Soluble analog of ApoER2 targeting beta2-glycoprotein I in immune complexes counteracts hypertension in lupus-prone mice with spontaneous antiphospholipid syndrome.

UNLABELLED: Essentials (NZWxBXSB)F1 male mice develop antibodies beta2-glycoprotein I (ß2GPI) and hypertension. A1-A1 is a soluble analogue of ApoE receptor 2 with a high affinity for ß2GPI/antibody complexes. A1-A1 improved blood pressure and arterial elastance in (NZWxBXSB)F1 male mice. A1-A1 had no adverse effects on the hemodynamics of healthy mice. SUMMARY: Background Antiphospholipid syndrome (APS) is diagnosed based on the presence of antiphospholipid antibodies and clinical thrombosis or fetal loss during pregnancy. Lupus-prone (NZWxBXSB)F1 male mice are the mouse model of spontaneous APS. They develop anti-ß2GPI antibodies, microinfarcts and hypertension. ApoER2 is a receptor that contributes to anti-ß2GPI-dependent thrombosis in APS by down-regulating endothelial nitric oxide synthase activation. Objectives A1-A1 is a small protein constructed from two identical ligand-binding modules from ApoER2, containing the binding site for ß2GPI. We studied how treatment with A1-A1 affects the development of hypertension in (NZWxBXSB)F1 male mice. Methods We treated (NZWxBXSB)F1 male mice with A1-A1 for up to 4 weeks and examined changes in hemodynamics by left ventricular pressure-volume loop measurements. Results We observed improvements in blood pressure in the A1-A1 treated mice. A1-A1 prevented the deterioration of arterial elastance by decreasing systemic resistance and improving vessel compliance. We did not detect any adverse effects of the treatment in either male mice or in apparently healthy female (NZWxBXSB)F1 mice. Conclusions We demonstrated that A1-A1, which is a soluble analog of ApoER2 that binds pathological ß2GPI/anti-ß2GPI complexes, has a positive impact on hemodynamics in lupus-prone mice with spontaneous anti-ß2GPI antibodies and hypertension.

Structural features of the low-density lipoprotein receptor facilitating ligand binding and release.

The LDLR (low-density lipoprotein receptor) is a modular protein built from several distinct structural units: LA (LDLR type-A), epidermal growth factor-like and beta-propeller modules. The low pH X-ray structure of the LDLR revealed long-range intramolecular contacts between the propeller domain and the central LA repeats of the ligand-binding domain, suggesting that the receptor changes its overall shape from extended to closed, in response to pH. Here we discuss how the LDLR uses flexibility and rigidity of linkers between modules to facilitate ligand binding and low-pH ligand release.

Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits.

Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor.

The ligand-binding domain of the LDL receptor consists of seven contiguous LDL-A modules. The fifth of these ligand-binding modules is absolutely required for recognition of both LDL and beta-VLDL particles. A four-residue linker of variable sequence connects each pair of modules, except for modules four and five, which are connected by a 12-residue linker. To provide a more detailed understanding of the structural relationship in a typical pair of functionally important LDL-A repeats of the LDLR, we investigated the backbone dynamics of repeats five (LR5) and six (LR6) alone and in the context of the covalently connected LR5-6 pair. Our results reveal substantial flexibility in the four-residue linker connecting the two repeats in the LR5-6 pair. The intrinsic dynamic behavior of each repeat is essentially unchanged when the repeats are covalently connected. These observations indicate that the relative orientation of repeats in LR5-6 is not fixed. Modeled in an extended conformation, the linker can separate LR5 and LR6 by up to 15 A, a distance that would allow substantial freedom of motion of each repeat with respect to the other in the pair.

Design and solution structure of functional peptide mimetics of nerve growth factor.

The C-D loop in nerve growth factor (NGF) is involved in binding to the NGF receptor, TrkA. It is flexible and adopts several different types conformations in different NGF crystal forms. We have previously shown that a small cyclic peptide derived from the C-D loop of NGF binds to the TrkA receptor by mimicking the structure of this loop. To understand structure-function relationships in NGF C-D loop mimetics, we have produced a series of peptides predicted to form different types of beta-turns. The peptides were tested for their ability to promote cell survival in serum-free medium and to induce TrkA tyrosine phosphorylation. NMR structural studies were used to determined the backbone conformation and the spatial orientation of side chains involved in binding to the TrkA receptor. Peptides that form type I or type gammaL-alphaR beta-turns were the most active. The variety of active loop conformations suggests that the mimetics (and NGF) accommodate the binding site on TrkA by an 'induced fit' mechanism. In agreement with this hypothesis, NMR relaxation measurements detected both fast and slow motion in the peptides. We also characterized a retro-inverso peptide derived from the NGF C-D loop. This D-amino acid cyclic peptide did not adopt a conformation homologous to the NGF C-D loop and was inactive. This may be representative of difficulties in producing structural and functional mimetics by retro-inverso schemes.

Rapid fold and structure determination of the archaeal translation elongation factor 1beta from Methanobacterium thermoautotrophicum.

The tertiary fold of the elongation factor, aEF-1beta, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1beta was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1beta structure revealed close similarity to its human analogue, eEF-1beta. In agreement with studies on EF-Ts and human EF-1beta, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1alpha. aEF-1beta was also found to bind calcium in the groove between helix alpha2 and strand beta4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.

Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action.

Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti-receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors.

Solution structure and internal motion of a bioactive peptide derived from nerve growth factor.

The conformation and internal dynamics of a bioactive cyclic peptide, N-acetyl-YCTDEKQCY, derived from the C-D loop of beta-nerve growth factor (beta-NGF) were analyzed by solution NMR spectroscopy. NMR experimental data were used to calculate an ensemble of peptide structures. All of the structures had a beta-turn at residues Asp4-Gln7 but could be divided into two families according the presence or absence of a hydrogen bond at Gln7. Comparison of the calculated structures with the corresponding C-D loops from the x-ray structures of the NGF revealed striking similarity. The orientation of Glu5, Lys6, and Gln7 side chains in the NGF mimetic was very similar to the C-D loop of NGF. These residues are known to participate in interactions with the TrkA receptor. Relaxation measurements of the peptidomimetic alpha-carbons at 13C natural abundance and calculated dynamic parameters suggest that the loop region of peptide is well structured but that residues Thr3, Asp4, Glu5, and Lys6 undergo slow conformational exchange. These results suggest that conformational similarity and possibly peptide dynamics are responsible for the bioactivity of the peptide.

1H, 15N and 13C NMR assignments, secondary structure and overall topology of the Escherichia coli GlgS protein.

GlgS is a 7892-Da protein which is involved in glycogen biosynthesis in bacteria. We report the 1H, 15N and 13C NMR assignments of the backbone and side-chain resonances at 25 degrees C and pH 6.7 from two-dimensional homonuclear and three-dimensional heteronuclear NMR experiments. The secondary structure of the protein was determined using sequential and medium-range NOE correlations, vicinal 3J(NH-H alpha) coupling values and amide proton exchange rates. The secondary structure obtained is consistent with the secondary chemical shifts of 1H alpha, 13C alpha and 13C = O. It was found that the secondary structure of GlgS comprises two amphipathic helices (Asn10-Met21 and Glu39-Arg60), one short highly hydrophobic helix (Ile30-Val33), a short extended beta-strand-like fragment (Arg26-Asp29) and two type I beta-turns (His22-Gly25 and Thr34-Met37). An overall topology of GlgS is suggested based on long-range NOEs. The elements of secondary structure form a sandwich in which the beta-strand and the short hydrophobic helix are positioned between the two amphipathic helices.