Matthias Stoldt

Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy.

Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The γ-aminobutyric acid type A (GABAA) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pulldown analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

Selection of D-Amino-Acid peptides that bind to Alzheimer's disease amyloid peptide A beta(1-42) by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimers disease amyloid peptide Aβ(1–42). A randomized 12‐mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Aβ(1–42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D‐pep) was shown to bind Aβ(1–42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimers disease, amyloid plaques and leptomeningeal vessels containing Aβ amyloid were stained specifically with a fluorescence‐labeled derivative of D‐pep. Fibrillar deposits derived from other amyloidosis were not labeled by D‐pep. Possible applications of this novel and highly specific Aβ ligand in diagnosis and therapy of Alzheimers disease are discussed.

The NMR structure of the 5S rRNA E-domain-protein L25 complex shows preformed and induced recognition

The structure of the complex between ribosomal protein L25 and a 37 nucleotide RNA molecule, which contains the E‐loop and helix IV regions of the E‐domain of Escherichia coli 5S rRNA, has been determined to an overall r.m.s. displacement of 1.08 Å (backbone heavy atoms) by heteronuclear NMR spectroscopy (Protein Databank code 1d6k). The interacting molecular surfaces are bipartite for both the RNA and the protein. One side of the six‐stranded β‐barrel of L25 recognizes the minor groove of the E‐loop with very little change in the conformations of either the protein or the RNA and with the RNA–protein interactions occurring mainly along one strand of the E‐loop duplex. This minor groove recognition module includes two parallel β‐strands of L25, a hitherto unknown RNA binding topology. Binding of the RNA also induces conversion of a flexible loop to an α‐helix in L25, the N‐terminal tip of which interacts with the widened major groove at the E‐loop/helix IV junction of the RNA. The structure of the complex reveals that the E‐domain RNA serves as a preformed docking partner, while the L25 protein has one preformed and one induced recognition module.

The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl‐tRNA synthetases

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 ± 0.14 Å by multi‐dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA‐binding proteins consisting of a six‐stranded β‐barrel and two α‐helices. A putative RNA‐binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E‐loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six‐stranded β‐barrels of L25 and of the tRNA anticodon‐binding domain of glutaminyl‐tRNA synthetase are similar.

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP′ part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of “natively unfolded” proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

Sequestration of a β-hairpin for control of α-synuclein aggregation.

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinsons disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β-wrapins (β-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:β-wrapin complex reveals a β-hairpin of α-syn comprising the sequence region α-syn(37-54). The β-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP

Cyclic nucleotide‐sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide‐binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide‐sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide anti‐parallel β‐roll topped by a helical bundle comprising five α‐helices and a short 310‐helix. In contrast to the dimeric arrangement (‘dimer‐of‐dimers’) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other.

Helix Formation in Arrestin Accompanies Recognition of Photoactivated Rhodopsin

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

NMR structural characterization of HIV-1 virus protein U cytoplasmic domain in the presence of dodecylphosphatidylcholine micelles.

The HIV‐1 encoded virus protein U (VpU) is required for efficient viral release from human host cells and for induction of CD4 degradation in the endoplasmic reticulum. The cytoplasmic domain of the membrane protein VpU (VpUcyt) is essential for the latter activity. The structure and dynamics of VpUcyt were characterized in the presence of membrane simulating dodecylphosphatidylcholine (DPC) micelles by high‐resolution liquid state NMR. VpUcyt is unstructured in aqueous buffer. The addition of DPC micelles induces a well‐defined membrane proximal α‐helix (residues I39–E48) and an additional helical segment (residues L64–R70). A tight loop (L73–V78) is observed close to the C‐terminus, whereas the interhelical linker (R49–E63) remains highly flexible. A 3D structure of VpUcyt in the presence of DPC micelles was calculated from a large set of proton–proton distance constraints. The topology of micelle‐associated VpUcyt was derived from paramagnetic relaxation enhancement of protein nuclear spins after the introduction of paramagnetic probes into the interior of the micelle or the aqueous buffer. Qualitative analysis of secondary chemical shift and paramagnetic relaxation enhancement data in conjunction with dynamic information from heteronuclear NOEs and structural insight from homonuclear NOE‐based distance constraints indicated that micelle‐associated VpUcyt retains a substantial degree of structural flexibility.