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Biomedical subjects

Michael Sattler

Publications and source records attributed to Michael Sattler.

At least 37 records · Page 2Linked to original sources
PubMed2005

Novel modes of protein-RNA recognition in the RNAi pathway.

Gene silencing mediated by RNA interference (RNAi) depends on short interfering RNAs (siRNAs) and micro RNAs (miRNAs). These RNAs have unique features, namely a defined size of 19-21 base pairs, and characteristic two-nucleotide single-stranded 3' overhangs and 5' monophosphate groups. These molecular features of siRNAs and miRNAs are produced by RNase III enzymes, which are a hallmark of gene silencing induced by double-stranded RNA. Recent structural studies of components of the RNAi pathway, including PAZ, Piwi and RNase III domains, as well as full-length Argonaute and viral p19 proteins, have revealed distinct and novel modes of sequence-independent recognition of the characteristic features of siRNAs and miRNAs in the RNAi pathway.

Binding Sites↗
PubMed2005

Structure and phosphatidylinositol-(3,4)-bisphosphate binding of the C-terminal PH domain of human pleckstrin.

Pleckstrin is the major target of protein kinase C (PKC) in blood platelets. Its phosphorylation triggers responses that ultimately lead to platelet activation and blood clot formation. Pleckstrin consists of three domains: a pleckstrin homology (PH) domain at both termini and a central DEP (Dishevelled, Egl-1, Pleckstrin) domain. Here, we report the solution nuclear magnetic resonance (NMR) structure of the C-terminal PH domain (C-PH) of human pleckstrin-1. We show that this PH domain binds phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) with high specificity in protein lipid overlay assays. Using NMR titration experiments and mutational analysis, residues involved in binding to PtdIns(3,4)P2 are identified. The binding site is formed by a patch of basic residues from the beta1 and beta2 strands and the beta1-beta2 loop. Since PtdIns(3,4)P2 is an important signaling molecule in platelets, our data suggest a C-PH dependent regulation of pleckstrin function in response to PtdIns(3,4)P2.

Amino Acid Sequence↗
PubMed2005

X-ray crystallographic and NMR studies of the third KH domain of hnRNP K in complex with single-stranded nucleic acids.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is implicated in multiple functions in the regulation of gene expression and acts as a hub at the intersection of signaling pathways and processes involving nucleic acids. Central to its function is its ability to bind both ssDNA and ssRNA via its KH (hnRNP K homology) domains. We determined crystal structures of hnRNP K KH3 domain complexed with 15-mer and 6-mer (CTC(4)) ssDNAs at 2.4 and 1.8 A resolution, respectively, and show that the KH3 domain binds specifically to both TCCC and CCCC sequences. In parallel, we used NMR to compare the binding affinity and mode of interaction of the KH3 domain with several ssRNA ligands and CTC(4) ssDNA. Based on a structure alignment of the KH3-CTC(4) complex with known structures of other KH domains in complex with ssRNA, we discuss recognition of tetranucleotide sequences by KH domains.

Amino Acid Sequence↗
PubMed2005

The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition.

We report the structure of the flock house virus B2 protein, a potent suppressor of RNA interference (RNAi) in animals and plants. The B2 protein is a homodimer in solution and contains three alpha-helices per monomer. Chemical shift perturbation shows that an antiparallel arrangement of helices (alpha2/alpha2') forms an elongated binding interface with double-stranded RNA (dsRNA). This implies a novel mode of dsRNA recognition and provides insights into the mechanism of RNAi suppression by B2.

Amino Acid Sequence↗
PubMed2004

Genetically encoded FRET probe for PKC activity based on pleckstrin.

We developed a probe for investigating protein kinase C (PKC) activity in living cells. The probe is based on a fragment of pleckstrin enclosed by two FRET-capable fluorophores. PKC activity modulation was reliably followed by FRET change in vitro and in vivo. The probe responds quickly to PKC activation by phorbol ester. FRET changes were reversible when PKC inhibitors were administered. Stimulation of cellular signaling pathways using histamine or bradykinin triggered PKC in a physiologically relevant way.

Bacterial Proteins↗
PubMed2004

Site-specific variations of carbonyl chemical shift anisotropies in proteins.

By combining molecular dynamics (MD) simulation with a novel extended density functional theory method, we calculate site-specific carbonyl chemical shift tensors in the SMN Tudor domain. We formulate a simple model for the C' chemical shift anisotropy (CSA) based solely on the isotropic chemical shift. Using this simple chemical shift tensor model and the MD simulation, an accurate prediction of transverse C'/N-H cross-correlated relaxation rates can be obtained.

Anisotropy↗
PubMed2004

Nucleic acid 3'-end recognition by the Argonaute2 PAZ domain.

We describe the solution structures of the Argonaute2 PAZ domain bound to RNA and DNA oligonucleotides. The structures reveal a unique mode of single-stranded nucleic acid binding in which the two 3'-terminal nucleotides are buried in a hydrophobic cleft. We propose that the PAZ domain contributes to the specific recognition of siRNAs by providing a binding pocket for their characteristic two-nucleotide 3' overhangs.

Argonaute Proteins↗
PubMed2004

Functional and structural characterization of a novel member of the natriuretic family of peptides from the venom of Pseudocerastes persicus.

A novel peptide, PNP (Pseudocerastes persicus natriuretic peptide), was isolated from the venom of the Iranian viper P. persicus. Amino acid sequencing revealed that the 37-residue peptide belongs to the family of natriuretic peptides. The physiological effects of intra-venously PNP infused into anesthetized rats on urine flow, sodium excretion and blood pressure were comparable to those of atrial natriuretic peptide (ANP). In PC12 cells that were treated with either PNP, ANP, or C-type natriuretic peptide, PNP induced a similar cGMP response as ANP. Since PC12 cells only express the natriuretic peptide receptor (NPR)-A receptor we conclude that PNP binds to the NPR-A receptor. The solution conformation of PNP was characterized using (1)H nuclear magnetic resonance spectroscopy and indicates a high degree of conformational flexibility.

Amino Acid Sequence↗
PubMed2004

Structural basis of single-stranded RNA recognition.

RNA is an ancient and highly versatile molecule that plays fundamental roles in all living organisms. Its molecular functions range from being a mediator of genetic information to the regulation of essential cellular processes. These functions are often accomplished in close association with RNA binding proteins. Over the past few years, a considerable number of high-resolution three-dimensional structures of important protein-RNA complexes have been determined. Here, we wish to discuss recent examples and highlight principles and distinct features of single-stranded RNA recognition by conserved RNA binding domains.

Models, Molecular↗
PubMed2004

Automated evaluation of chemical shift perturbation spectra: New approaches to quantitative analysis of receptor-ligand interaction NMR spectra.

This paper presents new methods designed for quantitative analysis of chemical shift perturbation NMR spectra. The methods automatically trace the displacements of cross peaks between a perturbed test spectrum and the reference spectrum (or among a series of titration spectra), and measure the changes of chemical shifts, heights, and widths of the altered peaks. The methods are primary aimed at the (1)H-(15)N HSQC spectra of relatively small proteins (<15 kDa) assuming fast exchange between free and ligand-bound states on the chemical shift time scale, or for comparing spectra of free and fully bound states in the slow exchange situation. Using the (1)H-(15)N HSQC spectra from a titration experiment of the 74-residue Pex13p SH3 domain with a Pex14p peptide ligand (14 residues, K (d)= approximately 40 microM), we demonstrate the scope and limits of our automatic peak tracing (APET) algorithm for efficient scoring of high-throughput SAR by NMR type HSQC spectra, and progressive peak tracing (PROPET) algorithm for detailed analysis of ligand titration spectra. Simulated spectra with low signal-to-noise ratios (S/N ranged from 20 to 1) were used to demonstrate the reliability and reproducibility of the results when dealing with poor quality spectra. These algorithms have been implemented in a new software module, FELIX-Autoscreen, for streamlined processing, analysis and visualization of SAR by NMR and other high-throughput receptor/ligand interaction experiments.

Algorithms↗
PubMed2003

Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain.

RNA interference is a conserved mechanism that regulates gene expression in response to the presence of double-stranded (ds)RNAs. The RNase III-like enzyme Dicer first cleaves dsRNA into 21-23-nucleotide small interfering RNAs (siRNAs). In the effector step, the multimeric RNA-induced silencing complex (RISC) identifies messenger RNAs homologous to the siRNAs and promotes their degradation. The Argonaute 2 protein (Ago2) is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. Here we present the three-dimensional nuclear magnetic resonance structure of the Drosophila melanogaster Ago2 PAZ domain. This domain adopts a nucleic-acid-binding fold that is stabilized by conserved hydrophobic residues. The nucleic-acid-binding patch is located in a cleft between the surface of a central beta-barrel and a conserved module comprising strands beta3, beta4 and helix alpha3. Because critical structural residues and the binding surface are conserved, we suggest that PAZ domains in all members of the Argonaute and Dicer families adopt a similar fold with nucleic-acid binding function, and that this plays an important part in gene silencing.

Animals↗
PubMed2003

Crystal structure and nuclear magnetic resonance analyses of the SAND domain from glucocorticoid modulatory element binding protein-1 reveals deoxyribonucleic acid and zinc binding regions.

The glucocorticoid-modulatory element-binding proteins, GMEB1 and GMEB2, are ubiquitous, multifunctional DNA-binding proteins with important roles in the modulation of transcription upon steroid hormone activation. The GMEB proteins have intrinsic transactivation ability, but also control the glucocorticoid response via direct binding to the glucocorticoid receptor. They are also mandatory host proteins for Parvovirus replication. Here we present the 1.55 A resolution crystal structure of a central portion of GMEB1, encompassing its SAND domain, which shares 80% sequence identity with the GMEB2 SAND domain. We demonstrate that this domain, also present in numerous proteins implicated in chromatin-associated transcriptional regulation, is necessary and sufficient to bind the glucocorticoid-modulatory element (GME) DNA target. We use nuclear magnetic resonance (NMR) and binding studies to map the DNA recognition surface to an alpha-helical region exposing the conserved KDWK motif. Using site-directed mutagenesis, key residues for DNA binding are identified. In contrast to the previously determined NMR structure of the Sp100b SAND domain, we find that the GMEB1 SAND domain also comprises a zinc-binding motif. Although the zinc ion is not necessary for DNA binding, it is found to determine the C-terminal conformation of the GMEB1 SAND domain. We also show that homologous zinc-binding motifs exist in a subset of SAND domain proteins and probe the roles of this novel motif.

Amino Acid Sequence↗
PubMed2003

High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues.

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.

Arginine↗
PubMed2003

Definition of domain boundaries and crystallization of the SMN Tudor domain.

Spinal muscular atropy (SMA) is the major genetic disease leading to childhood mortality and is caused by mutations in or deletions of the smn1 gene. The human survival of motor neurons (SMN) protein encoded by this gene plays an important role in the assembly of snRNPs (small nuclear ribonucleoprotein complexes) via binding to the spliceosomal Sm proteins. The tails of these Sm proteins contain symmetrically dimethylated arginines that are recognized by the central SMN Tudor domain. To gain insight in the molecular basis of this specific interaction, the SMN Tudor domain has been crystallized. The rapid crystallization of the protein and the high stability of the crystals is facilitated by redefinition of domain boundaries based on NMR relaxation experiments and the previously determined solution structure. The crystals diffract to high resolution (1.8 A) and a complete data set has been collected from a hexagonal crystal form (P6(1)/P6(5)), with unit-cell parameters a = b = 27.65, c = 110.30 A, alpha = beta = 90, gamma = 120 degrees. Crystal soaks and co-crystallization with peptides derived from the Sm protein tails have been initiated. Molecular replacement with the NMR coordinates is under way.

Crystallization↗