Hans Peter Bächinger

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin.

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming α-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the α-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 Å crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three α-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.

Hfq: A Bacterial Sm-like Protein that Mediates RNA-RNA Interaction

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

STRUCTURAL ANALYSIS OF PORCINE BRAIN NITRIC OXIDE SYNTHASE REVEALS A ROLE FOR TETRAHYDROBIOPTERIN AND L-ARGININE IN THE FORMATION OF AN SDS-RESISTANT DIMER

Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P‐450s, containing reductase and mono‐oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS‐polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha‐helix, 14% antiparallel beta‐sheet, 7% parallel beta‐sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS‐polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L‐arginine synergistically convert neuronal NOS into an exceptionally stable, non‐covalently linked homodimer surviving in 2% SDS and 5% 2‐mercaptoethanol. Ligand‐induced formation of an SDS‐resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L‐arginine in the allosteric regulation of protein subunit interactions.

The Prodomain of BMP-7 Targets the BMP-7 Complex to the Extracellular Matrix

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor β (TGFβ) complex. Because the prodomain of TGFβ interacts with latent TGFβ-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFβ-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFβ-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.

A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization.

Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of ∼20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins.

Targeting of Bone Morphogenetic Protein Growth Factor Complexes to Fibrillin

Both latent transforming growth factor-β (TGF-β)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-β family of growth factors. Interactions between latent TGF-β-binding protein-1 and TGF-β and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-β family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.

The Fate of Cartilage Oligomeric Matrix Protein Is Determined by the Cell Type in the Case of a Novel Mutation in Pseudoachondroplasia

We have identified a novel missense mutation in a pseudoachondroplasia (PSACH) patient in one of the type III repeats of cartilage oligomeric matrix protein (COMP). Enlarged lamellar rough endoplasmic reticulum vesicles were shown to contain accumulated COMP along with type IX collagen, a cartilage-specific component. COMP was secreted and assembled normally into the extracellular matrix of tendon, demonstrating that the accumulation of COMP in chondrocytes was a cell-specific phenomenon. We believe that the intracellular storage of COMP causes a nonspecific aggregation of cartilage-specific molecules and results in a cartilage matrix deficient in required structural components leading to impaired cartilage growth and maintenance. These data support a common pathogenetic mechanism behind two clinically related chondrodysplasias, PSACH and multiple epiphyseal dysplasia.

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap−/− mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti–TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap−/− mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Latent Transforming Growth Factor β-binding Proteins and Fibulins Compete for Fibrillin-1 and Exhibit Exquisite Specificities in Binding Sites

Latent transforming growth factor (TGF) β-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFβ. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit “exquisite specificities,” a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.