Concordance of residual dipolar couplings, backbone order parameters and crystallographic B-factors for a small alpha/beta protein: a unified picture of high probability, fast atomic motions in proteins

Using ensemble refinement of the third immunoglobulin binding domain (GB3) of streptococcal protein G (a small alpha/beta protein of 56 residues), we demonstrate that backbone (N-H, N-C', Calpha-Halpha, Calpha-C') residual dipolar coupling data in five independent alignment media, generalized order parameters from 15N relaxation data, and B-factors from a high-resolution (1.1A), room temperature crystal structure are entirely consistent with one another within experimental error. The optimal ensemble size representation is between four and eight, as assessed by complete cross-validation of the residual dipolar couplings. Thus, in the case of GB3, all three observables reflect the same low-amplitude anisotropic motions arising from fluctuations in backbone phi/psi torsion angles in the picosecond to nanosecond regime in both solution and crystalline environments, yielding a unified picture of fast, high-probability atomic motions in proteins. An understanding of these motions is crucial for understanding the impact of protein dynamics on protein function, since they provide part of the driving force for triggered conformational changes that occur, for example, upon ligand binding, signal transduction and enzyme catalysis.     

Solution structure of the IIAChitobiose-HPr complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.     

Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41

The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naïve human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.     

Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?

In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.     

转基因香石竹中F3’5’H基因的克隆、表达和免疫学鉴定

在研究转基因香石竹品系月之霓裳（Moonshade）、月之伊人（Moonlite）中外源基因F3’5’H的表达中,本文克隆了F3’5’H全长基因1.5kb,构建获得工程菌株Escherichia coli BL21（DE3）（＋F3＇5＇H）。SDS-PAGE分析的结果显示,该菌株高效表达出F3’5’H重组蛋白,约占菌体总蛋白的30%。用经纯化的F3’5’H重组蛋白作为抗原,制备F3’5’H重组蛋白的抗血清,经ELISA免疫学分析表明,该抗血清的效价为1：25600。Western blot结果表明F3’5’H重组蛋白具有良好的IgG结合活性,且抗血清与转基因香石竹品系月之霓裳和月之伊人中的外源基因F3’5’H所表达的蛋白发生明显的抗原抗体反应。这样,月之霓裳和月之伊人用于评价转基因香石竹品系的环境安全性在我国也得到了验证。     

Is serum HMGB1 a biomarker in ANCA-associated vasculitis?

Background

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease.

Methods

AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses.

Results

HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 ± 1.80 ng/ml vs. 2.39 ± 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 ± 1.83 ng/ml vs. MPA: 3.11 ± 1.91 ng/ml vs. RLV: 1.92 ± 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 ± 1.48 ng/ml vs. 3.52 ± 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (ρ = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients).

Conclusions

Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. In contrast to SLE, HMGB1 is not a useful biomarker in AAV.     

Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice

The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.     

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.     

Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation

Sparsely populated states of macromolecules, characterized by short lifetimes and high free-energies relative to the predominant ground state, often play a key role in many biological, chemical, and biophysical processes. In this review, we briefly summarize various new developments in NMR spectroscopy that permit these heretofore invisible, sparsely populated states to be detected, characterized, and in some instances visualized. Relaxation dispersion spectroscopy yields detailed kinetic information on processes involving species characterized by distinct chemical shifts with lifetimes in the ～50 μs-10 ms range and populations as low as 0.5%. In the fast exchange regime (time scale less than ～250-500 μs), the footprint of sparsely populated states can be observed on paramagnetic relaxation enhancement profiles measured on the resonances of the major species, thereby yielding structural information that is directly related to paramagnetic center-nuclei distances from which it is possible, under suitable circumstances, to compute a structure or ensemble of structures for the minor species. Finally, differential transverse relaxation measurements can be used to detect lifetime broadening effects that directly reflect the unidirectional rates for the conversion of NMR-visible into high-molecular weight NMR-invisible species. Examples of these various approaches are presented.     

BIGH3 modulates adhesion and migration of hematopoietic stem and progenitor cells

Cell adhesion and migration are important determinants of homing and development of hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) niches. The extracellular matrix protein transforming growth factor-β (TGF-β) inducible gene H3 (BIGH3) is involved in adhesion and migration, although the effect of BIGH3 is highly cell type-dependent. BIGH3 is abundantly expressed by mesenchymal stromal cells, while its expression in HSPCs is relatively low unless induced by certain BM stressors. Here, we set out to determine how BIGH3 modulates HSPC adhesion and migration. We show that primary HSPCs adhere to BIGH3-coated substrates, which is, in part, integrin-dependent. Overexpression of BIGH3 in HSPCs and HL60 cells reduced the adhesion to the substrate fibronectin in adhesion assays, which was even more profound in electrical cell-substrate impedance sensing (ECIS) assays. Accordingly, the CXCL12 induced migration over fibronectin-coated surface was reduced in BIGH3-expressing HSPCs. The integrin expression profile of HSPCs was not altered upon BIGH3 expression. Although expression of BIGH3 did not alter actin polymerization in response to CXCL12, it inhibited the PMA-induced activation of the small GTPase RAC1 as well as the phosphorylation and activation of extracellular-regulated kinases (ERKs). Reduced activation of ERK and RAC1 may be responsible for the inhibition of cell adhesion and migration by BIGH3 in HSPCs. Induced BIGH3 expression upon BM stress may contribute to the regulation of BM homeostasis.