Holger M. Strauss

Characterization of OPA1 isoforms isolated from mouse tissues

OPA1, a nuclear encoded mitochondrial protein causing autosomal dominant optic atrophy, is a key player in mitochondrial fusion and cristae morphology regulation. In the present study, we have compared the OPA1 transcription and translation products of different mouse tissues. Unlike in humans, we found only two exons (4b and 5b) to be involved in alternative splicing. The relative abundance of the resulting four different splice variants is tissue‐dependent. Proteolytic cleavage by mitochondrial processing peptidase generates two long forms, isoforms 1 and 7, which lead to three short forms representing the end products after further proteolytic processing. In contrast, isoforms 5 and 8 are directly processed into their corresponding short forms. Short form 1 molecules form 184 kDa dimers, whereas all other isoforms contribute to 285 kDa complexes. Coiled‐coil domains of the OPA1 protein specifically homo‐associate and may be involved in the formation of these complexes. Furthermore, the region encoded by exon 5b inhibits the self‐association of coiled‐coil domain‐I. Finally, our data pinpoint isoform 1 as the, by far, most abundant isoform in the nervous tissue. We postulate that manipulation of isoform 1 protein levels in relation to the other isoforms induces changes in the mitochondrial network in the cell and therefore, mutations affecting the level of functional isoform 1 could lead to devastating effects on retinal ganglion cells.

Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal α-helices (αE and αF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix αF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the αF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B β-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B β-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B β-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.

Pharmacological Interference with Protein-Protein Interactions Mediated by Coiled-Coil Motifs

Coiled coils are bundles of intertwined alpha-helices that provide protein-protein interaction sites for the dynamic assembly and disassembly of protein complexes. The coiled-coil motif combines structural versatility and adaptability with mechanical strength and specificity. Multimeric proteins that rely on coiled-coil interactions are structurally and functionally very diverse, ranging from simple homodimeric transcription factors to elaborate heteromultimeric scaffolding clusters. Several coiled-coil-bearing proteins are of outstanding pharmacological importance, most notably SNARE proteins involved in vesicular trafficking of neurotransmitters and viral fusion proteins. Together with their crucial roles in many physiological and pathological processes, the structural simplicity and reversible nature of coiled-coil associations render them a promising target for pharmacological interference, as successfully exemplified by botulinum toxins and viral fusion inhibitors. The alpha-helical coiled coil is a ubiquitous protein domain that mediates highly specific homo- and heteromeric protein-protein interactions among a wide range of proteins. The coiled-coil motif was first proposed by Crick on the basis of X-ray diffraction data on alpha-keratin more than 50 years ago (Crick 1952, 1953) and nowadays belongs to the best-characterized protein interaction modules. By definition, a coiled coil is an oligomeric protein assembly consisting of several right-handed amphipathic alpha-helices that wind around each other into a superhelix (or a supercoil) in which the hydrophobic surfaces of the constituent helices are in continuous contact, forming a hydrophobic core. Both homomeric and heteromeric coiled coils with different stoichiometries are possible, and the helices can be aligned in either a parallel or an antiparallel topology (Harbury et al. 1993, 1994). Stoichiometry and topology are governed by the primary structure, that is, the sequence of the polypeptide chains, and a given protein can participate in multiple assembly-disassembly equilibria among several coiled coils differing in stoichiometry and topology (Portwich et al. 2007). Protein complexes whose oligomeric quaternary structures - and, hence, biological activities - depend on coiled-coil interactions include transcription factors, tRNA synthetases (Biou et al. 1994; Cusack et al. 1990), cytoskeletal and signal-transduction proteins, enzyme complexes, proteins involved in vesicular trafficking, viral coat proteins, and membrane proteins (Langosch and Heringa 1998). It is thus not surprising that coiled-coil motifs have gained great attention as potential targets for modulating protein-protein interactions implicated in a large number of diseases. In this review, we will first discuss some fundamental functional and structural aspects of a simple and well-characterized representative of coiled-coil transcription factors (Sect. 1) before considering two more complex coiled coils found in scaffolding proteins involved in mitosis and meiosis and vesicular trafficking Sect. 2). This will set the stage for addressing the role of coiled coils in viral infection (Sect. 3) as well as strategies of interfering with such protein-protein interactions therapeutically (Sect. 4 and 5).

Performance of a fast fiber based UV/Vis multiwavelength detector for the analytical ultracentrifuge

The optical setup and the performance of a prototype UV/Vis multiwavelength analytical ultracentrifuge (MWL-AUC) is described and compared to the commercially available Optima XL-A from Beckman Coulter. Slight modifications have been made to the optical path of the MWL-AUC. With respect to wavelength accuracy and radial resolution, the new MWL-AUC is found to be comparable to the existing XL-A. Absorbance accuracy is dependent on the light intensity available at the detection wavelength as well as the intrinsic noise of the data. Measurements from single flashes of light are more noisy for the MWL-AUC, potentially due to the absence of flash-to-flash normalization in the current design. However, the possibility of both wavelength and scan averaging can compensate for this and still give much faster scan rates than the XL-A. Some further improvements of the existing design are suggested based on these findings.

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function

The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo- and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B β-sheet surface including a highly conserved tryptophan (Trp448mPER1, Trp419mPER2, Trp359mPER3). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.

Quantitative analyses of cryptochrome - mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock

The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.

Novel covalently linked insulin dimer engineered to investigate the function of insulin dimerization.

An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.

A Novel Subtype of AP-1-binding Motif within the Palmitoylated trans-Golgi Network/Endosomal Accessory Protein Gadkin/γ-BAR

Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1γ via degenerate peptide motifs that conform to the consensus sequence ØG(P/D/E)(Ø/L/M) (with Ø being a large hydrophobic amino acid). Recently, γ-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1γ has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the γ1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1γ binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.

Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may decrease the permeability. In conclusion, acylated sCT acts as its own in vitro intestinal permeation enhancer, with reversible effects on Caco-2 cells, indicating that acylation of sCT may represent a promising tool to increase intestinal permeability without adding oral permeation enhancers.

Expression, Receptor Binding, and Biophysical Characterization of Guinea Pig Insulin desB30: A Monomeric Insulin Variant

Here we report, for the first time, the heterologous expression of desB30 guinea pig insulin (GI desB30) in the yeast Saccharomyces cerevisiae. The affinities of GI desB30 for the insulin receptor A and the IGF‐I receptor were also quantified for the first time. Small‐angle X‐ray scattering and analytical ultracentrifugation studies confirmed that GI desB30 did not form dimers or hexamers, in contrast to human insulin. Sizeexclusion chromatography connected to inductively coupled plasma mass spectrometry revealed that GI desB30 has affinity towards several divalent metal ions. These studies did not indicate the formation of any larger structures of GI desB30 in the presence of various divalent metal ions, but did indicate that GI desB30 has an affinity towards Mn, Co, and Cu ions. Finally, the low affinity for the insulin receptor and the very low affinity for the IGF‐I receptor by GI desB30 were quantified.