

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification

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

Testosterone and Covid‐19: An update

Abstract: There is overwhelming evidence to suggest that male gender is at a higher risk of developing more severe Covid‐19 disease and thus having poorer clinical outcomes. However, the relationship between testosterone (T) and Covid‐19 remains unclear with both protective and deleterious effects on different aspects of the disease suggested. Here, we review the current epidemiological and biological evidence on the role of testosterone in the process of SARS‐CoV‐2 infection and in mediating Covid‐19 severity, its potential to serve as a biomarker for risk stratification and discuss the possibility of T supplementation as a treatment or preventative therapy for Covid‐19

Voiding function improves under long-term testosterone treatment (TTh) in hypogonadal men, independent of prostate size

Background Functional hypogonadism is a condition in which some, but not all, older men have low testosterone levels. Rather than chronological age per se, the causality of hypogonadism includes obesity and impaired general health (e.g., metabolic syndrome). An association between testosterone deficiency and lower urinary tract symptoms (LUTS) has been reported, yet due to prostate safety concerns, men with severe LUTS (IPSS score > 19) have invariably been excluded from entering testosterone trials. Irrespective, exogenous testosterone has not been demonstrated to cause de novo or worsen mild to moderate LUTS. Objective This study investigated whether long-term testosterone therapy (TTh) could have a protective effect on improving the symptoms of LUTS in hypogonadal men. However, the exact mechanism by which testosterone exerts is beneficial effect remains uncertain. Patients and methods In this study 321 hypogonadal patients with an average age of 58.9 ± 9.52 years received testosterone undecanoate in 12-week intervals for 12 years. One hundred and forty-seven of these males had the testosterone treatment interrupted for a mean of 16.9 months before it was resumed. Total testosterone, International Prostate Symptom Scale (IPSS), post-voiding residual bladder volume and aging male symptoms (AMS) were measured over the study period. Results Prior to TTh interruption, it was observed that testosterone stimulation improved the men’s IPSS, AMS and post-voiding residual bladder volume, while their prostate volume significantly increased. During the TTh interruption, there was a significant worsening in these parameters, although the increase in prostate volume continued. When TTh was resumed, these effects were reversed, implying that hypogonadism may require lifelong treatment

Long-term testosterone therapy improves liver parameters and steatosis in hypogonadal men: a prospective controlled registry study

Non-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease (CVD) and both are prevalent in men with testosterone deficiency. Long-term effects of testosterone therapy (TTh) on NAFLD are not well studied. This observational, prospective, cumulative registry study assesses long-term effects of testosterone undecanoate (TU) on hepatic physiology and function in 505 hypogonadal men (T levels ≤350 ng/dL). Three hundred and twenty one men received TU 1000 mg/12 weeks for up to 12 years following an initial 6-week interval (T-group), while 184 who opted against TTh served as controls (C-group). T-group patients exhibited decreased fatty liver index (FLI, calculated according to Mayo Clinic guidelines) (83.6 ± 12.08 to 66.91 ± 19.38), γ-GT (39.31 ± 11.62 to 28.95 ± 7.57 U/L), bilirubin (1.64 ± 4.13 to 1.21 ± 1.89 mg/dL) and triglycerides (252.35 ± 90.99 to 213 ± 65.91 mg/dL) over 12 years. Waist circumference and body mass index were also reduced in the T-group (107.17 ± 9.64 to 100.34 ± 9.03 cm and 31.51 ± 4.32 to 29.03 ± 3.77 kg/m2). There were 25 deaths (7.8%) in the T-group of which 11 (44%) were cardiovascular related. In contrast, 28 patients (15.2%) died in C-group, and all deaths (100%) were attributed to CVD. These data suggest that long-term TTh improves hepatic steatosis and liver function in hypogonadal men. Improvements in liver function may have contributed to reduced CVD-related mortality