Structural basis of semaphorin-plexin signalling.

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed ß-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.

A dual binding mode for RhoGTPases in plexin signalling.

Plexins are cell surface receptors for the semaphorin family of cell guidance cues. The cytoplasmic region comprises a Ras GTPase-activating protein (GAP) domain and a RhoGTPase binding domain. Concomitant binding of extracellular semaphorin and intracellular RhoGTPase triggers GAP activity and signal transduction. The mechanism of this intricate regulation remains elusive. We present two crystal structures of the human Plexin-B1 cytoplasmic region in complex with a constitutively active RhoGTPase, Rac1. The structure of truncated Plexin-B1-Rac1 complex provides no mechanism for coupling RhoGTPase and Ras binding sites. On inclusion of the juxtamembrane helix, a trimeric structure of Plexin-B1-Rac1 complexes is stabilised by a second, novel, RhoGTPase binding site adjacent to the Ras site. Site-directed mutagenesis combined with cellular and biophysical assays demonstrate that this new binding site is essential for signalling. Our findings are consistent with a model in which extracellular and intracellular plexin clustering events combine into a single signalling output.

FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis.

Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.

Using structural information to change the phosphotransfer specificity of a two-component chemotaxis signalling complex.

Two-component signal transduction pathways comprising histidine protein kinases (HPKs) and their response regulators (RRs) are widely used to control bacterial responses to environmental challenges. Some bacteria have over 150 different two-component pathways, and the specificity of the phosphotransfer reactions within these systems is tightly controlled to prevent unwanted crosstalk. One of the best understood two-component signalling pathways is the chemotaxis pathway. Here, we present the 1.40 A crystal structure of the histidine-containing phosphotransfer domain of the chemotaxis HPK, CheA(3), in complex with its cognate RR, CheY(6). A methionine finger on CheY(6) that nestles in a hydrophobic pocket in CheA(3) was shown to be important for the interaction and was found to only occur in the cognate RRs of CheA(3), CheY(6), and CheB(2). Site-directed mutagenesis of this methionine in combination with two adjacent residues abolished binding, as shown by surface plasmon resonance studies, and phosphotransfer from CheA(3)-P to CheY(6). Introduction of this methionine and an adjacent alanine residue into a range of noncognate CheYs, dramatically changed their specificity, allowing protein interaction and rapid phosphotransfer from CheA(3)-P. The structure presented here has allowed us to identify specificity determinants for the CheA-CheY interaction and subsequently to successfully reengineer phosphotransfer signalling. In summary, our results provide valuable insight into how cells mediate specificity in one of the most abundant signalling pathways in biology, two-component signal transduction.

High throughput FAMS - A fatty acid mass spectrometry method for monitoring polysorbate hydrolysis in QC.

Surfactant degradation in biopharmaceuticals has recently gained significant attention in the pharmaceutical industry. Specifically, hydrolytic degradation of polysorbates, leading to the release of free fatty acids potentially forming visible particles, is a key theme in technical development. To address this emerging topic, we present the development of a fully automated liquid-chromatography single quad mass detector method for the quantification of free fatty acids in biopharmaceuticals. For the first time, we have quantified the longer chain fatty acid degradation products of polysorbate, palmitic and stearic acid, allowing reliable detection and early critical insights for process improvements. This high-throughput method was validated underlining its robust performance in an interlaboratory trial as well as high flexibility allowing different robotic platforms and preparation techniques. The combination of automated sample preparation, separation by liquid chromatography and single quad mass detection makes the validated fatty acid mass spectrometry assay ready for routine use in a regulated environment.

Simultaneous quantification of polysorbate 20 and poloxamer 188 in biopharmaceutical formulations using evaporative light scattering detection.

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method's practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate.

Monitoring polysorbate hydrolysis in biopharmaceuticals using a QC-ready free fatty acid quantification method.

Hydrolysis of the non-ionic surfactant polysorbate upon long-term storage poses significant challenges to development of biopharmaceutical liquid formulations. Low concentrations of intact surfactant may compromise its protective properties and thus affect protein stability. In addition, accumulation of polysorbate hydrolysis products is increasingly put into context with the formation of visible and subvisible particulates based on the low solubility of the main degradation products. Despite of this potential negative impact on product quality, quantification of the released free fatty acids is performed commonly in an indirect and consequently insensitive manner by determining the remaining PS content or by cumbersome methods, which are unsuitable for routine testing in quality control laboratories. For this purpose, this study describes the development and qualification of a label-free, reliable liquid-chromatography single quad mass detector (LC-QDa)-based method capable of resolving slight changes in the free fatty acid profile which can be readily integrated into quality control facilities. The practical utility of the herein described method is outlined by a case study on the real-time storage stability of a formulated monoclonal antibody.

(1)H, (13)C and (15)N resonance assignments for the response regulator CheY3 from Rhodobacter sphaeroides.

Rhodobacter sphaeroides has emerged as a model system for studies of the complex chemotaxis pathways that are a hallmark of many non-enteric bacteria. The genome of R. sphaeroides encodes two sets of flagellar genes, fla1 and fla2, that are controlled by three different operons. Each operon encodes homologues of most of the proteins required for the well-studied E. coli chemotaxis pathway. R. sphaeroides has six homologues of the response regulator CheY that are localized to and are regulated by different clusters of chemosensory proteins in the cell and have different effects on chemotaxis. CheY6 is the major CheY stopping the fla1 flagellar motor and associated with a cytoplasmically localised chemosensory pathway. CheY3 and CheY4 are associated with a membrane localised polar chemosensory cluster, and can bind to but not stop the motor. CheY6 and either CheY3 or CheY4 are required for chemotaxis. We are using NMR spectroscopy to characterise and compare the structure and dynamics of CheY3 and CheY6 in solution. We are interested in defining the conformational changes that occur upon activation of these two proteins and to identify differences in their properties that can explain the different functions they play in chemotaxis in R. sphaeroides. Here we present the (1)H, (13)C and (15)N assignments for CheY3 in its active, inactive and Mg(2+)-free apo form. These assignments provide the starting point for detailed investigations of the structure and function of CheY3.

Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein.

Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2-RGM-NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM-NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways.

Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub.

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways.

Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization.

F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab' fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 A resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue alpha-turn around the conserved GPGRA apex of the beta-hairpin loop. In agreement with previous mutagenesis analyses, the Fab' interacts primarily with V3 through side-chain contacts with just two residues, Ile(P309) and Arg(P315), while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.