Residual flexibility in the topologically constrained multivalent complex between the GKAP scaffold and LC8 hub proteins.

Guanylate kinase-associated protein (GKAP) is a large postsynaptic scaffold protein bearing two closely spaced noncanonical binding sites for the bivalent dynein light chain LC8 hub protein. This might allow the formation of heterogeneous complexes with different sizes and topologies. Here, we show that a well-defined hexameric complex is formed, composed of two GKAP molecules and two LC8 dimers.

Reassessing the substrate specificities of the major peptidoglycan hydrolases lysostaphin and LytM.

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined.

Chemical shift assignments of the catalytic domain of Staphylococcus aureus LytM.

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called "last-resort" drugs.

H, C, and N NMR chemical shift assignment of LytM N-terminal domain (residues 26-184).

Antibiotic resistance is a growing problem and a global threat for modern healthcare. New approaches complementing the traditional antibiotic drugs are urgently needed to secure the ability to treat bacterial infections also in the future. Among the promising alternatives are bacteriolytic enzymes, such as the cell wall degrading peptidoglycan hydrolases.

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs.

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity.

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage.

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain.

Decreased temperature increases the expression of a disordered bacterial late embryogenesis abundant (LEA) protein that enhances natural transformation.

Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently.

H, C, and N NMR chemical shift assignment of the complex formed by the first EPEC EspF repeat and N-WASP GTPase binding domain.

LEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli's (EPEC's) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein's complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection.

HACANCOi: a new H-detected experiment for backbone resonance assignment of intrinsically disordered proteins.

Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. H-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs.

Dispersion from C or N: 4D experiments for backbone resonance assignment of intrinsically disordered proteins.

Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three H-detected 4D experiments with C as an additional dimension.

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors.

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017.

NMR structure of a non-conjugatable, ADP-ribosylation associated, ubiquitin-like domain from Tetrahymena thermophila polyubiquitin locus.

Background: Ubiquitin-like domains (UbLs), in addition to being post-translationally conjugated to the target through the E1-E2-E3 enzymatic cascade, can be translated as a part of the protein they ought to regulate. As integral UbLs coexist with the rest of the protein, their structural properties can differ from canonical ubiquitin, depending on the protein context and how they interact with it. In this work, we investigate T.

Structural and Functional Insights Into Lysostaphin-Substrate Interaction.

Lysostaphin from and its family enzymes rapidly acquire prominence as the next generation agents in treatment of infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity toward pentaglycine bridges in cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase.

Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family.

We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis.

H, C and N NMR chemical shift assignments of A. thaliana RCD1 RST.

The A. thaliana RCD1 (radical-induced cell death1) protein is a cellular signaling hub protein which interacts with numerous plant transcription factors from different families. It consists of three conserved domains and intervening unstructured regions, the C-terminal RST domain being responsible for the interactions with the transcription factors.

H, C and N resonance assignments of the new lysostaphin family endopeptidase catalytic domain from Staphylococcus aureus.

Lysostaphin family endopeptidases, produced by Staphylococcus genus, are zinc-dependent enzymes that cleave pentaglycine bridges of cell wall peptidoglycan. They act as autolysins to maintain cell wall metabolism or as toxins and weapons against competing strains. Consequently, these enzymes are compelling targets for new drugs as well as are potential antimicrobial agents themselves against Staphylococcus pathogens, which depend on cell wall to retain their immunity against antibiotics.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity.

Redox-dependent disulfide bond formation in SAP30L corepressor protein: Implications for structure and function.

Sin3A-associated protein 30-like (SAP30L) is one of the key proteins in a multi-subunit protein complex involved in transcriptional regulation via histone deacetylation. SAP30L, together with a highly homologous SAP30 as well as other SAP proteins (i.e.

Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20-21.

Filamins (FLNs) are large, multidomain actin cross-linking proteins with diverse functions. Besides regulating the actin cytoskeleton, they serve as important links between the extracellular matrix and the cytoskeleton by binding cell surface receptors, functioning as scaffolds for signaling proteins, and binding several other cytoskeletal proteins that regulate cell adhesion dynamics. Structurally, FLNs are formed of an amino terminal actin-binding domain followed by 24 immunoglobulin-like domains (IgFLNs).

H(N), N, C(α), C(β) and C' assignments of the intrinsically disordered C-terminus of human adenosine A2A receptor.

The C-terminus of the human adenosine A2A receptor differs from the other human adenosine receptors by its exceptional length and lack of a canonical cysteine residue. We have previously structurally characterized this C-terminal domain and its interaction with calmodulin. It was shown to be structurally disordered and flexible, and to bind calmodulin with high affinity in a calcium-dependent manner.

Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus.

Chimeric Avidin--NMR structure and dynamics of a 56 kDa homotetrameric thermostable protein.

Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin-bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin-free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability.

A novel structural unit in the N-terminal region of filamins.

Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces.

H(N), N(H), C (α), C (β), and methyl group assignments of filamin multidomain fragments IgFLNc4-5 and IgFLNa3-5.

Filamins regulate the actin cytoskeleton by cross-linking actin filaments, linking the cytoskeleton to the cell membrane, and through interaction with numerous binding partners such as intracellular signalling molecules, ion channels, receptors, enzymes and transcription factors. The rod region of filamins consists of 24 immunoglubulin (Ig)-like repeats, for some of which the functional unit is a domain pair. Our aim is to study filamin Ig domain-domain interactions and quaternary arrangement as well as to locate peptide binding sites on domain assemblies.

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors.