Biomedical subjects

Holger Sondermann

Publications and source records attributed to Holger Sondermann.

11 recordsLinked to original sources

PubMed2006

A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases.

The p21-activated kinases (PAKs) are serine/threonine kinases that are involved in a wide variety of cellular functions including cytoskeletal motility, apoptosis, and cell cycle regulation. PAKs are inactivated by blockage of the active site of the kinase domain by an N-terminal regulatory domain. GTP-bound forms of Cdc42 and Rac bind to the regulatory domain and displace it, thereby allowing phosphorylation of the kinase domain and maximal activation. A key step in the activation process is the phosphorylation of the activation loop of one PAK kinase domain by another, but little is known about the underlying recognition events that make this phosphorylation specific. We show that the phosphorylated kinase domain of PAK2 dimerizes in solution and that this association is prevented by addition of a substrate peptide. We have identified a crystallographic dimer in a previously determined crystal structure of activated PAK1 in which two kinase domains are arranged face to face and interact through a surface on the large lobe of the kinase domain that is exposed upon release of the auto-inhibitory domain. The crystallographic dimer is suggestive of an engagement that mediates trans-autophosphorylation. Mutations at the predicted dimerization interface block dimerization and reduce the rate of autophosphorylation, supporting the role of this interface in PAK activation.

Amino Acid Sequence↗

PubMed2005

Computational docking and solution x-ray scattering predict a membrane-interacting role for the histone domain of the Ras activator son of sevenless.

The Ras-specific nucleotide exchange factor son of sevenless (SOS) is a large, multidomain protein with complex regulation, including a Ras-dependent allosteric mechanism. The N-terminal segment of SOS, the histone domain, contains two histone folds, which is highly unusual for a cytoplasmic protein. Using a combination of computational docking, small-angle x-ray scattering, mutagenesis, and calorimetry, we show that the histone domain folds into the rest of SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and Dbl-homology (DH) domains of SOS to the catalytic domain. In this model, a positively charged surface region on the histone domain is positioned so as to provide a fourth potential anchorage site on the membrane for SOS in addition to the PH domain, the allosteric Ras molecule, and the C-terminal adapter-binding site. The histone domain in SOS interacts with the helical linker, using a region of the surface that in nucleosomes is involved in histone tetramerization. Adjacent surface elements on the histone domain that correspond to the DNA-binding surface of nucleosomes form the predicted interaction site with the membrane. The orientation and position of the histone domain in the SOS model implicates it as a potential mediator of membrane-dependent activation signals.

Allosteric Regulation↗

PubMed2005

C2 can do it, too.

In this issue of Cell, report that the C2 domain of the serine/threonine protein kinase Cdelta is a phosphotyrosine binding domain and present the crystal structure of this C2 domain bound to a peptide containing phosphotyrosine. Prior to this work, C2 domains were thought to bind only to phospholipids or to unphosphorylated proteins, and the SH2 and PTB domains were the only signaling domains known to recognize phosphotyrosine. This new role for the C2 domain links phosphotyrosine recognition directly to serine/threonine kinase activity and reveals an unexpected mechanism for crosstalk between distinct signaling pathways.

Animals↗

PubMed2005

Structural bases of unphosphorylated STAT1 association and receptor binding.

The crystal structure has been determined at 3.0 A resolution for an unphosphorylated STAT1 (1-683) complexed with a phosphopeptide derived from the alpha chain of interferon gamma (IFNgamma) receptor. Two dimer interfaces are seen, one between the N domains (NDs) (amino acid residues 1-123) and the other between the core fragments (CFs) (residues 132-683). Analyses of the wild-type (wt) and mutant STAT1 proteins by static light scattering, analytical ultracentrifugation, and coimmunoprecipitation suggest that STAT1 is predominantly dimeric prior to activation, and the dimer is mediated by the ND interactions. The connecting region between the ND and the CF is flexible and allows two interconvertable orientations of the CFs, termed "antiparallel" or "parallel," as determined by SH2 domain orientations. Functional implications of these dimer conformations are discussed. Also revealed in this structure is the detailed interaction between STAT1 SH2 domain and its docking site on IFNgamma receptor.

Crystallization↗

PubMed2005

Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering.

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality

Animals↗

PubMed2004

Structural analysis of autoinhibition in the Ras activator Son of sevenless.

The classical model for the activation of the nucleotide exchange factor Son of sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that Ras*GTP is an allosteric activator of SOS indicated that the regulation of SOS is more complex than originally envisaged. We now present crystallographic and biochemical analyses of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains and show that the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of Ras*GDP binding, and maximal activity, which requires Ras*GTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low to high activity forms as Ras*GDP is converted to Ras*GTP by SOS.

Allosteric Site↗

PubMed2003

Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76.

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS.

Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

Catalytic Domain↗

PubMed2003

Tandem histone folds in the structure of the N-terminal segment of the ras activator Son of Sevenless.

The Ras activator Son of Sevenless (Sos) contains a Cdc25 homology domain, responsible for nucleotide exchange, as well as Dbl/Pleckstrin homology (DH/PH) domains. We have determined the crystal structure of the N-terminal segment of human Sos1 (residues 1-191) and show that it contains two tandem histone folds. While the N-terminal domain is monomeric in solution, its structure is surprisingly similar to that of histone dimers, with both subunits of the histone "dimer" being part of the same peptide chain. One histone fold corresponds to the region of Sos that is clearly similar in sequence to histones (residues 91-191), whereas the other is formed by residues in Sos (1-90) that are unrelated in sequence to histones. Residues that form a contiguous patch on the surface of the histone domain of Sos are conserved from C. elegans to humans, suggesting a potential role for this domain in protein-protein interactions.

Amino Acid Sequence↗

PubMed2002

Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae.

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro. Based on the strict positional conservation of the Arg-237 residue, several Bag domain proteins were predicted from various eukaryotic genomes. One candidate, Snl1p from Saccharomyces cerevisiae, was confirmed as a Bag domain co-chaperone. Snl1p bound specifically to the Ssa and Ssb forms of yeast cytosolic Hsp70, as revealed by two-hybrid screening and co-precipitations from yeast lysate. In vitro, Snl1p also recognized mammalian Hsp70 and regulated the Hsp70 ATPase activity identically to Bag-1M. Point mutations in Snl1p that disrupted the conserved residues Glu-112 and Arg-141, equivalent to Glu-212 and Arg-237 in Bag-1M, abolished the interaction with Hsp70 proteins. In live yeast, mutated Snl1p could not substitute for wild-type Snl1p in suppressing the lethal defect caused by truncation of the Nup116p nuclear pore component. Thus, Snl1p is the first Bag domain protein identified in S. cerevisiae, and its interaction with Hsp70 is essential for biological activity.

Adenosine Triphosphatases↗

PubMed2002

The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.

Adaptor Proteins, Signal Transducing↗