The PASTA domain: a β-lactam-binding domain

The PASTA domain (for penicillin-binding protein and serine/threonine kinase associated domain) is found in the high molecular weight penicillin-binding proteins and eukaryotic-like serine/threonine kinases of a range of pathogens. We describe this previously uncharacterized domain and infer that it binds β-lactam antibiotics and their peptidoglycan analogues. We postulate that PknB-like kinases are key regulators of cell-wall biosynthesis. The essential function of these enzymes suggests an additional pathway for the action of β-lactam antibiotics.     

Reuse of structural domain–domain interactions in protein networks

Background  

Protein interactions are thought to be largely mediated by interactions between structural domains. Databases such as iPfam relate interactions in protein structures to known domain families. Here, we investigate how the domain interactions from the iPfam database are distributed in protein interactions taken from the HPRD, MPact, BioGRID, DIP and IntAct databases.     

1H, 13C and 15N assignments of Sgt2 N-terminal dimerisation domain and its binding partner, Get5 Ubiquitin-like domain

The first stage of the GET (guided entry of tail-anchored proteins) mechanism for tail-anchored (TA) membrane protein insertion is thought to occur when Sgt2 (small, glutamine-rich, tetratricopeptide repeat-containing protein 2) binds TA proteins upon their release from the ribosome. It sorts them and passes the majority over to a complex of Get5 and Get4 for transmission along the GET pathway and delivery to their membrane destination. Sgt2 is a 38 kDa protein consisting of three domains. The N-terminal domain effects tight dimerisation of the protein and is also the site for binding with the ubiquitin-like (UBL) domain of Get5. Here we have expressed and purified uniformly-¹⁵N/¹³C-labelled N-terminal Sgt2 (Sgt2_NT) and its binding partner, Get5 UBL domain (Get5_UBL) and assigned the backbone and side-chain resonances as a basis for structure solution of the individual components and, ultimately, the complex. This will provide detailed molecular insight into the early stages of the GET pathway.     

The sterol-sensing domain: multiple families, a unique role?

The "sterol-sensing domain" (SSD) is conserved across phyla and is present in several membrane proteins, such as Patched (a Hedgehog receptor) and NPC-1 (the protein defective in Niemann-Pick type C1 disease). The role of the SSD is perhaps best understood from the standpoint of its involvement in cholesterol homeostasis. This article discusses how the SSD appears to function as a regulatory domain involved in linking vesicle trafficking and protein localization with such varied processes as cholesterol homeostasis, cell signalling and cytokinesis.     

BAR domain proteins—a linkage between cellular membranes,signaling pathways,and the actin cytoskeleton

Actin filament assembly typically occurs in association with cellular membranes. A large number of proteins sit at the interface between actin networks and membranes, playing diverse roles such as initiation of actin polymerization, modulation of membrane curvature, and signaling. Bin/Amphiphysin/Rvs (BAR) domain proteins have been implicated in all of these functions. The BAR domain family of proteins comprises a diverse group of multi-functional effectors, characterized by their modular architecture. In addition to the membrane-curvature sensing/inducing BAR domain module, which also mediates antiparallel dimerization, most contain auxiliary domains implicated in protein-protein and/or protein-membrane interactions, including SH3, PX, PH, RhoGEF, and RhoGAP domains. The shape of the BAR domain itself varies, resulting in three major subfamilies: the classical crescent-shaped BAR, the more extended and less curved F-BAR, and the inverse curvature I-BAR subfamilies. Most members of this family have been implicated in cellular functions that require dynamic remodeling of the actin cytoskeleton, such as endocytosis, organelle trafficking, cell motility, and T-tubule biogenesis in muscle cells. Here, we review the structure and function of mammalian BAR domain proteins and the many ways in which they are interconnected with the actin cytoskeleton.     

The β1 domain of protein G can replace the chorismate mutase domain of the T-protein

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.     

A segmental labeling strategy for unambiguous determination of domain–domain interactions of large multi-domain proteins

NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain–domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain–domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain–domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues α-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was ²H(99%)/¹⁵N-labeled whereas the C-terminal (CT-) domain was either ¹⁵N- or ¹⁵N/¹³C-labeled. 3-D ¹⁵N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.     

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.     

Backbone 1H, 15N, and 13C resonance assignments for the NOXO1β PX domain

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47 ^phox . NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

Chromatin domain boundaries： insulators and beyond

The eukaryotic genome is organized into functionally and structurally distinct domains, representing regulatory units for gene expression and chromosome behavior. DNA sequences that mark the border between adjacent domains are the insulators or boundary elements, which are required in maintenance of the function of different domains. Some insulators need others enable to play insulation activity. Chromatin domains are defined by distinct sets of post-translationally modified histones. Recent studies show that these histone modifications are also involved in establishment of sharp chromatin boundaries in order to prevent the spreading of distinct domains. Additionally, in some loci, the high-order chromatin structures for long-range looping interactions also have boundary activities, suggesting a correlation between insulators and chromatin loop domains. In this review, we will discuss recent progress in the field of chromatin domain boundaries.     

Is the myonuclear domain size fixed?

It has been suggested that the number of myonuclei in a muscle fibre changes in proportion to the change in fibre size, resulting in a constant myonuclear domain size, defined as the cytoplasmic volume per myonucleus. The myonuclear domain size varies, however, between fibre types and is inversely related with the oxidative capacity of a fibre. Overall, the observations of an increase in myonuclear domain size during both maturational growth and overload-induced hypertrophy, and the decrease in myonuclear domain size during disuse- and ageing-associated muscle atrophy suggest that the concept of a constant myonuclear domain size needs to be treated cautiously. It also suggests that only when the myonuclear domain size exceeds a certain threshold during growth or overload-induced hypertrophy acquisition of new myonuclei is required for further fibre hypertrophy.     

Understanding the role of domain–domain linkers in the spatial orientation of domains in multi-domain proteins

Inter-domain linkers (IDLs)’ bridge flanking domains and support inter-domain communication in multi-domain proteins. Their sequence and conformational preferences enable them to carry out varied functions. They also provide sufficient flexibility to facilitate domain motions and, in conjunction with the interacting interfaces, they also regulate the inter-domain geometry (IDG). In spite of the basic intuitive understanding of the inter-domain orientations with respect to linker conformations and interfaces, we still do not entirely understand the precise relationship among the three. We show that IDG is evolutionarily well conserved and is constrained by the domain–domain interface interactions. The IDLs modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. Results of our analysis provide guidelines in modelling of multi-domain proteins from the tertiary structures of constituent domain components.     

Anti-TNFα domain antibody construct CEP-37247

We report preclinical data for CEP-37247, the first human framework domain antibody construct to enter the clinic. At approximately 11 – 13kDa, domain antibodies or dAbs are the smallest antibody domain able to demonstrate the antigen-recognition function of an antibody, e.g. high selectivity and affinity for target antigen. CEP-37247 is a bivalent anti-tumor necrosis factor (TNF)α domain antibody protein construct combining the antigen-recognition function of a dAb with the pharmacological advantages of an antibody Fc region. As a homodimer, with each chain comprising V_L dAb, truncated C_H1, hinge, C_H2 and C_H3 domains, CEP-37247 has a molecular mass of approximately 78kDa, which is about half the size of a conventional IgG molecule. Surface plasmon resonance data demonstrate that CEP-37247 possesses high selectivity and affinity for TNFα. CEP-37247 is a potent neutralizer of TNFα activity in vitro in the L929 TNF-mediated cytotoxicity assay. In a human TNFα-over-expressing mouse model of polyarthritis, CEP-37247 prevents development of disease, and is at least as effective as the marketed product etanercept. Fc functionality is intact – CEP-37247 is capable of mediating antibody-dependent cell-mediated cytotoxicity and has a circulating half-life of approximately 4.5 days in cynomolgus macaques. Given the favorable properties outlined above, and its high expression levels (approaching 7 g/L) in a CHOK1 based-expression system, CEP-37247 is progressing into the clinic, where other potential advantages such as enhanced efficacy due to improved tissue distribution, and beneficial immunogenicity profile, will be evaluated.     

Characterization of the functional domain of β2-microglobulin from the Asian seabass, Lates calcarifer

Background

β2-Microglobulin (β₂M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism.

Methodology/Principal Findings

In this study, a cDNA sequence encoding β₂M in the Asian seabass (Lates calcarifer) was identified and analyzed using in silico approaches to predict and characterize its functional domain. The β₂M cDNA contains an open reading frame (ORF) of 351 bases with a coding capacity of 116 amino acids. A large portion of the protein consists of the IG constant domain (IGc1), similar to β₂M sequences from other species studied thus far. Alignment of the IGc1 domains of β₂M from L. calcarifer and other species shows a high degree of overall conservation. Seven amino acids were found to be conserved across taxa whereas conservation between L. calcarifer and other fish species was restricted to 14 amino acids at identical conserved positions.

Conclusion/Significance

As the L. calcarifer β₂M protein analyzed in this study contains a functional domain similar to that of β₂M proteins in other species, it can be postulated that the β₂M proteins from L. calcarifer and other organisms are derived from a common ancestor and thus have a similar immune function. Interestingly, fish β₂M genes could also be classified according to the ecological habitat of the species, i.e. whether it is from a freshwater, marine or euryhaline environment.