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Abstract: The desmoglein-specific cytoplasmic region (DSCR) is a conserved region of unknown structure and function that uniquely defines the desmoglein family of cell adhesion molecules. It is the site of caspase cleavage during apoptosis, and its mutation is linked to cardiomyopathy. Here, we reveal that a 276-residue DSCR construct of human desmoglein 1 is intrinsically disordered and forms an interaction hub for desmosomal proteins. In solution, it contains 6.5% helical and 10.3% β-strand structure based on circular dichroism spectroscopy. A single monomeric state with a predominantly unfolded structure is found by size-exclusion chromatography and analytical ultracentrifugation. Thermal stability assays and nuclear magnetic resonance spectroscopy reveal a nonglobular structure under a range of solution conditions. However, the introduction of detergent micelles increases structure to 18% helical and 16% β-strand character, suggesting an inducible structure. The DSCR exhibits weak but specific interactions with plakoglobin, the plakin domain of desmoplakin, plakophilin 1, and the cytoplasmic domain of desmocollin 1. The desmoglein 1 membrane proximal region also interacts with all four DSCR ligands, strongly with plakoglobin and plakophilin and more weakly with desmoplakin and desmocollin 1. Thus, the DSCR is an intrinsically disordered functional domain with an inducible structure that, along with the membrane proximal region, forms a flexible scaffold for cytoplasmic assembly at the desmosome. [Copyright &y& Elsevier]

Abstract: NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91 phox /Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22 phox , a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22 phox and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3–PRR interaction in Noxo1. [Copyright &y& Elsevier]

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47phox, binds to the C-terminal SH3 domain from p67phox. We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67phox, Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67Pphox and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47phox binds to the p67Pphox SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67phox SH3 in a compact helix-turn-helix structure (PDB entry 1K4U). [ABSTRACT FROM AUTHOR]

Examines the role of human homologue p47 and p67 adapter proteins in the activation of superoxide-producing NAD(P)H oxidases. Structure and domain architecture of p41, a novel homologue of p47; Interaction of human p41 with other oxidase factors; Effects of p41 and p51 on activation of the phagocyte NAD(P)H oxidase.

Identification of the interfaces of large (Mr > 50,000) protein?protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein?protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system. [ABSTRACT FROM AUTHOR]

Abstract: Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the “plakin domain,” which includes residues 180–1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin''s plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an “L” shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function. [Copyright &y& Elsevier]

The causative agent of Legionnaires disease, Legionellapneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (1cm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (P1) metabolism. The 1cm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (Ptdlns(4)P). Using a nonbiased screen for novel L. pneumophila P1-binding proteins, we identified the Rabi guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant Ptdlns(4)P-binding protein. Purified SidM specifically and directly bound to Ptdlns(4)P, whereas the SidM-interacting 1cm/Dot substrate LidA preferentially bound Ptdlns(3)P but also Ptdlns(4)P, and the L. pneumophila Arfi GEF Ra1F did not bind to any PIs. The Ptdlns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (tdIns P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher Ptdlns(4)P binding activity in the context of the a-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by 1cm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to Ptdlns(4)P on LCVs via its P4M domain. An L. pneumophila ΔsidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of Ptdlns(4)P on the vacuole. Finally, RNA interference revealed that Ptdlns(4)P on LCVs is specifically formed by host Ptdlns 4-kinase IIIβ. Thus, L. pneumophila exploits Ptdlns(4)P produced by Ptdlns 4-kinase IIIβ to anchor the effectors SidC and SidM to LCVs. [ABSTRACT FROM AUTHOR]

An analysis of the backbone dynamics of the C-terminal Src homology 3 (SH3) domain of p67phoxSH3(C), in complex with a 32-residue high-affinity (Kd = 24 nM) peptide, Pf, from the C-terminal region of p47phox is presented. This paper represents the first detailed analysis of the backbone dynamics and the ligand-induced changes therein of a high-affinity, high-specificity interaction involving an SH3 domain. The dynamic features are compared with those in the high-affinity, highly specific interaction between the SH3 domain of C-terminal Src kinase (Csk-SH3) and a proline-rich peptide from proline-enriched phosphatase (PEP). Both systems share common dynamic features especially in the canonical PxxP motif recognition surface where slow micro- to millisecond time scale dynamics persist on complex formation especially in several residues that are implicated in ligand recognition and in stabilizing the SH3 fold. These residues are highly conserved in SH3 domains. Ile505, which lies outside the PxxP recognition motif on p67phoxSH3(C) and is key in conferring high specificity to the p67phoxSH3(C)/Pf interaction, becomes more disordered upon complex formation. This behavior is similar to that seen in the residues that constitute the specificity surface in Csk-SH3. [ABSTRACT FROM AUTHOR]