Structural Studies of Immunoglobulin-Binding Domains of Streptococcal Protein G

The solution and X-ray structures of the IgG-binding domains of streptococcal protein G are described and compared. Each domain comprises a core of 56 residues and exhibits extreme thermal stability (∼900C), despite the absence of any disulfide bridges. The structure has an unusual fold comprising a four-stranded β-sheet with a −1, +3x, −1 topology on top of which lies an α-helix. The central two strands, comprising the N- and C-termini, are parallel; the outer two strands, which are anti-parallel to the inner strands, are connected by the helix in a +3x crossover. This fold is also found in ubiquitin, a protein with no sequence similarity or functional relationship to the IgG-binding domains of protein G. The thermal stability of the domains can be accounted for by the unusual topology, coupled with an extensive hydrogen bonding network and a tightly packed and buried hydrophobic core. Possible sites of interaction with IgG are discussed in the light of the structure.     

链球菌G蛋白IgG结合域（GBx）的融合表达及其IgG结合活性的比较分析

链球菌G蛋白的IgG结合域能够特异性地结合IgG 的Fc区,是制备免疫微阵列的一种理想的IgG固定材料。克隆表达了具有IgG结合活性的3种IgG结合域的GST融合蛋白（GST-GBx）,该3种蛋白分别含有1个、2个和3个IgG结合域。采用ELISA对三蛋白IgG结合能力进行了比较分析。结果表明在含B-Domain的量相同的情况下,GST-GB3蛋白固定IgG的量最多,其次为GST-GB2,GST-GB1最弱;对IgG的灵敏度也是GST-GB3最强,GST-GB1最弱,提示GST-GB3固定IgG的能力较其他两蛋白具有明显优势。     

Pharmacokinetic and Pharmacodynamic Characterisation of an Anti-Mouse TNF Receptor 1 Domain Antibody Formatted for In Vivo Half-Life Extension

Tumour Necrosis Factor-α (TNF-α) inhibition has been transformational in the treatment of patients with inflammatory disease, e.g. rheumatoid arthritis. Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively. To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed. Receptor-deficient mice have been very insightful, but are not reversible and could distort receptor cross-talk, while inhibitory anti-TNFR1 monoclonal antibodies have a propensity to induce receptor agonism. Therefore, we set out to characterise a monovalent anti-TNFR1 domain antibody (dAb) formatted for in vivo use. The mouse TNFR1 antagonist (DMS5540) is a genetic fusion product of an anti-TNFR1 dAb with an albumin-binding dAb (AlbudAb). It bound mouse TNFR1, but not human TNFR1, and was an antagonist of TNF-α-mediated cytotoxicity in a L929 cell assay. Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding. This was supported by additional data showing the anti-TNFR1 epitope mapped to a single residue in the first domain of TNFR1. Pharmacokinetic studies of DMS5540 in mice over three doses (0.1, 1.0 and 10 mg/kg) confirmed extended in vivo half-life, mediated by the AlbudAb, and demonstrated non-linear clearance of DMS5540. Target engagement was further confirmed by dose-dependent increases in total soluble TNFR1 levels. Functional in vivo activity was demonstrated in a mouse challenge study, where DMS5540 provided dose-dependent inhibition of serum IL-6 increases in response to bolus mouse TNF-α injections. Hence, DMS5540 is a potent mouse TNFR1 antagonist with in vivo pharmacokinetic and pharmacodynamic properties compatible with use in pre-clinical disease models and could provide a useful tool to dissect the individual contributions of TNFR1 and TNFR2 in homeostasis and disease.     

Novel Functional Features of the LIS-H Domain: Role in Protein Dimerization,Half-Life and Cellular Localization

The presence of a conserved protein motif usually implies common functional features. Here, we focused on the LisH (LIS1 homology) domain, which is found in multiple proteins, and have focused on three involved in human genetic diseases; LIS1, Transducin beta-like 1X (TBL1) and Oral-facial-digital type 1 (OFD1). The recently solved structure of the LisH domain in the N-terminal region of LIS1 depicted it as a novel dimerization motif. Our findings indicated that the LisH domain of both LIS1 and TBL1 is essential for in vitro oligomerization. Furthermore, our study disclosed novel in vivo features of the LisH motif. Mutationsin conserved LisH amino acids significantly reduced both the protein half-life of LIS1, TBL1, and OFD1, and dramatically affected specific intracellular localizations of these proteins. LIS1 mutated in the LisH domain induced its localization to the actin filaments. TBL1 mutated in the LisH domain was not imported into the nucleus. Mutations in OFD1 modified its localization to the Golgi apparatus and in some cases also to the nucleus. In summary, the LisH domain may participate in protein dimerization, affect protein half-life, and may influence specific cellular localizations. Our results allow the prediction that mutations withinthe LisH motif are likely to result in pathogenic consequences in genes associated with genetic diseases.     

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.     

The Proline-rich N-terminal Domain of G18 Exhibits a Novel G Protein Regulatory Function

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Gα_i, whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Gα_i1 and Gα_o. Surprisingly, in the presence of fluoroaluminate (AlF₄⁻) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18ΔC) but not to its GoLoco region (ΔNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Gα_i/o. Neither G18wt nor any G18 mutant affected the GTPase activity of Gα_i1 or Gα_o. In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [³⁵S]GTPγS (guanosine 5′-O-[γ-thio]triphosphate) to Gα_i1, the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [³⁵S]GTPγS binding to Gα_o, albeit to a lesser extent than the effect of the GoLoco region on Gα_i1. Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the G_i/o subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.     

蛋白G IgG Fc段结合域的克隆表达及功能研究

旨在研究蛋白G IgG Fc段结合域(PGFB)的克隆、表达及其抗体结合功能,用于抗体的纯化.根据PGFB的氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了4个寡核苷酸片段.通过重叠延伸PCR方法合成了PGFB DNA片段,测序鉴定后克隆至原核表达系统pET-28a-c(+)上,转化大肠杆菌,获得表达菌株;IPTG诱导表达PGFB,经Ni+-NTA琼脂糖凝胶层析纯化后偶联到琼脂糖凝胶6B上,用其纯化多克隆抗体.结果显示,PGFB在大肠杆菌BL21(DE3)中获得高效表达,纯化后纯度达到90%以上,相对分子量为12.25 kD,与预期值相符.此外,偶联产物纯化多克隆抗体达到了良好的效果,每毫升基质可结合20 mg抗体.本研究克隆构建并高效表达了具有较好抗体亲和能力的PGFB,为多克隆抗体的快速纯化提供了方便.     

Fusion of a Short Peptide that Binds Immunoglobulin G to a Recombinant Protein Substantially Increases Its Plasma Half-Life in Mice

We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287∶1279–1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.     

Domain Organization and Conformational Plasticity of the G Protein Effector,PDE6

The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration.     

Purification and Properties of Streptococcal Competence Factor Isolated from Chemically Defined Medium

A procedure for the isolation and purification of competence factor produced in a defined medium by group H streptococci, strain Challis-6, is presented. Partial characterization and chemical analysis of the product are described. The procedure yields competence factor of high purity, as shown by homogeneity in electrofocusing, by electrophoresis in sodium dodecyl sulfate polyacrylamide gels, and by chemical analysis. The data indicate that competence factor is a small, dialyzable, highly basic compound. It is free from lipids, phosphorus, and carbohydrates, and is colorless and thermoresistant. Its biological activity is destroyed by trypsin but not by deoxyribonuclease, ribonuclease, lipase, or lysozyme. Its high isoelectric point of above pH 11.0 suggests that competence factor may be a protamine or a polymer of basic amino acids. The possibility that a polyamine may be an integral part of the polypeptide molecule has not been excluded.     

一种简便的考马斯亮蓝G250蛋白质染色方法

介绍一种快速、简便、几乎无背景的考马斯亮蓝G250(CBB G250)染色方法.该方法所用试剂仅为稀盐酸和CBB G250, CBB G250的工作浓度为0.0015%,灵敏度达0.02 μg/带, 染色2 h达70%,4 h以上或染色过夜即可充分染色.与以往的考马斯亮蓝染色方法相比,该方法有经济方便、灵敏度高、几乎无背景等优点,便于推广应用.     

呼吸道合胞病毒G蛋白基因片段的克隆及高效表达

呼吸道合胞病毒(RSV)感染遍布全球,并可导致严重的疾病,但目前尚无成功的疫苗问世。为寻求可能用于RSV疫苗研制的重组蛋白抗原,我们在克隆RSV-A全长G蛋白基因的基础上,构建了多种共表达载体蛋白和G蛋白片段的表达载体,并从中筛选出能以可溶形式高效表达抗原蛋白的原核表达体系。通过亲和层析纯化了重组蛋白抗原DsbA-G101,将其免疫Balb／c小鼠后获得了相应的抗血清。经ELISA检测表明DsbA-G101具有良好的免疫原性。基于本研究所构建的系列表达载体,可以比较不同的G蛋白片段免疫原性的强弱及载体蛋白的优劣,从中发现最佳的RSV抗原蛋白。     

呼吸道合胞病毒G蛋白基因片段的克隆及高效表达

呼吸道合胞病毒(RSV)感染遍布全球,并可导致严重的疾病,但目前尚无成功的疫苗问世.为寻求可能用于RSV疫苗研制的重组蛋白抗原,我们在克隆RSV-A全长G蛋白基因的基础上,构建了多种共表达载体蛋白和G蛋白片段的表达载体,并从中筛选出能以可溶形式高效表达抗原蛋白的原核表达体系.通过亲和层析纯化了重组蛋白抗原DsbA-G101,将其免疫Balb/c小鼠后获得了相应的抗血清.经ELISA检测表明DsbA-G101具有良好的免疫原性.基于本研究所构建的系列表达载体,可以比较不同的G蛋白片段免疫原性的强弱及载体蛋白的优劣,从中发现最佳的RSV抗原蛋白.     

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates

DnaJ from Escherichia coli is a Type I Hsp40 that functions as a cochaperone of DnaK (Hsp70), stimulating its ATPase activity and delivering protein substrates. How DnaJ binds protein substrates is still poorly understood. Here we have studied the role of DnaJ G/F-rich domain in binding of several substrates with different conformational properties (folded, partially (un)folded and unfolded). Using partial proteolysis we find that RepE, a folded substrate, contacts a wide DnaJ area that involves part of the G/F-rich region and Zn-binding domain. Deletion of G/F-rich region hampers binding of native RepE and reduced the affinity for partially (un)folded substrates. However, binding of completely unfolded substrates is independent on the G/F-rich region. These data indicate that DnaJ distinguishes the substrate conformation and is able to adapt the use of the G/F-rich region to form stable substrate complexes.     

A Conserved Streptococcal Membrane Protein,LsrS, Exhibits a Receptor-Like Function for Lantibiotics

Streptococcus mutans strain GS-5 produces a two-peptide lantibiotic, Smb, which displays inhibitory activity against a broad spectrum of bacteria, including other streptococci. For inhibition, lantibiotics must recognize specific receptor molecules present on the sensitive bacterial cells. However, so far no such receptor proteins have been identified for any lantibiotics. In this study, using a powerful transposon mutagenesis approach, we have identified in Streptococcus pyogenes a gene that exhibits a receptor-like function for Smb. The protein encoded by that gene, which we named LsrS, is a membrane protein belonging to the CAAX protease family. We also found that nisin, a monopeptide lantibiotic, requires LsrS for its optimum inhibitory activity. However, we found that LsrS is not required for inhibition by haloduracin and galolacticin, both of which are two-peptide lantibiotics closely related to Smb. LsrS appears to be a well-conserved protein that is present in many streptococci, including S. mutans. Inactivation of SMU.662, an LsrS homolog, in S. mutans strains UA159 and V403 rendered the cells refractory to Smb-mediated killing. Furthermore, overexpression of LsrS in S. mutans created cells more susceptible to Smb. Although LsrS and its homolog contain the CAAX protease domain, we demonstrate that inactivation of the putative active sites on the LsrS protein has no effect on its receptor-like function. This is the first report describing a highly conserved membrane protein that displays a receptor-like function for lantibiotics.     

Requirement for C-terminal Extension to the RNA Binding Domain for Efficient RNA Binding by Ribosomal Protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

A Self-Scaffolding Model for G Protein Signaling

Activation of heterotrimeric G proteins is generally believed to induce dissociation of Gα and Gβγ subunits, which are then free to bind to and change the catalytic activity of a variety of intracellular enzymes. We have previously found that in cells, Gαq subunits remain complexed with its major effector, phospholipase Cβ1, through the activation cycle. To determine whether this behavior may be operative in other systems, we carried out Förster resonance energy transfer studies and found that eYFP-Gαi and eCFP-Gβγ remain associated after stimulation in HEK293 cells. We also found that the level of Forster resonance energy transfer between Alexa546-phospholipase Cβ2 and eGFP-Gβγ is significant and unchanged upon activation in HEK293 cells, thus showing that these proteins can localize into stable signaling complexes. To understand the basis for this stabilization, we carried out in vitro studies using a series of single-Cys mutants labeled with fluorescence tags and monitored their interaction with Gβγ subunits and changes in their fluorescence properties and accessibility upon activation and Gβγ binding. Our studies suggest a significant change in the orientation between G protein subunits upon activation that allows the G proteins to remain complexed while activating effectors.